{Next, to examine whether expression of the gene for Hes1 is regulated by miR-23, we introduced synthetic miR-23 or mutant miR-23 (Fig. 2a) into undifferentiated NT2 cells. When synthetic miR-23 was introduced at 2 mMinto undifferentiated NT2 cells,the intracellular level of Hes1 fell significantly (Fig. 2b).By contrast,in the presence of synthetic mutant miR-23,the level of Hes1 in undifferentiated NT2 cells remained unchanged and similar to that in untreated wild-type NT2 cells (Fig. 2b)}{ To examine the function of miR-23 further, we tried to reduce the level of endogenous miR-23 using synthetic siRNA (siRNA-miR-23) targeted to a loop region of the precursor to miR-23 (Fig. 2d).siRNAs can induce the RNAi-mediated sequence-specific silencing of gene expression in mammalian cells32,33. RNAi refers to the sequence-specific silencing of gene expression that is induced by double-stranded RNAs in animals and plants34,35. When 100nM synthetic siRNA-miR-23 was introduced into differentiated NT2 cells, the intracellular level of precursor and mature miR-23 fell significantly (Fig. 2e). By contrast, the level of Hes1 protein increased in the presence of siRNA-miR-23 (Fig. 2f).}
{For all eight predicted human targets of endogenous HeLa miRNAs that responded to mutations, the increase in expression seen when disrupting the pairing to the miRNA seed transwas at least as high as that seen for mutations in the outlet-7 target sites of lin-41 (Figure 3B).}
{For all eight predicted human targets of endogenous HeLa miRNAs that responded to mutations, the increase in expression seen when disrupting the pairing to the miRNA seed transwas at least as high as that seen for mutations in the outlet-7 target sites of lin-41 (Figure 3B).}
{At LNA concentrations of 10 nM, accumulation of PFV-1 mRNAs was higher in the anti-miR-32-treated cells than in the anti-miR-23-treated cells (Fig. 2C). Use of a luciferasebased assay also indicated that the antimiR-32, unlike the anti-miR-23, almost doubled progeny virus production (Fig. 2D).}
{We treated Ramos cells with 0.1 mM of 5-Aza-CdR because they are more sensitive to this drug compared to T24 cells, which caused a reduction of DNA methylation at the promoter region of miR-127 from 75% to 57% (data not shown). We confirmed that miR-127 was substantially induced by the low-dose combination treatment (Figures 2A and 2B). We found a decrease in BCL6 protein expression in T24 and Ramos cells treated with 5-Aza-CdR and PBA (Figure 6A).}{Furthermore, Ramos cells were transfected with miR-127 precursor molecules, which are designed to directly enter the miRNA processing pathway and mimic endogenous miR-127 in the cells. Overexpression of miR-127 induced a reduction of BCL6 protein level (Figure 6B)}
{The correspondence between the time-dependent increase of miR-122 and the decrease of CAT-1 was supportive of the prediction that CAT-1 was a direct target of miR-122.}
{In contrast, an equivalent amount of the anti-miR-124 LNA control oligo had no effect (Fig. 5A, lane 3). Treatment with a combination of three LNA oligos targeting miR-133, miR-1, and miR-206 increased nPTB levels slightly more than the miR-133 LNA alone (Fig. 5A, lane 5). Although PTB protein is not eliminated upon differentiation (Fig. 5A, lane 2), similar increases were seen in the PTB protein level upon LNA treatment (Fig. 5A, middle panel).}{Two days after transfection of wild-type miR-133, nPTB protein was reduced by 50% compared with control cells treated with buffer or transfected with a luciferase small interfering RNA (siRNA) (Fig. 4A). This effect was dose dependent and saturated at higher amounts of miRNA (data not shown). The mutant miR-133 gave weaker repression, at about half that of the wild-type RNA.}
{In contrast, an equivalent amount of the anti-miR-124 LNA control oligo had no effect (Fig. 5A, lane 3). Treatment with a combination of three LNA oligos targeting miR-133, miR-1, and miR-206 increased nPTB levels slightly more than the miR-133 LNA alone (Fig. 5A, lane 5). Although PTB protein is not eliminated upon differentiation (Fig. 5A, lane 2), similar increases were seen in the PTB protein level upon LNA treatment (Fig. 5A, middle panel).}{Two days after transfection of wild-type miR-133, nPTB protein was reduced by 50% compared with control cells treated with buffer or transfected with a luciferase small interfering RNA (siRNA) (Fig. 4A). This effect was dose dependent and saturated at higher amounts of miRNA (data not shown). The mutant miR-133 gave weaker repression, at about half that of the wild-type RNA.}
{Then we investigated whether both miRNAs affect Bcl2 protein expression or this effect is restricted to only one of them.We have transiently transfected miR-15a and miR-16-1 sense and antisense oligo RNAs into the same MEG-01 cell line. As shown in Fig. 2B, separate transfections of miR-15a-sense and miR-16-1-sense, respectively, completely abolished BCL2 expression,whereas transfection of antisense RNAs did not. Similar data were obtained by cotransfection with both sense RNAs or both antisense RNAs, confirming that both miR-15a and miR-16-1 influence Bcl2 protein expression.}
{Then we investigated whether both miRNAs affect Bcl2 protein expression or this effect is restricted to only one of them.We have transiently transfected miR-15a and miR-16-1 sense and antisense oligo RNAs into the same MEG-01 cell line. As shown in Fig. 2B, separate transfections of miR-15a-sense and miR-16-1-sense, respectively, completely abolished BCL2 expression,whereas transfection of antisense RNAs did not. Similar data were obtained by cotransfection with both sense RNAs or both antisense RNAs, confirming that both miR-15a and miR-16-1 influence Bcl2 protein expression.}
{Finally, we analyzed kit expression in E culture overexpressing miR 221 and_or 222, or at 2 and 4 days after transfection. Western blotting analysis showed a marked decrease of kit protein expression in the transfected cells as compared with controls (Fig. 4 Bottom).}
{Finally, we analyzed kit expression in E culture overexpressing miR 221 and_or 222, or at 2 and 4 days after transfection. Western blotting analysis showed a marked decrease of kit protein expression in the transfected cells as compared with controls (Fig. 4 Bottom).}
{We thus investigated whether miR-10a could target a HOX gene. We performed real-time PCR for the predicted HOX targets of miR-10: HOXA1, HOXA3, and HOXD10. After normalization with 18S RNA, we found that HOXA1 mRNA is up-regulated 7-fold during megakaryocytic differentiation compared with CD34 progenitors (Figs. 3A). HOXA1 protein levels were also up-regulated during megakaryocytic differentiation (Fig. 3B).These results are in a sharp contrast with the down-regulation of miR-10a in megakaryocytic differentiation, suggesting that miR- 10a could be an inhibitor of HOXA1 expression.}
{In contrast, AIB1 protein levels decreased substantially after treatment with Mir-17-5p, as shown by Western blotting (Fig. 2B) and by pulse-labeling (Fig. 2C); the reduction level was comparable to that using AIB1-specific siRNA, which occasionally decreased AIB1 protein to almost undetectable levels (data not shown).}{To further examine the function of Mir-17-5p, we tested whether the levels of endogenous Mir-17-5p in HeLa cells could be reduced by using synthetic siRNA targeted to the Mir-17-5p precursor. We selected HeLa cells for their high expression of endogenous mature Mir-17-5p. The intracellular levels of Mir-17-5p decreased significantly after treatment with Mir-17-5p-specific siRNA (Fig. 2D). As expected, the levels of endogenous AIB1 protein increased in the presence of Mir-17-5p-specific siRNA, whereas mutant siRNA did not affect the expression of Mir-17-5p or AIB1 protein (Fig. 2E). These results suggest that synthetic Mir-17-5p suppresses the expression of AIB1, primarily at the level of translation.}
{In contrast, AIB1 protein levels decreased substantially after treatment with Mir-17-5p, as shown by Western blotting (Fig. 2B) and by pulse-labeling (Fig. 2C); the reduction level was comparable to that using AIB1-specific siRNA, which occasionally decreased AIB1 protein to almost undetectable levels (data not shown).}{To further examine the function of Mir-17-5p, we tested whether the levels of endogenous Mir-17-5p in HeLa cells could be reduced by using synthetic siRNA targeted to the Mir-17-5p precursor. We selected HeLa cells for their high expression of endogenous mature Mir-17-5p. The intracellular levels of Mir-17-5p decreased significantly after treatment with Mir-17-5p-specific siRNA (Fig. 2D). As expected, the levels of endogenous AIB1 protein increased in the presence of Mir-17-5p-specific siRNA, whereas mutant siRNA did not affect the expression of Mir-17-5p or AIB1 protein (Fig. 2E). These results suggest that synthetic Mir-17-5p suppresses the expression of AIB1, primarily at the level of translation.}
{Therefore, hPFBs were transfected with the appropriate miRNAs, and AT1R levels were quantitated by performing radioreceptor binding assays. AT1R binding assays demonstrated that only hPFBs transfected with miR-155 showed a significant reduction in the expression of hAT1Rs (48.3 _ 6.2%) compared with controls (Fig. 3A).}
{We cotransfected this construct with miR-29b,miR-181b, and pre-miR negative control (scramble) into 293 cells as indicated in Fig. 2B. These experiments revealed that coexpression of Tcl1 with miR-29 and miR-181 significantly decreased Tcl1 expression (Fig. 2B, lane 2 and 4 versus lanes 1 and 3). }
{We cotransfected this construct with miR-29b,miR-181b, and pre-miR negative control (scramble) into 293 cells as indicated in Fig. 2B. These experiments revealed that coexpression of Tcl1 with miR-29 and miR-181 significantly decreased Tcl1 expression (Fig. 2B, lane 2 and 4 versus lanes 1 and 3). }
As shown in the Western blots of Fig. 2B, ERBB2 protein levels were reduced to 40 and 65% that of control (SKBR3-puro) levels in SKBR3-125a and SKBR3-125b cells, respectively, whereas ERBB3 protein levels were reduced to 60 and 80% that of control levels, respectively, after loading normalization by _-actin levels.
{As shown in the Western blots of Fig. 2B, ERBB2 protein levels were reduced to 40 and 65% that of control (SKBR3-puro) levels in SKBR3-125a and SKBR3-125b cells, respectively, whereas ERBB3 protein levels were reduced to 60 and 80% that of control levels, respectively, after loading normalization by _-actin levels.}
{As shown in the Western blots of Fig. 2B, ERBB2 protein levels were reduced to 40 and 65% that of control (SKBR3-puro) levels in SKBR3-125a and SKBR3-125b cells, respectively, whereas ERBB3 protein levels were reduced to 60 and 80% that of control levels, respectively, after loading normalization by _-actin levels.}
{As shown in the Western blots of Fig. 2B, ERBB2 protein levels were reduced to 40 and 65% that of control (SKBR3-puro) levels in SKBR3-125a and SKBR3-125b cells, respectively, whereas ERBB3 protein levels were reduced to 60 and 80% that of control levels, respectively, after loading normalization by _-actin levels.}
{We analysed the level of HDAC4 by Western blot after transfecting 3T3 cells with siRNA-140 or a non-specific siRNA. Fig. 2B shows that the control siRNA did not affect the level of HDAC4 but siRNA-140 significantly downregulated the accumulation of HDAC4 protein, confirming that it is indeed a target of miR-140, at least in vitro.}
{Overexpression of miR132 reduced levels of p250GAP-GFP whereas miR1-1 did not (Fig. 6B). Furthermore, miR132 specifically reduced endogenous p250GAP levels in primary neurons (Fig. 6C), confirming that p250GAP is a target of miR132 in vivo.}{To examine the specificity of miR132 regulation of p250GAP protein expression, we blocked miR132 function in primary neurons by using an antisense 2_O-methyl oligoribonucleotide.We found that this sequence-specific miRNA inhibitor increased levels of the p250GAP-GFP and endogenous p250GAP,whereas a sense strand 2_O-methyl oligoribonucleotide did not (Fig.6DandE).}
{Immunofluorescence demonstrated decreased Mcl-1 reactivity in mir-29b-transfected cells (Figure 3c).}{Similarly, KMCH cells transfected with the mir-29b expression plasmid p29b had reduced Mcl-1 protein by Western blot (Figure 3d).}
{Therefore, we tested these three genes by cloning their UTRs into a luciferase reporter. Interestingly, Western blot analysis of the tumor samples also indicated that the endogenous TPM1 was increased in the anti-mir-21-treated tumors by almost 2-fold (Fig. 1, C and D).}
{The results corresponded to those observed in vitro with an increase in basal expression of p-Stat-3 and decrease in NF2 in Mz-IL-6 xenografts when compared with control cell xenografts (Fig.8A). Intratumoral administration of anti-let-7a increased NF2 and decreased p-Stat-3 expression in Mz-IL-6 xenografts in vivo (Fig. 8, B and C).}
{Western blot analysis of SK-N-AS cells transfected with miR-34a showed a significant reduction in E2F3 protein in cells overexpressing miR-34a, confirming that miR-34a targets E2F3 mRNA (Figure 4b)}
{However, of the miR-143 targets proposed by Lewis et al. (4), only antibodies for ERK5/BMK1 were commercially available. Levels of ERK5 protein in normal adipocytes were compared with adipocytes that had been treated with miR-143 ASO before induction of differentiation. After 7 or 10 days of differentiation, the level of ERK5 protein, normalized to G3PDH levels, was 2-fold higher in the miR-143 ASO-treated cells (Fig. 4).}
{Co-transfection of these plasmids with miR-17-5p or miR-20a antisense oligonucleotides, but not scrambled oligonucleotides, enhanced expression of the sensor constructs, indicating inhibition of these miRNAs (Fig. 4a). Because of nucleotide similarity between miR-17-5p and miR-20a, both were inhibited by antisense oligonucleotides directed against either miRNA. Transfection with miR-17-5p and miR-20a antisense oligonucleotides, but not scrambled oligonucleotides, resulted in an approximately fourfold increase in E2F1 protein levels without altering E2F1 mRNA abundance (Fig. 4b, c).}
{[assessed from the table in the paper]}
{[assessed from the table in the paper]}
{Western blots of transgenic heart protein overexpressing miR-1 postnatally showed a significant decrease in Hand2 protein levels compared with non-transgenic littermates, confirming Hand2 as a miR-1 target in vivo (Fig. 4e). No change in mRNA levels of Hand2 was noted (Fig. 4e).}
{In addition, overexpression of miR-1 led to the underexpression of endogenous HDAC4 protein in C2C12 cells in both the growth condition(Fig.4c) and the differentiation condition(Fig.4e),wheras overexpression of miR-133 repressed the expression of endogenous SRF proteins(Fig.4c,e)}{The application of 2'-O-methyl-antisense oligobibonucleotides targeted towards miR-1 or miR-133 relieved the repression ot HDAC4 or SRF protein, respectively(Fig-4g),but had no effect on their mRNA levels(Fig.4f)}
{In addition, overexpression of miR-1 led to the underexpression of endogenous HDAC4 protein in C2C12 cells in both the growth condition(Fig.4c) and the differentiation condition(Fig.4e),wheras overexpression of miR-133 repressed the expression of endogenous SRF proteins(Fig.4c,e)}{The application of 2'-O-methyl-antisense oligobibonucleotides targeted towards miR-1 or miR-133 relieved the repression ot HDAC4 or SRF protein, respectively(Fig-4g),but had no effect on their mRNA levels(Fig.4f)}
{In addition, overexpression of miR-1 led to the underexpression of endogenous HDAC4 protein in C2C12 cells in both the growth condition(Fig.4c) and the differentiation condition(Fig.4e),wheras overexpression of miR-133 repressed the expression of endogenous SRF proteins(Fig.4c,e)}{The application of 2'-O-methyl-antisense oligobibonucleotides targeted towards miR-1 or miR-133 relieved the repression ot HDAC4 or SRF protein, respectively(Fig-4g),but had no effect on their mRNA levels(Fig.4f)/}
{In addition, overexpression of miR-1 led to the underexpression of endogenous HDAC4 protein in C2C12 cells in both the growth condition(Fig.4c) and the differentiation condition(Fig.4e),wheras overexpression of miR-133 repressed the expression of endogenous SRF proteins(Fig.4c,e)}{The application of 2'-O-methyl-antisense oligobibonucleotides targeted towards miR-1 or miR-133 relieved the repression ot HDAC4 or SRF protein, respectively(Fig-4g),but had no effect on their mRNA levels(Fig.4f)/}
{For expression studies, the human cervical cancer cell line HeLa was used because it expresses neither Cx43 nor miR-206. HeLa cells were grown in culture and transfected with pcDNA Cx43 and either pSUPER 206pri or pSUPER as a negative control.Forty-eight hours after transfection, protein extracts and total RNA were prepared. Western blot analysis against Cx43 shows a significant inhibition of Cx43 expression as shown in Figure 3A.}
{For expression studies, the human cervical cancer cell line HeLa was used because it expresses neither Cx43 nor miR-206. HeLa cells were grown in culture and transfected with pcDNA Cx43 and either pSUPER 206pri or pSUPER as a negative control.Forty-eight hours after transfection, protein extracts and total RNA were prepared. Western blot analysis against Cx43 shows a significant inhibition of Cx43 expression as shown in Figure 3A.}
{To verify that GJA1 and KCNJ2 are targets of miR-1 for posttranscriptional repression, we determined the effects of miR-1 on the expression of the protein products of these genes by western blotting. The levels of Cx43 and Kir2.1 were significantly (P o 0.05) diminished in rats that had undergone MI, consistent with our previous findings16, and this reduction was reversed by pretreatment with AMO-1.}{When mutant miR-1 (MT miR-1) was injected, there was little or no repression of GJA1 and KCNJ2, and co-application of AMO-1 with miR-1 nearly abolished the repressing effects of miR-1 on GJA1 and KCNJ2 (Fig. 2a,b). Comparison with the data in Fig. 1h indicates an inverse correlation between miR-1 levels and Cx43 and Kir2.1 expression. The hearts of humans with CAD also consistently showed reduced levels of Cx43 and Kir2.1}
{To verify that GJA1 and KCNJ2 are targets of miR-1 for posttranscriptional repression, we determined the effects of miR-1 on the expression of the protein products of these genes by western blotting. The levels of Cx43 and Kir2.1 were significantly (P o 0.05) diminished in rats that had undergone MI, consistent with our previous findings16, and this reduction was reversed by pretreatment with AMO-1.}{When mutant miR-1 (MT miR-1) was injected, there was little or no repression of GJA1 and KCNJ2, and co-application of AMO-1 with miR-1 nearly abolished the repressing effects of miR-1 on GJA1 and KCNJ2 (Fig. 2a,b). Comparison with the data in Fig. 1h indicates an inverse correlation between miR-1 levels and Cx43 and Kir2.1 expression. The hearts of humans with CAD also consistently showed reduced levels of Cx43 and Kir2.1}
{To investigate the possibility that miR-372 and miR-373 suppress the expression of LATS2, we performed immunoblot analysis of cells expressing wt and mutant miR-372&3, the cluster and the controls p53kd and empty vector. Both in the absence of RASV12 and in its presence,a significant reduction in LATS2 protein level was observed upon miR-372&3 expression (Figure 5B).}{To investigate the possibility that miR-372 and miR-373 suppress the expression of LATS2, we performed immunoblot analysis of cells expressing wt and mutant miR-372&3, the cluster and the controls p53kd and empty vector. Both in the absence of RASV12 and in its presence,a significant reduction in LATS2 protein level was observed upon miR-372&3 expression (Figure 5B).}
{To investigate the possibility that miR-372 and miR-373 suppress the expression of LATS2, we performed immunoblot analysis of cells expressing wt and mutant miR-372&3, the cluster and the controls p53kd and empty vector. Both in the absence of RASV12 and in its presence,a significant reduction in LATS2 protein level was observed upon miR-372&3 expression (Figure 5B).}{To investigate the possibility that miR-372 and miR-373 suppress the expression of LATS2, we performed immunoblot analysis of cells expressing wt and mutant miR-372&3, the cluster and the controls p53kd and empty vector. Both in the absence of RASV12 and in its presence,a significant reduction in LATS2 protein level was observed upon miR-372&3 expression (Figure 5B).}
{Since miRNAs can repress protein production by affecting either mRNA stability or translation, we assessed both mRNA and protein levels in miR-1-2 mutant hearts. qPCR revealed a nearly 2-fold increase in Irx5 mRNA levels in miR-1-2-null hearts compared to wild-type, and western blots showed an approximately 5-fold increase in Irx5 protein accumulation by densitometry (Figure 4).}
{The mRNA levels of Hand2 were unchanged in miR-1-2 mutants, but the protein levels were increased approximately 4-fold as seen by western blots (Figure 3),consistent with Hand2 being a physiologic miR-1 target in vivo.}
{To see if KCNQ1 repression by miR-133 reported by luciferase assays has functional implications, we determined the effects of miR-133 on endogenous expression of KCNQ1 at protein levels by Western blot with HEK293 membrane protein samples. Our data showed that transfection of miR-133 at 10 nM remarkably reduced KCNQ1 protein level, and as a negative control the mutant miR-133 did not cause any appreciable changes (Fig. 4E).}{Transfection of miR-1 (100 nM) failed to affectKCNQ1 protein level, validating base-pairing as a major criterion for miRNA targeting. Moreover, transfection of AMO-133 alone significantly increased KCNQ1 protein level, presumably by removing the inhibitory effect of endogenous miR-133}{[assessed from the westernblot picture in the paper]}
{To see if KCNQ1 repression by miR-133 reported by luciferase assays has functional implications, we determined the effects of miR-133 on endogenous expression of KCNQ1 at protein levels by Western blot with HEK293 membrane protein samples. Our data showed that transfection of miR-133 at 10 nM remarkably reduced KCNQ1 protein level, and as a negative control the mutant miR-133 did not cause any appreciable changes (Fig. 4E).}{Transfection of miR-1 (100 nM) failed to affectKCNQ1 protein level, validating base-pairing as a major criterion for miRNA targeting. Moreover, transfection of AMO-133 alone significantly increased KCNQ1 protein level, presumably by removing the inhibitory effect of endogenous miR-133}{[assessed from the westernblot picture in the paper]}
{To see if KCNQ1 repression by miR-133 reported by luciferase assays has functional implications, we determined the effects of miR-133 on endogenous expression of KCNQ1 at protein levels by Western blot with HEK293 membrane protein samples. Our data showed that transfection of miR-133 at 10 nM remarkably reduced KCNQ1 protein level, and as a negative control the mutant miR-133 did not cause any appreciable changes (Fig. 4E).}{Transfection of miR-1 (100 nM) failed to affectKCNQ1 protein level, validating base-pairing as a major criterion for miRNA targeting. Moreover, transfection of AMO-133 alone significantly increased KCNQ1 protein level, presumably by removing the inhibitory effect of endogenous miR-133}{[assessed from the westernblot picture in the paper]}
{To see if KCNQ1 repression by miR-133 reported by luciferase assays has functional implications, we determined the effects of miR-133 on endogenous expression of KCNQ1 at protein levels by Western blot with HEK293 membrane protein samples. Our data showed that transfection of miR-133 at 10 nM remarkably reduced KCNQ1 protein level, and as a negative control the mutant miR-133 did not cause any appreciable changes (Fig. 4E).}{Transfection of miR-1 (100 nM) failed to affectKCNQ1 protein level, validating base-pairing as a major criterion for miRNA targeting. Moreover, transfection of AMO-133 alone significantly increased KCNQ1 protein level, presumably by removing the inhibitory effect of endogenous miR-133}{[assessed from the westernblot picture in the paper]}
{Our experiments demonstrated that KCNE1 is critically regulated by miR-1, but not by miR-133; miR-1 (10 nM) produced some 79% abrogation of the luciferase activity reported by the vectors carrying the 30UTR of KCNE1 and 70% reduction of KCNE1 protein level (Fig. 5C,E).}{Notably, transfection of AMO-1 alone significantly increased KCNE1 protein level (Fig. 5E).}{[assessed from the westernblot picture in the paper]}
{Our experiments demonstrated that KCNE1 is critically regulated by miR-1, but not by miR-133; miR-1 (10 nM) produced some 79% abrogation of the luciferase activity reported by the vectors carrying the 30UTR of KCNE1 and 70% reduction of KCNE1 protein level (Fig. 5C,E).}{Notably, transfection of AMO-1 alone significantly increased KCNE1 protein level (Fig. 5E).}{[assessed from the westernblot picture in the paper]}
{Our experiments demonstrated that KCNE1 is critically regulated by miR-1, but not by miR-133; miR-1 (10 nM) produced some 79% abrogation of the luciferase activity reported by the vectors carrying the 30UTR of KCNE1 and 70% reduction of KCNE1 protein level (Fig. 5C,E).}{Notably, transfection of AMO-1 alone significantly increased KCNE1 protein level (Fig. 5E).}{[assessed from the westernblot picture in the paper]}
{Western blots for CDK6 in original HCT-116 cells and HCT-116 cells transfected with miR-124a precursor, showing CDK6 down-regulation. RT-PCR analyses of CDK6 mRNA do not show any change.}{Using CDK6 Western blot analyses, we observed that whereas the original HCT-116 cells with miR-124a methylation-associated silencing strongly expressed CDK6, the cells treated with the demethylating agent or the DKO cells showed CDK6 down-regulation (Fig. 3A). On the other hand, there was no difference in the CDK6 mRNA expression levels in any of the described cells (Fig. 3A), suggesting translational inhibition of CDK6 by miR-124a, rather than mRNA degradation or transcriptional repression.}
{Co-transfection of the chimeric constructs with miR-1 or miR-133 (Fig. 5) into HEK293 cells, consistently resulted in smaller luciferase activity relative to transfection of the chimeric plasmid alone.}{Co-application of miR-1 or miR-133 with their respective AMOs eliminated the silencing effects.}
{Co-transfection of the chimeric constructs with miR-1 or miR-133 (Fig. 5) into HEK293 cells, consistently resulted in smaller luciferase activity relative to transfection of the chimeric plasmid alone.}{Co-application of miR-1 or miR-133 with their respective AMOs eliminated the silencing effects.}
{Co-transfection of the chimeric constructs with miR-1 or miR-133 (Fig. 5) into HEK293 cells, consistently resulted in smaller luciferase activity relative to transfection of the chimeric plasmid alone.}{Co-application of miR-1 or miR-133 with their respective AMOs eliminated the silencing effects.}
{Co-transfection of the chimeric constructs with miR-1 or miR-133 (Fig. 5) into HEK293 cells, consistently resulted in smaller luciferase activity relative to transfection of the chimeric plasmid alone.}{Co-application of miR-1 or miR-133 with their respective AMOs eliminated the silencing effects.}
{Co-transfection of the chimeric constructs with miR-1 or miR-133 (Fig. 5) into HEK293 cells, consistently resulted in smaller luciferase activity relative to transfection of the chimeric plasmid alone.}{Co-application of miR-1 or miR-133 with their respective AMOs eliminated the silencing effects.}
{We first determined the effects of the two miRNAs on protein levels, by western blotting and caspase activity assays. miR-1 remarkably reduced the levels of HSP60 proteins by ~73% in H9c2 cells both in the absence and presence of H2O2 (Fig. 3A).HSP70 protein level was similarly reduced, albeit to a less extent (~53%; Fig. 3B). Co-application of miR-1 and AMO-1 almost abolished the effects.}{We first determined the effects of the two miRNAs on protein levels, by western blotting and caspase activity assays. miR-1 remarkably reduced the levels of HSP60 proteins by ~73% in H9c2 cells both in the absence and presence of H2O2 (Fig. 3A).HSP70 protein level was similarly reduced, albeit to a less extent (~53%; Fig. 3B). Co-application of miR-1 and AMO-1 almost abolished the effects.}
{We first determined the effects of the two miRNAs on protein levels, by western blotting and caspase activity assays. miR-1 remarkably reduced the levels of HSP60 proteins by ~73% in H9c2 cells both in the absence and presence of H2O2 (Fig. 3A).HSP70 protein level was similarly reduced, albeit to a less extent (~53%; Fig. 3B). Co-application of miR-1 and AMO-1 almost abolished the effects.}{We first determined the effects of the two miRNAs on protein levels, by western blotting and caspase activity assays. miR-1 remarkably reduced the levels of HSP60 proteins by ~73% in H9c2 cells both in the absence and presence of H2O2 (Fig. 3A).HSP70 protein level was similarly reduced, albeit to a less extent (~53%; Fig. 3B). Co-application of miR-1 and AMO-1 almost abolished the effects.}
{By comparison, miR-133 decreased total Casp9 protein level by ~89% (Fig. 3C), an effect eliminated by co-transfection with AMO-133.}
{By comparison, miR-133 decreased total Casp9 protein level by ~89% (Fig. 3C), an effect eliminated by co-transfection with AMO-133.}
{By comparison, miR-133 decreased total Casp9 protein level by ~89% (Fig. 3C), an effect eliminated by co-transfection with AMO-133.}
{By comparison, miR-133 decreased total Casp9 protein level by ~89% (Fig. 3C), an effect eliminated by co-transfection with AMO-133.}
{By comparison, miR-133 decreased total Casp9 protein level by ~89% (Fig. 3C), an effect eliminated by co-transfection with AMO-133.}
{By comparison, miR-133 decreased total Casp9 protein level by ~89% (Fig. 3C), an effect eliminated by co-transfection with AMO-133.}
{[assessed from the westernblot picture in the paper]}
{[assessed from the westernblot picture in the paper]}
{[assessed from the westernblot picture in the paper]}
{[assessed from the westernblot picture in the paper]}
{[assessed from the westernblot picture in the paper]}
{[assessed from the westernblot picture in the paper]}
{Third, no additive effects of the miRNA-masking antisense ODNs and AMOs are expected if the action of the former is to block the miRNA that is antagonized by the AMOs. To test this point, we compared the effects of the miRNA-masking antisense ODNs for HCNs or AMOs alone with those of co-transfection with the miRNA-masking antisense ODNs and AMOs. The results showed no significant difference among the different treatments (Fig. 3E).}{Luciferase reporter systemwas alsoemployed to verify the protein expression (nU5 for each group) and the core promoter fragments (S191/S1 forHCN2 and S267/R157 for HCN4, relative to the transcription start sites; see Supplementary Data) were used}
{Third, no additive effects of the miRNA-masking antisense ODNs and AMOs are expected if the action of the former is to block the miRNA that is antagonized by the AMOs. To test this point, we compared the effects of the miRNA-masking antisense ODNs for HCNs or AMOs alone with those of co-transfection with the miRNA-masking antisense ODNs and AMOs. The results showed no significant difference among the different treatments (Fig. 3E).}{Luciferase reporter systemwas alsoemployed to verify the protein expression (nU5 for each group) and the core promoter fragments (S191/S1 forHCN2 and S267/R157 for HCN4, relative to the transcription start sites; see Supplementary Data) were used}
{Third, no additive effects of the miRNA-masking antisense ODNs and AMOs are expected if the action of the former is to block the miRNA that is antagonized by the AMOs. To test this point, we compared the effects of the miRNA-masking antisense ODNs for HCNs or AMOs alone with those of co-transfection with the miRNA-masking antisense ODNs and AMOs. The results showed no significant difference among the different treatments (Fig. 3E).}{Luciferase reporter systemwas alsoemployed to verify the protein expression (nU5 for each group) and the core promoter fragments (S191/S1 forHCN2 and S267/R157 for HCN4, relative to the transcription start sites; see Supplementary Data) were used}
{For example, the increase in Nedd4 protein level in the miR-1 mutant embryos is most easily explained if Nedd4 is a genuine target in miR-1 expressing cells.}
{We also examined the effect of miR-124 on endogenous PTBP1 and PTBP2 protein levels. The PTBP1 levels decreased_4.6-fold in CAD cells transfected with RIPmiR-124 (Figure 1F, lanes 1 and 2).}
{As shown in Fig. 3B, the CYP1B1 protein level was significantly increased by the transfection of the AsO for miR-27b. That there was no change of the CYP1A1 protein level indicated that the effects of the AsO for miR-27b were specific for CYP1B1 protein.}{As shown in Fig. 4B, the CYP1B1 protein level was significantly decreased by the transfection of the precursor for miR-27b. P450-Glo assays showed that the enzymatic activity of CYP1B1 was decreased by the electroporation of the precursor for miR-27b in HEK293/1B1 cells in a concentration-dependent manner (Fig. 4C). These results suggest that miR-27b regulates the protein level and enzymatic activity of CYP1B1.}
{Our data showed that transfection of miR-133 reduced HERG protein level down to _10% of control value,and as a negative control the mutant miR-133 did not cause any appreciable changes (Fig. 2C).}{Co-application of AMO-133 nearly abolished the effects of miR-133, verifying the specificity of the miR-133 action.}{Co-application of AMO-133 nearly abolished the effects of miR-133, verifying the specificity of the miR-133 action.}{Cotransfection of miR-133 and the chimeric luciferase-HERG vector into HEK293 cells consistently demonstrated smaller luciferase activities relative to transfection of the chimeric plasmid alone, but co-transfection of the mutant miR-133 (M-miR-133) failed to produce any effects (Fig. 2A).}{Co-application of miR-133 with its antisense inhibitor AMO-133 eliminated the silencing effect on luciferase reporter activities (16, 19, 20). As an additional negative control, application of miR-1 failed to affect luciferase reporter activity.}{Cotransfection of miR-133 and the chimeric luciferase-HERG vector into HEK293 cells consistently demonstrated smaller luciferase activities relative to transfection of the chimeric plasmid alone, but co-transfection of the mutant miR-133 (M-miR-133) failed to produce any effects (Fig. 2A).}
{Our data showed that transfection of miR-133 reduced HERG protein level down to _10% of control value,and as a negative control the mutant miR-133 did not cause any appreciable changes (Fig. 2C).}{Co-application of AMO-133 nearly abolished the effects of miR-133, verifying the specificity of the miR-133 action.}{Co-application of AMO-133 nearly abolished the effects of miR-133, verifying the specificity of the miR-133 action.}{Cotransfection of miR-133 and the chimeric luciferase-HERG vector into HEK293 cells consistently demonstrated smaller luciferase activities relative to transfection of the chimeric plasmid alone, but co-transfection of the mutant miR-133 (M-miR-133) failed to produce any effects (Fig. 2A).}{Co-application of miR-133 with its antisense inhibitor AMO-133 eliminated the silencing effect on luciferase reporter activities (16, 19, 20). As an additional negative control, application of miR-1 failed to affect luciferase reporter activity.}{Cotransfection of miR-133 and the chimeric luciferase-HERG vector into HEK293 cells consistently demonstrated smaller luciferase activities relative to transfection of the chimeric plasmid alone, but co-transfection of the mutant miR-133 (M-miR-133) failed to produce any effects (Fig. 2A).}
{Our data showed that transfection of miR-133 reduced HERG protein level down to _10% of control value,and as a negative control the mutant miR-133 did not cause any appreciable changes (Fig. 2C).}{Co-application of AMO-133 nearly abolished the effects of miR-133, verifying the specificity of the miR-133 action.}{Co-application of AMO-133 nearly abolished the effects of miR-133, verifying the specificity of the miR-133 action.}{Cotransfection of miR-133 and the chimeric luciferase-HERG vector into HEK293 cells consistently demonstrated smaller luciferase activities relative to transfection of the chimeric plasmid alone, but co-transfection of the mutant miR-133 (M-miR-133) failed to produce any effects (Fig. 2A).}{Co-application of miR-133 with its antisense inhibitor AMO-133 eliminated the silencing effect on luciferase reporter activities (16, 19, 20). As an additional negative control, application of miR-1 failed to affect luciferase reporter activity.}{Cotransfection of miR-133 and the chimeric luciferase-HERG vector into HEK293 cells consistently demonstrated smaller luciferase activities relative to transfection of the chimeric plasmid alone, but co-transfection of the mutant miR-133 (M-miR-133) failed to produce any effects (Fig. 2A).}
{Our data showed that transfection of miR-133 reduced HERG protein level down to _10% of control value,and as a negative control the mutant miR-133 did not cause any appreciable changes (Fig. 2C).}{Co-application of AMO-133 nearly abolished the effects of miR-133, verifying the specificity of the miR-133 action.}{Co-application of AMO-133 nearly abolished the effects of miR-133, verifying the specificity of the miR-133 action.}{Cotransfection of miR-133 and the chimeric luciferase-HERG vector into HEK293 cells consistently demonstrated smaller luciferase activities relative to transfection of the chimeric plasmid alone, but co-transfection of the mutant miR-133 (M-miR-133) failed to produce any effects (Fig. 2A).}{Co-application of miR-133 with its antisense inhibitor AMO-133 eliminated the silencing effect on luciferase reporter activities (16, 19, 20). As an additional negative control, application of miR-1 failed to affect luciferase reporter activity.}{Cotransfection of miR-133 and the chimeric luciferase-HERG vector into HEK293 cells consistently demonstrated smaller luciferase activities relative to transfection of the chimeric plasmid alone, but co-transfection of the mutant miR-133 (M-miR-133) failed to produce any effects (Fig. 2A).}
{Our data showed that transfection of miR-133 reduced HERG protein level down to _10% of control value,and as a negative control the mutant miR-133 did not cause any appreciable changes (Fig. 2C).}{Co-application of AMO-133 nearly abolished the effects of miR-133, verifying the specificity of the miR-133 action.}{Co-application of AMO-133 nearly abolished the effects of miR-133, verifying the specificity of the miR-133 action.}{Cotransfection of miR-133 and the chimeric luciferase-HERG vector into HEK293 cells consistently demonstrated smaller luciferase activities relative to transfection of the chimeric plasmid alone, but co-transfection of the mutant miR-133 (M-miR-133) failed to produce any effects (Fig. 2A).}{Co-application of miR-133 with its antisense inhibitor AMO-133 eliminated the silencing effect on luciferase reporter activities (16, 19, 20). As an additional negative control, application of miR-1 failed to affect luciferase reporter activity.}{Cotransfection of miR-133 and the chimeric luciferase-HERG vector into HEK293 cells consistently demonstrated smaller luciferase activities relative to transfection of the chimeric plasmid alone, but co-transfection of the mutant miR-133 (M-miR-133) failed to produce any effects (Fig. 2A).}
{Our data showed that transfection of miR-133 reduced HERG protein level down to _10% of control value,and as a negative control the mutant miR-133 did not cause any appreciable changes (Fig. 2C).}{Co-application of AMO-133 nearly abolished the effects of miR-133, verifying the specificity of the miR-133 action.}{Co-application of AMO-133 nearly abolished the effects of miR-133, verifying the specificity of the miR-133 action.}{Cotransfection of miR-133 and the chimeric luciferase-HERG vector into HEK293 cells consistently demonstrated smaller luciferase activities relative to transfection of the chimeric plasmid alone, but co-transfection of the mutant miR-133 (M-miR-133) failed to produce any effects (Fig. 2A).}{Co-application of miR-133 with its antisense inhibitor AMO-133 eliminated the silencing effect on luciferase reporter activities (16, 19, 20). As an additional negative control, application of miR-1 failed to affect luciferase reporter activity.}{Cotransfection of miR-133 and the chimeric luciferase-HERG vector into HEK293 cells consistently demonstrated smaller luciferase activities relative to transfection of the chimeric plasmid alone, but co-transfection of the mutant miR-133 (M-miR-133) failed to produce any effects (Fig. 2A).}
{In addition, THRAP1 protein expression was increased in cardiac protein lysates from miR-208?/? mice compared with that in wild-type littermates (Fig. 5D), whereas THRAP1 mRNA was comparable in hearts of the two genotypes (fig. S6), consistent with the conclusion that miR-208 acts as a negative regulator of THRAP1 translation in vivo.}{In addition, THRAP1 protein expression was increased in cardiac protein lysates from miR-208?/- mice compared with that in wild-type littermates (Fig. 5D), whereas THRAP1 mRNA was comparable in hearts of the two genotypes (fig. S6), consistent with the conclusion that miR-208 acts as a negative regulator of THRAP1 translation in vivo.}
{Some transcripts showed a modest increase in the total input mRNA when miR-30a-3p was knocked down (Fig. 5). This is consistent with a recent report suggesting that miRNAs overexpression could also reduce target mRNA abundance (21).}
{Some transcripts showed a modest increase in the total input mRNA when miR-30a-3p was knocked down (Fig. 5). This is consistent with a recent report suggesting that miRNAs overexpression could also reduce target mRNA abundance (21).}
{Some transcripts showed a modest increase in the total input mRNA when miR-30a-3p was knocked down (Fig. 5). This is consistent with a recent report suggesting that miRNAs overexpression could also reduce target mRNA abundance (21).}
{Some transcripts showed a modest increase in the total input mRNA when miR-30a-3p was knocked down (Fig. 5). This is consistent with a recent report suggesting that miRNAs overexpression could also reduce target mRNA abundance (21).}
{Some transcripts showed a modest increase in the total input mRNA when miR-30a-3p was knocked down (Fig. 5). This is consistent with a recent report suggesting that miRNAs overexpression could also reduce target mRNA abundance (21).}
{Some transcripts showed a modest increase in the total input mRNA when miR-30a-3p was knocked down (Fig. 5). This is consistent with a recent report suggesting that miRNAs overexpression could also reduce target mRNA abundance (21).}
{Some transcripts showed a modest increase in the total input mRNA when miR-30a-3p was knocked down (Fig. 5). This is consistent with a recent report suggesting that miRNAs overexpression could also reduce target mRNA abundance (21).}
{Some transcripts showed a modest increase in the total input mRNA when miR-30a-3p was knocked down (Fig. 5). This is consistent with a recent report suggesting that miRNAs overexpression could also reduce target mRNA abundance (21).}
{We then experimentally confirmed the in-silico predictions for three different cancer genes, Retinoblastoma (RB1), TGF_-2 receptor (TGFBR2), and PLAG1. Three of four miRNA::mRNA predicted interactions (75%) were found to be positive in a luciferase assay by showing a significant reduction of protein translation in respect to the scrambled control oligoRNAs (Fig. 3A). Retinoblastoma 3_UTR, for example, was found to be functionally interacting strongly with miR-106a.}
{We then experimentally confirmed the in-silico predictions for three different cancer genes, Retinoblastoma (RB1), TGF_-2 receptor (TGFBR2), and PLAG1. Three of four miRNA::mRNA predicted interactions (75%) were found to be positive in a luciferase assay by showing a significant reduction of protein translation in respect to the scrambled control oligoRNAs (Fig. 3A). Retinoblastoma 3_UTR, for example, was found to be functionally interacting strongly with miR-106a.}
{We then experimentally confirmed the in-silico predictions for three different cancer genes, Retinoblastoma (RB1), TGF_-2 receptor (TGFBR2), and PLAG1. Three of four miRNA::mRNA predicted interactions (75%) were found to be positive in a luciferase assay by showing a significant reduction of protein translation in respect to the scrambled control oligoRNAs (Fig. 3A). Retinoblastoma 3_UTR, for example, was found to be functionally interacting strongly with miR-106a.}
{Whether or not miR-181 could affect Hox-A11 cellular protein levels was examined and it was found that Hox-A11 protein was downregulated by ectopic miR-181a in proliferating myoblasts (Fig. 4a).}{Conversely, Hox-A11 was upregulated by inhibition of miR-181; absolute levels of Hox-A11 protein were higher in cells treated with the LNA antisense oligonucleotides than in control cells (Fig. 4b). Twofold upregulation of the Hox-A11 protein was estimated by semi-quantitative analysis of the western blots, similarly to previously published data on miR-375 and its target, myotrophin25.}
{Peptide-mediated delivery of miR-134 into neurons led to a dosedependent decrease in the level of endogenous Limk1 protein, whereas delivery of its inhibitor 20-O-Me-134 led to an increase in protein level (Fig. 4c, d), suggesting that miR-134 inhibits translation of the endogenous Limk1 mRNA. Taken together, these data suggest that endogenous miR-134 inhibits Limk1 mRNA translation in neurons by binding to a single site present in the Limk1 30 UTR.}
{Western blots from protein extracts obtained from the NIH 3T3 cells after treatment of PMA revealed dramatically reduced activation of Fos after si-miR-7b transfection (Fig. 4A).}
{Western blots from protein extracts obtained from the NIH 3T3 cells after treatment of PMA revealed dramatically reduced activation of Fos after si-miR-7b transfection (Fig. 4A).}
{miR-206 alone is suffi cient to decrease Pola1 at the mRNA and protein levels as early as 24 h after transfection (Fig. 4 A). During differentiation of MBs to MTs, Pola1 mRNA and protein were also decreased (Fig. 4 A).}
{miR-206 alone is suffi cient to decrease Pola1 at the mRNA and protein levels as early as 24 h after transfection (Fig. 4 A). During differentiation of MBs to MTs, Pola1 mRNA and protein were also decreased (Fig. 4 A).}
{Although all of them have predicted target sites (Table S3), fusion to luciferase reporter reveals that only B-ind1, Cx43, and Mmd are direct targets of miR-206 like DNA pol ? (Fig. 6 D).}
{Although all of them have predicted target sites (Table S3), fusion to luciferase reporter reveals that only B-ind1, Cx43, and Mmd are direct targets of miR-206 like DNA pol ? (Fig. 6 D).}
{Although all of them have predicted target sites (Table S3), fusion to luciferase reporter reveals that only B-ind1, Cx43, and Mmd are direct targets of miR-206 like DNA pol ? (Fig. 6 D).}
{Although all of them have predicted target sites (Table S3), fusion to luciferase reporter reveals that only B-ind1, Cx43, and Mmd are direct targets of miR-206 like DNA pol ? (Fig. 6 D).}
{In agreement with this finding, Western blot analysis confirmed a reduced expression of OC2 in cells transfected with the mir-9 duplex (Fig. 6C).}
{In agreement with this finding, Western blot analysis confirmed a reduced expression of OC2 in cells transfected with the mir-9 duplex (Fig. 6C).}
{In agreement with this finding, Western blot analysis confirmed a reduced expression of OC2 in cells transfected with the mir-9 duplex (Fig. 6C).}
{To assess the function of the predicted bantam target sites, we produced a tubulin-EGFP sensor transgene using the 3_UTR of the hid mRNA. The resulting GFP pattern was identical to that produced by the bantam sensor (Figure 6B, compare with Figure 3C). In addition, the hid UTR sensor was downregulated when EP(3)3622 was overexpressed under ptc-Gal4 control (Figure 6C), indicating that the hid UTR confers bantam-dependent regulation on the transgene. }
{To assess the function of the predicted bantam target sites, we produced a tubulin-EGFP sensor transgene using the 3_UTR of the hid mRNA. The resulting GFP pattern was identical to that produced by the bantam sensor (Figure 6B, compare with Figure 3C). In addition, the hid UTR sensor was downregulated when EP(3)3622 was overexpressed under ptc-Gal4 control (Figure 6C), indicating that the hid UTR confers bantam-dependent regulation on the transgene. }
{To assess the function of the predicted bantam target sites, we produced a tubulin-EGFP sensor transgene using the 3_UTR of the hid mRNA. The resulting GFP pattern was identical to that produced by the bantam sensor (Figure 6B, compare with Figure 3C). In addition, the hid UTR sensor was downregulated when EP(3)3622 was overexpressed under ptc-Gal4 control (Figure 6C), indicating that the hid UTR confers bantam-dependent regulation on the transgene. }
{To assess the function of the predicted bantam target sites, we produced a tubulin-EGFP sensor transgene using the 3_UTR of the hid mRNA. The resulting GFP pattern was identical to that produced by the bantam sensor (Figure 6B, compare with Figure 3C). In addition, the hid UTR sensor was downregulated when EP(3)3622 was overexpressed under ptc-Gal4 control (Figure 6C), indicating that the hid UTR confers bantam-dependent regulation on the transgene. }
{To assess the function of the predicted bantam target sites, we produced a tubulin-EGFP sensor transgene using the 3_UTR of the hid mRNA. The resulting GFP pattern was identical to that produced by the bantam sensor (Figure 6B, compare with Figure 3C). In addition, the hid UTR sensor was downregulated when EP(3)3622 was overexpressed under ptc-Gal4 control (Figure 6C), indicating that the hid UTR confers bantam-dependent regulation on the transgene. }
{We measured Drice levels in adults by using an antiDrice antibody. Drice was elevated in mir-14_1 flies as compared to the wild-type, and this increase was suppressed in the presence of two copies of the mir-14-containing 3.4 kb genomic DNA fragment (Figure 3D). }
{The bristle defect was rescued to different degrees with two different UAS-pre-miR-9a transgenic lines (lines #3 and #8) (Fig. 4H). As a control, overexpression of the mutant miR-9a precursor with a 7-nt deletion failed to rescue the sens overexpression phenotype (Fig. 4H). These data provide further evidence that miR-9a and sens function in the same genetic pathway in controlling SOP formation.}
{The bristle defect was rescued to different degrees with two different UAS-pre-miR-9a transgenic lines (lines #3 and #8) (Fig. 4H). As a control, overexpression of the mutant miR-9a precursor with a 7-nt deletion failed to rescue the sens overexpression phenotype (Fig. 4H). These data provide further evidence that miR-9a and sens function in the same genetic pathway in controlling SOP formation.}
{The bristle defect was rescued to different degrees with two different UAS-pre-miR-9a transgenic lines (lines #3 and #8) (Fig. 4H). As a control, overexpression of the mutant miR-9a precursor with a 7-nt deletion failed to rescue the sens overexpression phenotype (Fig. 4H). These data provide further evidence that miR-9a and sens function in the same genetic pathway in controlling SOP formation.}
{We found that reporter gene activity is downregulated around the L4 stage (Figures 2D-2F), around the same time that let-7 is expressed in the seam cells (Johnson et al., 2003). In contrast, the same reporter gene fused to an unregulated control3'UTR was expressed at all stages (Figure 2F; Reinhart et al., 2000; Slack et al., 2000; Vella et al., 2004; Wight man et al., 1993).}
{We searched for LCSs in the 3'UTRs of all genes known to play a role in vulval development (Table S1). LCSs have the potential to bind all members of the let-7 family, including mir-84. Approximately 11 vulval genes contained at least one LCS (Table S1), raising the possiprecocious bility that the let-7 family may regulate multiple genes in the vulva. In this analysis though, let-60/RAS stood out due to the high number of LCS sites.}
{We searched for LCSs in the 3'UTRs of all genes known to play a role in vulval development (Table S1). LCSs have the potential to bind all members of the let-7 family, including mir-84. Approximately 11 vulval genes contained at least one LCS (Table S1), raising the possiprecocious bility that the let-7 family may regulate multiple genes in the vulva. In this analysis though, let-60/RAS stood out due to the high number of LCS sites.}
{We searched for LCSs in the 3'UTRs of all genes known to play a role in vulval development (Table S1). LCSs have the potential to bind all members of the let-7 family, including mir-84. Approximately 11 vulval genes contained at least one LCS (Table S1), raising the possiprecocious bility that the let-7 family may regulate multiple genes in the vulva. In this analysis though, let-60/RAS stood out due to the high number of LCS sites.}
{We searched for LCSs in the 3'UTRs of all genes known to play a role in vulval development (Table S1). LCSs have the potential to bind all members of the let-7 family, including mir-84. Approximately 11 vulval genes contained at least one LCS (Table S1), raising the possiprecocious bility that the let-7 family may regulate multiple genes in the vulva. In this analysis though, let-60/RAS stood out due to the high number of LCS sites.}
{We searched for LCSs in the 3'UTRs of all genes known to play a role in vulval development (Table S1). LCSs have the potential to bind all members of the let-7 family, including mir-84. Approximately 11 vulval genes contained at least one LCS (Table S1), raising the possiprecocious bility that the let-7 family may regulate multiple genes in the vulva. In this analysis though, let-60/RAS stood out due to the high number of LCS sites.}
{We searched for LCSs in the 3'UTRs of all genes known to play a role in vulval development (Table S1). LCSs have the potential to bind all members of the let-7 family, including mir-84. Approximately 11 vulval genes contained at least one LCS (Table S1), raising the possiprecocious bility that the let-7 family may regulate multiple genes in the vulva. In this analysis though, let-60/RAS stood out due to the high number of LCS sites.}
{We searched for LCSs in the 3'UTRs of all genes known to play a role in vulval development (Table S1). LCSs have the potential to bind all members of the let-7 family, including mir-84. Approximately 11 vulval genes contained at least one LCS (Table S1), raising the possiprecocious bility that the let-7 family may regulate multiple genes in the vulva. In this analysis though, let-60/RAS stood out due to the high number of LCS sites.}
{We searched for LCSs in the 3'UTRs of all genes known to play a role in vulval development (Table S1). LCSs have the potential to bind all members of the let-7 family, including mir-84. Approximately 11 vulval genes contained at least one LCS (Table S1), raising the possiprecocious bility that the let-7 family may regulate multiple genes in the vulva. In this analysis though, let-60/RAS stood out due to the high number of LCS sites.}
{The fact that the let-7 null mutant phenotype depends on a wild-type copy of daf-12 supports the idea that daf-12 acts downstream of let-7.}
{The fact that the let-7 null mutant phenotype depends on a wild-type copy of daf-12 supports the idea that daf-12 acts downstream of let-7.}
{In the nervous system, hbl-1::gfp::hbl-1 deregulation was more pronounced, with 35% of animals exhibiting increased expression in the VNC of let-7 mutant adults Reduring blind scoring tests (Figures 4G and 4H). Similar deregulation was also observed in the anterior nerve ring (Figures 4J and 4K). These results suggest that let-7 plays a role in controlling hbl-1::gfp::hbl-1 expression in the nervous system.}
{In the nervous system, hbl-1::gfp::hbl-1 deregulation was more pronounced, with 35% of animals exhibiting increased expression in the VNC of let-7 mutant adults Reduring blind scoring tests (Figures 4G and 4H). Similar deregulation was also observed in the anterior nerve ring (Figures 4J and 4K). These results suggest that let-7 plays a role in controlling hbl-1::gfp::hbl-1 expression in the nervous system.}
{In the nervous system, hbl-1::gfp::hbl-1 deregulation was more pronounced, with 35% of animals exhibiting increased expression in the VNC of let-7 mutant adults Reduring blind scoring tests (Figures 4G and 4H). Similar deregulation was also observed in the anterior nerve ring (Figures 4J and 4K). These results suggest that let-7 plays a role in controlling hbl-1::gfp::hbl-1 expression in the nervous system.}
{In the nervous system, hbl-1::gfp::hbl-1 deregulation was more pronounced, with 35% of animals exhibiting increased expression in the VNC of let-7 mutant adults Reduring blind scoring tests (Figures 4G and 4H). Similar deregulation was also observed in the anterior nerve ring (Figures 4J and 4K). These results suggest that let-7 plays a role in controlling hbl-1::gfp::hbl-1 expression in the nervous system.}
{In the nervous system, hbl-1::gfp::hbl-1 deregulation was more pronounced, with 35% of animals exhibiting increased expression in the VNC of let-7 mutant adults Reduring blind scoring tests (Figures 4G and 4H). Similar deregulation was also observed in the anterior nerve ring (Figures 4J and 4K). These results suggest that let-7 plays a role in controlling hbl-1::gfp::hbl-1 expression in the nervous system.}
{In the nervous system, hbl-1::gfp::hbl-1 deregulation was more pronounced, with 35% of animals exhibiting increased expression in the VNC of let-7 mutant adults Reduring blind scoring tests (Figures 4G and 4H). Similar deregulation was also observed in the anterior nerve ring (Figures 4J and 4K). These results suggest that let-7 plays a role in controlling hbl-1::gfp::hbl-1 expression in the nervous system.}
{In the nervous system, hbl-1::gfp::hbl-1 deregulation was more pronounced, with 35% of animals exhibiting increased expression in the VNC of let-7 mutant adults Reduring blind scoring tests (Figures 4G and 4H). Similar deregulation was also observed in the anterior nerve ring (Figures 4J and 4K). These results suggest that let-7 plays a role in controlling hbl-1::gfp::hbl-1 expression in the nervous system.}
{In the nervous system, hbl-1::gfp::hbl-1 deregulation was more pronounced, with 35% of animals exhibiting increased expression in the VNC of let-7 mutant adults Reduring blind scoring tests (Figures 4G and 4H). Similar deregulation was also observed in the anterior nerve ring (Figures 4J and 4K). These results suggest that let-7 plays a role in controlling hbl-1::gfp::hbl-1 expression in the nervous system.}
{This partial restoration of the adult seam cell program to lin-4 mutants indicates that hbl-1 acts downstream of lin-4 and is partially redundant with lin-41.}
{This partial restoration of the adult seam cell program to lin-4 mutants indicates that hbl-1 acts downstream of lin-4 and is partially redundant with lin-41.}
{The similar timing of these two events suggested that let-7 might be involved in underexpression of hbl-1 in the VNC. Indeed, we found that, while 45% (n_33) of let-7(n2853) adults expressed intense HBL-1/GFP in the VNC, only 4% (n _ 45) of wild-type animals did the same (Figures 3I and 3J). lin-4 RNA is also present in the L4 stage. We observed intense HBLwith 1/GFP expression in the VNC of 100% (n_30) of lin-4(e912) adult animals (data not shown). Thus, both wildgenome type let-7 and lin-4 RNAs are required for proper hbl-1 underexpression in the VNC.}
{The similar timing of these two events suggested that let-7 might be involved in underexpression of hbl-1 in the VNC. Indeed, we found that, while 45% (n_33) of let-7(n2853) adults expressed intense HBL-1/GFP in the VNC, only 4% (n _ 45) of wild-type animals did the same (Figures 3I and 3J). lin-4 RNA is also present in the L4 stage. We observed intense HBLwith 1/GFP expression in the VNC of 100% (n_30) of lin-4(e912) adult animals (data not shown). Thus, both wildgenome type let-7 and lin-4 RNAs are required for proper hbl-1 underexpression in the VNC.}
{The similar timing of these two events suggested that let-7 might be involved in underexpression of hbl-1 in the VNC. Indeed, we found that, while 45% (n_33) of let-7(n2853) adults expressed intense HBL-1/GFP in the VNC, only 4% (n _ 45) of wild-type animals did the same (Figures 3I and 3J). lin-4 RNA is also present in the L4 stage. We observed intense HBLwith 1/GFP expression in the VNC of 100% (n_30) of lin-4(e912) adult animals (data not shown). Thus, both wildgenome type let-7 and lin-4 RNAs are required for proper hbl-1 underexpression in the VNC.}
{The similar timing of these two events suggested that let-7 might be involved in underexpression of hbl-1 in the VNC. Indeed, we found that, while 45% (n_33) of let-7(n2853) adults expressed intense HBL-1/GFP in the VNC, only 4% (n _ 45) of wild-type animals did the same (Figures 3I and 3J). lin-4 RNA is also present in the L4 stage. We observed intense HBLwith 1/GFP expression in the VNC of 100% (n_30) of lin-4(e912) adult animals (data not shown). Thus, both wildgenome type let-7 and lin-4 RNAs are required for proper hbl-1 underexpression in the VNC.}
{The similar timing of these two events suggested that let-7 might be involved in underexpression of hbl-1 in the VNC. Indeed, we found that, while 45% (n_33) of let-7(n2853) adults expressed intense HBL-1/GFP in the VNC, only 4% (n _ 45) of wild-type animals did the same (Figures 3I and 3J). lin-4 RNA is also present in the L4 stage. We observed intense HBLwith 1/GFP expression in the VNC of 100% (n_30) of lin-4(e912) adult animals (data not shown). Thus, both wildgenome type let-7 and lin-4 RNAs are required for proper hbl-1 underexpression in the VNC.}
{The similar timing of these two events suggested that let-7 might be involved in underexpression of hbl-1 in the VNC. Indeed, we found that, while 45% (n_33) of let-7(n2853) adults expressed intense HBL-1/GFP in the VNC, only 4% (n _ 45) of wild-type animals did the same (Figures 3I and 3J). lin-4 RNA is also present in the L4 stage. We observed intense HBLwith 1/GFP expression in the VNC of 100% (n_30) of lin-4(e912) adult animals (data not shown). Thus, both wildgenome type let-7 and lin-4 RNAs are required for proper hbl-1 underexpression in the VNC.}
{The similar timing of these two events suggested that let-7 might be involved in underexpression of hbl-1 in the VNC. Indeed, we found that, while 45% (n_33) of let-7(n2853) adults expressed intense HBL-1/GFP in the VNC, only 4% (n _ 45) of wild-type animals did the same (Figures 3I and 3J). lin-4 RNA is also present in the L4 stage. We observed intense HBLwith 1/GFP expression in the VNC of 100% (n_30) of lin-4(e912) adult animals (data not shown). Thus, both wildgenome type let-7 and lin-4 RNAs are required for proper hbl-1 underexpression in the VNC.}
{The similar timing of these two events suggested that let-7 might be involved in underexpression of hbl-1 in the VNC. Indeed, we found that, while 45% (n_33) of let-7(n2853) adults expressed intense HBL-1/GFP in the VNC, only 4% (n _ 45) of wild-type animals did the same (Figures 3I and 3J). lin-4 RNA is also present in the L4 stage. We observed intense HBLwith 1/GFP expression in the VNC of 100% (n_30) of lin-4(e912) adult animals (data not shown). Thus, both wildgenome type let-7 and lin-4 RNAs are required for proper hbl-1 underexpression in the VNC.}
{The similar timing of these two events suggested that let-7 might be involved in underexpression of hbl-1 in the VNC. Indeed, we found that, while 45% (n_33) of let-7(n2853) adults expressed insense HBL-1/GFP in the VNC, only 4% (n_45) of wild-type animals did the same (Figures 3I and 3J). lin-4 RNA is also present in the L4 stage. We observed intense HBLwith 1/GFP expression in the VNC of 100% (n_30) of lin-4(e912) adult animals (data not shown).Thus, both wildgenome type let-7 and lin-4 RNAs are required for proper hbl-1 underexpression in the VNC.}
{The similar timing of these two events suggested that let-7 might be involved in underexpression of hbl-1 in the VNC. Indeed, we found that, while 45% (n_33) of let-7(n2853) adults expressed insense HBL-1/GFP in the VNC, only 4% (n_45) of wild-type animals did the same (Figures 3I and 3J). lin-4 RNA is also present in the L4 stage. We observed intense HBLwith 1/GFP expression in the VNC of 100% (n_30) of lin-4(e912) adult animals (data not shown).Thus, both wildgenome type let-7 and lin-4 RNAs are required for proper hbl-1 underexpression in the VNC.}
{The gfp::lin41 30-UTR mRNA levels remained unaffected, whereas GFP protein levels were dramatically reduced upon let-7 overexpression (Figure 3C).}{The phenotypic effects caused by let-7 misexpression in zebrafish could be specifically inhibited by co-injection with a 2'-O-methyl oligonucleotides complementary to the let-7 miRNA, but not by a control 20-O-methyl oligonucleotides unrelated in sequence to let-7 (Figure 3B) (35). Injection of the oligonucleotides alone did not cause any developmental abnormalities.}
{The gfp::lin41 30-UTR mRNA levels remained unaffected, whereas GFP protein levels were dramatically reduced upon let-7 overexpression (Figure 3C).}{The phenotypic effects caused by let-7 misexpression in zebrafish could be specifically inhibited by co-injection with a 2'-O-methyl oligonucleotides complementary to the let-7 miRNA, but not by a control 20-O-methyl oligonucleotides unrelated in sequence to let-7 (Figure 3B) (35). Injection of the oligonucleotides alone did not cause any developmental abnormalities.}
{When 100 nM synthetic siRNA-miR-23 was introduced into differentiated NT2 cells, the intracellular level of precursor and mature miR-23 fell significantly. By contrast, the level of Hesl protein increased in the presence of siRNA-miR-23. Importantly, mutant siRNAmiR-23 did not affect expression of miR-23 or the level of Hesl in NT2 cells.}{Importantly, mutant siRNAmiR- 23 did not affect expression of miR-23 or the level of Hesl in NT2 cells.}
{By inhibiting miR-21 using 2'-O-methyl (2'OMe-) ribonucleotides14 in HCT-116 cells, which are known to express miR-21 at high levels,29 a specific elevation of the NFIB protein levels was detected (Fig. 3a).}
{The next step in determining the inhibitory effect of mir-128a activity on SNAP25 3 UTR was to evaluate expression of SNAP25 protein upon forced expression of mir-128a. For these experiments we utilized primary cultures of mouse neuronal progenitors (Neuronal Stem Cells, NSC), which can be efficiently nucleofected. Basal expression levels of mir-128 were determined by qRT-PCR on RNA extracts from mouse NSC (data not shown). The activity of mir-128a on SNAP25 protein was determined 24 h after nucleofection (see Materials and Methods Section) with pSIL-mir-128a expression vector. Figure 4 shows underexpression of SNAP25 protein levels in mir-128a transfected cells but not in cells transfected with a control plasmid (pSIL-NC) or in untransfected cells. Expression of SNAP25 protein did not change in cells transfected with a non-relevant microRNA, mir-146a, confirming the specificity of mir-128a on its target sequence. }
{We first performed Western blot analysis of these predicted targets 48 h after transfection of dsRNA mimicking either miR-137 or miR-193a into OSCC lines lacking the expression of these miRNAs (Fig. 5A). Protein expression levels of CDK6, E2F6, and NCOA2/TIF2 were clearly reduced in all miR-137 transfectants compared with their control counterparts. In all miR-193a transfectants, protein levels of E2F6 and PTK2/FAK were remarkably decreased compared with their control counterparts.}
{We first performed Western blot analysis of these predicted targets 48 h after transfection of dsRNA mimicking either miR-137 or miR-193a into OSCC lines lacking the expression of these miRNAs (Fig. 5A). Protein expression levels of CDK6, E2F6, and NCOA2/TIF2 were clearly reduced in all miR-137 transfectants compared with their control counterparts. In all miR-193a transfectants, protein levels of E2F6 and PTK2/FAK were remarkably decreased compared with their control counterparts.}
{We first performed Western blot analysis of these predicted targets 48 h after transfection of dsRNA mimicking either miR-137 or miR-193a into OSCC lines lacking the expression of these miRNAs (Fig. 5A). Protein expression levels of CDK6, E2F6, and NCOA2/TIF2 were clearly reduced in all miR-137 transfectants compared with their control counterparts. In all miR-193a transfectants, protein levels of E2F6 and PTK2/FAK were remarkably decreased compared with their control counterparts.}
{We first performed Western blot analysis of these predicted targets 48 h after transfection of dsRNA mimicking either miR-137 or miR-193a into OSCC lines lacking the expression of these miRNAs (Fig. 5A). Protein expression levels of CDK6, E2F6, and NCOA2/TIF2 were clearly reduced in all miR-137 transfectants compared with their control counterparts. In all miR-193a transfectants, protein levels of E2F6 and PTK2/FAK were remarkably decreased compared with their control counterparts.}
{We first performed Western blot analysis of these predicted targets 48 h after transfection of dsRNA mimicking either miR-137 or miR-193a into OSCC lines lacking the expression of these miRNAs (Fig. 5A). Protein expression levels of CDK6, E2F6, and NCOA2/TIF2 were clearly reduced in all miR-137 transfectants compared with their control counterparts. In all miR-193a transfectants, protein levels of E2F6 and PTK2/FAK were remarkably decreased compared with their control counterparts.}
{In addition, down-regulation of MCL1, an antiapoptotic Bcl-2 family member, was also detected in all OSCC cell lines transfected with miR-193a compared with negative control dsRNA}
{When mir-21 expression was inhibited by anti-miR-21 in HEK-293T cells (B50%; Figure 2a), the protein level of Pdcd4 was increased B3-fold while that of RASA1 was unchanged (Figure 2a).}
{We observed a marked reduction of 60% of GFP-DNMT3b1 expression, but not of cotransfected H2B-GFP, in cells expressing miR-148 (Fig. 2C). As expected from the sequence similarity between miR-148a and miR-148b, a similar reduction of GFP-DNMT3b1 expression in cells expressing miR-148b was observed (Fig. 2D). These results suggest that the presence of miR-148 represses the expression of DNMT3b1.}
{We observed a marked reduction of 60% of GFP-DNMT3b1 expression, but not of cotransfected H2B-GFP, in cells expressing miR-148 (Fig. 2C). As expected from the sequence similarity between miR-148a and miR-148b, a similar reduction of GFP-DNMT3b1 expression in cells expressing miR-148b was observed (Fig. 2D). These results suggest that the presence of miR-148 represses the expression of DNMT3b1.}
{Despite the pre-senescent phenotype of these cells and their high p16 levels, miR-24 overexpression (Fig. 4A, right) markedly reduced p16 protein abundance (Fig. 4B), but not p16 mRNA levels (Fig. 4A, left).}{Conversely, Y HDFs were transfected with AS-miR-24 in order to reduce the function of miR-24 in these populations with high endogenous miR-24 levels (Fig. 3A). The intervention to reduce miR-24 (Fig. 4C, right) caused no significant changes in p16 mRNA levels (Fig. 4C, left), but it strongly increased p16 protein abundance (Fig. 4D).}
{Despite the pre-senescent phenotype of these cells and their high p16 levels, miR-24 overexpression (Fig. 4A, right) markedly reduced p16 protein abundance (Fig. 4B), but not p16 mRNA levels (Fig. 4A, left).}{Conversely, Y HDFs were transfected with AS-miR-24 in order to reduce the function of miR-24 in these populations with high endogenous miR-24 levels (Fig. 3A). The intervention to reduce miR-24 (Fig. 4C, right) caused no significant changes in p16 mRNA levels (Fig. 4C, left), but it strongly increased p16 protein abundance (Fig. 4D).}
{Treating BxPC-3 cells with 10 Amol/L curcumin downregulates both ESR1 (48.6%; Fig. 4A)and SP1 (42.4%; P V 0.05; Fig. 5A). Transfection of miRNA-22 oligonucleotides significantly altered both ESR1 and SP1 (P V 0.05; Figs. 4B and 5B).When miRNA-22 was inhibited by transfecting the antisense RNA oligonucleotides of miRNA-22 into BxPC-3 cells, ESR1 and SP1 proteins were significantly upregulated by 196% and 132%, respectively (P V 0.05). However, transfecting the RNA oligonucleotides of miRNA-22 down-regulated ESR1 and SP1 by 65% and 61%, respectively (P V 0.05; Figs. 4B and 5B). }
{Treating BxPC-3 cells with 10 Amol/L curcumin downregulates both ESR1 (48.6%; Fig. 4A)and SP1 (42.4%; P V 0.05; Fig. 5A). Transfection of miRNA-22 oligonucleotides significantly altered both ESR1 and SP1 (P V 0.05; Figs. 4B and 5B).When miRNA-22 was inhibited by transfecting the antisense RNA oligonucleotides of miRNA-22 into BxPC-3 cells, ESR1 and SP1 proteins were significantly upregulated by 196% and 132%, respectively (P V 0.05). However, transfecting the RNA oligonucleotides of miRNA-22 down-regulated ESR1 and SP1 by 65% and 61%, respectively (P V 0.05; Figs. 4B and 5B). }
{Transfection of LNA antisense oligonucleotides (ASOs) against miR-106b and miR-93 induced an accumulation of E2F1 protein in Snu-16 cells, indicating that endogenous levels of these miRNAs control its expression (Figure 3B).}{Also, overexpression of these miRNAs by either oligonucleotides transfection or lentiviral transduction (Figure S1D) clearly decreased E2F1 protein levels in Snu-16 and AGS gastric cancer cell lines (Figures 3C and 3D) and inhibited the expression of a reporter vector containing E2F1 30UTR}
{Transfection of LNA antisense oligonucleotides (ASOs) against miR-106b and miR-93 induced an accumulation of E2F1 protein in Snu-16 cells, indicating that endogenous levels of these miRNAs control its expression (Figure 3B).}{Also, overexpression of these miRNAs by either oligonucleotides transfection or lentiviral transduction (Figure S1D) clearly decreased E2F1 protein levels in Snu-16 and AGS gastric cancer cell lines (Figures 3C and 3D) and inhibited the expression of a reporter vector containing E2F1 30UTR}
{In fact, their inhibition by ASOs enhanced the expression of p21 protein (Figure 4A). Conversely, upregulation of miR-106b and miR-93 achieved by either oligonucleotides transfection (Figure 4B) or lentiviral transduction (Figure 4C) repressed p21 protein expression without significant changes in p21 mRNA levels (Figure 4D).}{In fact, their inhibition by ASOs enhanced the expression of p21 protein (Figure 4A). Conversely, upregulation of miR-106b and miR-93 achieved by either oligonucleotides transfection (Figure 4B) or lentiviral transduction (Figure 4C) repressed p21 protein expression without significant changes in p21 mRNA levels (Figure 4D).}
{In fact, their inhibition by ASOs enhanced the expression of p21 protein (Figure 4A). Conversely, upregulation of miR-106b and miR-93 achieved by either oligonucleotides transfection (Figure 4B) or lentiviral transduction (Figure 4C) repressed p21 protein expression without significant changes in p21 mRNA levels (Figure 4D).}{In fact, their inhibition by ASOs enhanced the expression of p21 protein (Figure 4A). Conversely, upregulation of miR-106b and miR-93 achieved by either oligonucleotides transfection (Figure 4B) or lentiviral transduction (Figure 4C) repressed p21 protein expression without significant changes in p21 mRNA levels (Figure 4D).}
{The experiments indicated that 23 of the 30 miRNAs significantly repressed VEGF expression by over 20%, compared to negative controls (Figure 1).}
{The experiments indicated that 23 of the 30 miRNAs significantly repressed VEGF expression by over 20%, compared to negative controls (Figure 1).}
{The experiments indicated that 23 of the 30 miRNAs significantly repressed VEGF expression by over 20%, compared to negative controls (Figure 1).}
{The experiments indicated that 23 of the 30 miRNAs significantly repressed VEGF expression by over 20%, compared to negative controls (Figure 1).}
{The experiments indicated that 23 of the 30 miRNAs significantly repressed VEGF expression by over 20%, compared to negative controls (Figure 1).}
{The experiments indicated that 23 of the 30 miRNAs significantly repressed VEGF expression by over 20%, compared to negative controls (Figure 1).}
{The experiments indicated that 23 of the 30 miRNAs significantly repressed VEGF expression by over 20%, compared to negative controls (Figure 1).}
{The experiments indicated that 23 of the 30 miRNAs significantly repressed VEGF expression by over 20%, compared to negative controls (Figure 1).}
{The experiments indicated that 23 of the 30 miRNAs significantly repressed VEGF expression by over 20%, compared to negative controls (Figure 1).}
{The experiments indicated that 23 of the 30 miRNAs significantly repressed VEGF expression by over 20%, compared to negative controls (Figure 1).}
{The experiments indicated that 23 of the 30 miRNAs significantly repressed VEGF expression by over 20%, compared to negative controls (Figure 1).}
{The experiments indicated that 23 of the 30 miRNAs significantly repressed VEGF expression by over 20%, compared to negative controls (Figure 1).}
{The experiments indicated that 23 of the 30 miRNAs significantly repressed VEGF expression by over 20%, compared to negative controls (Figure 1).}
{The experiments indicated that 23 of the 30 miRNAs significantly repressed VEGF expression by over 20%, compared to negative controls (Figure 1).}
{The experiments indicated that 23 of the 30 miRNAs significantly repressed VEGF expression by over 20%, compared to negative controls (Figure 1).}
{The experiments indicated that 23 of the 30 miRNAs significantly repressed VEGF expression by over 20%, compared to negative controls (Figure 1).}
{The experiments indicated that 23 of the 30 miRNAs significantly repressed VEGF expression by over 20%, compared to negative controls (Figure 1).}
{The experiments indicated that 23 of the 30 miRNAs significantly repressed VEGF expression by over 20%, compared to negative controls (Figure 1).}
{The experiments indicated that 23 of the 30 miRNAs significantly repressed VEGF expression by over 20%, compared to negative controls (Figure 1).}
{The experiments indicated that 23 of the 30 miRNAs significantly repressed VEGF expression by over 20%, compared to negative controls (Figure 1).}
{By the transfection of the precursor for miR-148a into HepG2 cells that harbor the increased level of mature miR-148a, the PXR protein level was significantly (p _0.05) decreased compared with the control (Fig. 3A).}{Conversely, by the transfection of the AsO for miR-148a into HepG2 cells, where the expression of mature miR-148a was extinguished, the PXR protein level was significantly (p _0.05) increased compared with the control (Fig. 3B).}
{Western blotting indicated that the suppression of mir-21 by anti-mir-21 in MDA-MB-231 cells causes upregulation of endogenous PDCD4 and maspin (Figure 5C).}
{Western blotting indicated that the suppression of mir-21 by anti-mir-21 in MDA-MB-231 cells causes upregulation of endogenous PDCD4 and maspin (Figure 5C).}
{To determine whether miR- 26a acts on Ezh2 at the mRNA level, miR-26a was transiently transfected into C2C12 myoblasts, and endogenous Ezh2 mRNA and protein expression was measured 48 h after transfection. In the presence of miR-26a, Ezh2 mRNA levels decreased significantly by 1.6-fold when compared with the control (Fig. 4e), and Ezh2 protein levels decreased by 1.5-fold (Fig. 4f). This result indicates that miR-26a is able to decrease Ezh2 mRNA levels.}
{To corroborate this result, we generated a siRNA construct containing two hairpin structures complementary to Fus-1 sequences (SI Fig. 16A). Down-regulation of Fus-1 was confirmed by RT-PCR (SI Fig. 16B) and Western blot (SI Fig. 16C).}{Cotransfection of miR-378 with FusUTR produced lower levels of Fus-1 protein than cotransfection of GFP vector with FusUTR (Fig. 5D Upper).}
{Repression of endogenous Sufu expression was confirmed by Western blot probed with anti-Sufu monoclonal antibody (Novus Biologicals). We detected a clear reduction of Sufu expression in cells transfected with miR-378 compared with the control group (Fig. 4B).}{Immunocytochemical staining further confirmed the repression of Sufu expression (SI Fig. 10D).}
{Moreover, AldoA protein levels were 3-fold increased in LNAantimiR- treated mice compared to saline control mice (Figure 3D), concurring with the observed 2-fold derepression of AldoA message levels in LNA-treated mice (Figure 3B).}
{Overexpression of 40pg HoxB3a RNA induces a very strong phenotype with both anterior and posterior truncations of the embryo (figure 2D). These defects are completely rescued by co-injection of miR-10 siRNA, indicating absence of overexpressed HoxB3a protein. These experiments identify the predicted HoxB1a and HoxB3a 39UTR and ORF target sites as mediators of miR-10 repression.}
{The results show that ZO-1 protein levels are significantly decreased in Caco-2 cells transfected with miR-212 precursors compared with cells transfected with control constructs (Fig. 3D, p < 0.05). These studies indicate that over-expression of miR-212 can decrease ZO-1 protein levels.}
{To investigate CD44, we initially verified changes in its protein expression level by immunoblot analysis using an anti-human-CD44 antibody (Hermes-3) that recognizes all CD44 isoform variants33,34.Indeed, expression of all CD44 isoforms was lower in MCF-7 cells that expressed miR-373 and miR-520c (Fig. 3c), compared with control cells.}{To investigate CD44, we initially verified changes in its protein expression level by immunoblot analysis using an anti-human-CD44 antibody (Hermes-3) that recognizes all CD44 isoform variants33,34.Indeed, expression of all CD44 isoforms was lower in MCF-7 cells that expressed miR-373 and miR-520c (Fig. 3c), compared with control cells.}
{To investigate CD44, we initially verified changes in its protein expression level by immunoblot analysis using an anti-human-CD44 antibody (Hermes-3) that recognizes all CD44 isoform variants33,34.Indeed, expression of all CD44 isoforms was lower in MCF-7 cells that expressed miR-373 and miR-520c (Fig. 3c), compared with control cells.}
{We then showed that introduction of exogenous synthetic miR-126*-mimic in LNCaP cells silenced prostein protein expression, while the mRNA level remained unaffected (Fig. 3a).}
{We then showed that introduction of exogenous synthetic miR-126*-mimic in LNCaP cells silenced prostein protein expression, while the mRNA level remained unaffected (Fig. 3a).}
{Western blot analysis showed that expression of the SMAD1 protein was complementary to that of miR-26a.}{Whether miR-26a could affect SMAD1 cellular protein levels by modulating osteoblastic differentiation was examined firstly by Western blot of cellular extracts from differentiating osteoblasts transfected with miR-26a antisense 2'-O-methyl RNA. SMAD1 protein was upregulated by the inhibition of miR-26a, because absolute levels of SMAD1 protein were higher in cells treated with the 26a antisense 2'-O-methyl RNA than in control cells (Fig. 3A).}
{Immunoblotting and RT-PCR analyses revealed that PTEN protein but not mRNA was considerably decreased in miR-214-transfected HIOSE-80 cells (Fig. 2B).}{In contrast, knockdown of miR-214 by 2-O-me miR-214 in A2780CP cells, which express high levels of endogenous miR-214 (Fig. 3A), increased the protein level of PTEN (Fig. 2B).}
{Anti-miR-106b increased p21 protein levels by 53% (Fig. 4B), consistent with p21 being a direct target of miR-106b.}
{Transfection of AS-miR-126 oligonucleotides leads to a dose-dependent increase in VCAM-1 expression without affecting other adhesion molecules such as intercellular adhesion molecule 1 (ICAM-1) [Fig. 3B and supporting information (SI) Fig. 5].}{Transfection of AS-miR-126 oligonucleotides leads to a dose-dependent increase in VCAM-1 expression without affecting other adhesion molecules such as intercellular adhesion molecule 1 (ICAM-1) [Fig. 3B and supporting information (SI) Fig. 5].}
{One of the predicted microRNA-target pairs, miR-450 and HNRPK, was further tested in HK-2 cells. The pair was selected in part because of our ongoing interest in HNRPK. As shown in Figure 4B, blocking endogenous miR-450 with anti-miR-450 slightly but significantly increased the protein expression level of HNRPK.}{One of the predicted microRNA-target pairs, miR-450 and HNRPK, was further tested in HK-2 cells. The pair was selected in part because of our ongoing interest in HNRPK. As shown in Figure 4B, blocking endogenous miR-450 with anti-miR-450 slightly but significantly increased the protein expression level of HNRPK.}
{we found that transfection of 2.3 _ 105 rat 1a-c-Myc cells with pre-let-7a resulted in an increase at 48 h posttransfection of let-7a miRNA and a concomitant decrease in Myc mRNA (Table 2). Duplicate cultures were harvested for the analysis of Myc protein by Western blot and densitometry. Scanning of the resultant autoradiograpy indicated that the protein was decreased by 25% (Fig. 4A, lane 5).}
{By both QRT-PCR (Figure 4A) and Western blot (Figure 4B), miR-130a strongly down-regulated endogenous GAX expression.}
{As in the case of GAX, we observed significant down-regulation of HOXA5 expression in response to incubation in medium supplemented with a relatively modestly increased serum concentration (Figure 6A). We also observed that miR-130a down-regulated HOXA5 expression but not as strongly as it did GAX (Figure 6B,C).}
{As a control, PTEN, a known target of miR-21, was also induced (Figure 4c). In parallel, we observed a significant( B50%) underexpression of invasion into Matrigel in anti-miR-21-transfected RKO cells (Figure 4d).}{Whereas Pdcd4 mRNA was almost unaltered in pre-miR-21-transfected cells (Figure 4f), there was a significant reduction of Pdcd4 protein amounts in pre-miR-21-transfected cells (Figure 4g),}
{At 3 months, RT-PCR, realtime PCR and western blot analysis of the clone cells demonstrated decreased PRDX6 expression compared with parental cells (P < 0.05; Additional file 2).}
{To investigate miR-21 regulation of endogenous target proteins, three targets for which functional antibodies could be obtained were confirmed by Western blot analysis.Upon transfection with the miR-21 precursor, PDCD4, CDK6, and cofilin 2 protein levels were all reduced relative to the lin-4 control. In addition,miR-21 inhibition led to a corresponding increase in endogenous protein levels relative to the effect of the scrambled control (Fig. 4C).}{To investigate miR-21 regulation of endogenous target proteins, three targets for which functional antibodies could be obtained were confirmed by Western blot analysis.Upon transfection with the miR-21 precursor, PDCD4, CDK6, and cofilin 2 protein levels were all reduced relative to the lin-4 control. In addition,miR-21 inhibition led to a corresponding increase in endogenous protein levels relative to the effect of the scrambled control (Fig. 4C).}
{To investigate miR-21 regulation of endogenous target proteins, three targets for which functional antibodies could be obtained were confirmed by Western blot analysis.Upon transfection with the miR-21 precursor, PDCD4, CDK6, and cofilin 2 protein levels were all reduced relative to the lin-4 control. In addition,miR-21 inhibition led to a corresponding increase in endogenous protein levels relative to the effect of the scrambled control (Fig. 4C).}
{To investigate miR-21 regulation of endogenous target proteins, three targets for which functional antibodies could be obtained were confirmed by Western blot analysis.Upon transfection with the miR-21 precursor, PDCD4, CDK6, and cofilin 2 protein levels were all reduced relative to the lin-4 control. In addition,miR-21 inhibition led to a corresponding increase in endogenous protein levels relative to the effect of the scrambled control (Fig. 4C).}
{Consistent with this idea, the introduction of miR132 into primary cortical neurons decreased MeCP2 protein levels (Fig. 1a and Supplementary Fig. 2 online).}{These effects were post-transcriptional, as no change was observed in MeCP2 mRNA levels (Supplementary Fig. 6 online). Conversely, a 2'-O-methyl oligoribonucleotide antisense to miR132 (AS 2'-O-me) increased MeCP2 protein levels under basal conditions (Fig. 1b, Supplementary Fig. 7 online) and blocked the decrease induced by forskolin (Fig. 1a and Supplementary Fig. 8 online).}{An LNA designed to block the interaction of miR132 with the MeCP2 MRE (AS LNA) increased MeCP2 protein in cortical neurons under basal conditions, as compared with a control LNA corresponding to a non-MRE sequence in the MeCP2 3'UTR (Fig. 1b).}
{We also investigated the effects of miR-27a on expression of Sp proteins and Sp-regulated proteins. Results in Fig. 3A show that in MDA-MB-231 cells transfected with the as-miR-27a (100 nmol/mL), there was decreased expression of Sp1, Sp3, and Sp4 proteins (Fig. 3A).}{In contrast, transfection with miR-27a mimic enhanced expression of Sp1, Sp3, and Sp4 proteins (Fig. 3B) and showed a direct correlation between miR-27a and Sp protein expression. Previous studies have shown that Sp proteins regulate expression of survival and angiogenic genes (20-25, 34).}
{We also investigated the effects of miR-27a on expression of Sp proteins and Sp-regulated proteins. Results in Fig. 3A show that in MDA-MB-231 cells transfected with the as-miR-27a (100 nmol/mL), there was decreased expression of Sp1, Sp3, and Sp4 proteins (Fig. 3A).}{In contrast, transfection with miR-27a mimic enhanced expression of Sp1, Sp3, and Sp4 proteins (Fig. 3B) and showed a direct correlation between miR-27a and Sp protein expression. Previous studies have shown that Sp proteins regulate expression of survival and angiogenic genes (20-25, 34).}
{We also investigated the effects of miR-27a on expression of Sp proteins and Sp-regulated proteins. Results in Fig. 3A show that in MDA-MB-231 cells transfected with the as-miR-27a (100 nmol/mL), there was decreased expression of Sp1, Sp3, and Sp4 proteins (Fig. 3A).}{In contrast, transfection with miR-27a mimic enhanced expression of Sp1, Sp3, and Sp4 proteins (Fig. 3B) and showed a direct correlation between miR-27a and Sp protein expression. Previous studies have shown that Sp proteins regulate expression of survival and angiogenic genes (20-25, 34).}
{Whole cell lysates of transduced K562 cells were analyzed for E2F-1 expression by immunoblotting. As shown in Figure 3A and Table 3, expression of both miR-20a (lane 8) and the miR-17-19b polycistron (lane 2) reduce, whereas anti-miR-20a antagomirs (lane 5) increase E2F-1 levels.}{Whole cell lysates of transduced K562 cells were analyzed for E2F-1 expression by immunoblotting. As shown in Figure 3A and Table 3, expression of both miR-20a (lane 8) and the miR-17-19b polycistron (lane 2) reduce, whereas anti-miR-20a antagomirs (lane 5) increase E2F-1 levels.}
{However, we could easily detect Pu.1 expressed in miR-155-deficient B cells (Figure 7B), consistent with Pu.1 being a direct target of miR-155. }
{Next, we examined the level of Bak1 protein in miR-125bm- and antimiR-125b-treated LNCaP and/or cds1 cells. Transfection with miR-125bm led to _70% down-regulation of Bak1 in both cell lines (Fig. 6B), whereas treatment with anti-miR-125b induced 200% increase in the Bak1 in LNCaP cells (Fig. 6C).}
{Knockdown of endogenous Mirn140 elevated Pdgfra protein amounts (Fig. 3a) and GFP fluorescence compared to controls (Fig. 3c,f; see g for quantification). These experiments show that the 3'UTR of pdgfra is a target of Mirn140.}{[accessed from the pictures]}
{Both the miRNA 221 and 222 antagonists increased p27Kip1 levels above the levels seen in mock or control oligonucleotides-transfected cells in U87MG cells (Fig. 3B) and in A172 cells (not shown). The miR-221 antagonist also selectively decreased cell proliferation (Fig. 3C) without an increase in non-viable cells, as seen with Dicer depletion.}
{Both the miRNA 221 and 222 antagonists increased p27Kip1 levels above the levels seen in mock or control oligonucleotides-transfected cells in U87MG cells (Fig. 3B) and in A172 cells (not shown). The miR-221 antagonist also selectively decreased cell proliferation (Fig. 3C) without an increase in non-viable cells, as seen with Dicer depletion.}
{To assess the ability of endogenous miR-14 to regulate EcR expression, we examined EcR protein levels in extracts from control pupae and pupae lacking miR-14. EcR protein was more abundant in the miR-14 mutant samples (Fig. 2A). }
{To assess the ability of endogenous miR-14 to regulate EcR expression, we examined EcR protein levels in extracts from control pupae and pupae lacking miR-14. EcR protein was more abundant in the miR-14 mutant samples (Fig. 2A). }
{To assess the ability of endogenous miR-14 to regulate EcR expression, we examined EcR protein levels in extracts from control pupae and pupae lacking miR-14. EcR protein was more abundant in the miR-14 mutant samples (Fig. 2A). }
{Fluorescence-activated cell sorter (FACS) analysis of permeablized primary rat midbrain cells with an antibody that recognizes Pitx3 protein revealed that miR-133b overexpression induced a reduction in Pitx3 protein levels in TH+ cells (Fig. 4A), whereas miR-133b reduction led to an increase in Pitx3 protein in TH+ cells (Fig. 4B).}{Fluorescence-activated cell sorter (FACS) analysis of permeablized primary rat midbrain cells with an antibody that recognizes Pitx3 protein revealed that miR-133b overexpression induced a reduction in Pitx3 protein levels in TH+ cells (Fig. 4A), whereas miR-133b reduction led to an increase in Pitx3 protein in TH+ cells (Fig. 4B).}
{Fig. 3 shows that miR145 decreases IRS-1 levels 96 h after transfection.}
{Fig. 3 shows that miR145 decreases IRS-1 levels 96 h after transfection.}
{Because of the lack of a mouse uterine cell line that spontaneously expresses Cox-2 protein, we overexpressed miR-101a and miR-199a* in Cox-2-expressing HeLa cells, originally derived from human cervical carcinoma, and examined whether overexpression of these miRNAs reduces endogenous Cox-2 levels. Indeed, these miRNAs caused dose-dependent decreases in Cox-2 protein levels within 72 h of transfection (Fig. 6D).}
{Because of the lack of a mouse uterine cell line that spontaneously expresses Cox-2 protein, we overexpressed miR-101a and miR-199a* in Cox-2-expressing HeLa cells, originally derived from human cervical carcinoma, and examined whether overexpression of these miRNAs reduces endogenous Cox-2 levels. Indeed, these miRNAs caused dose-dependent decreases in Cox-2 protein levels within 72 h of transfection (Fig. 6D).}
{We have monitored c-MYC expression at the mRNA and protein levels following the inhibition of let-7 with the antisense synthetic let-7 oligo (anti-let-7c from Dharmacon) in HeLa cells. 48 h post-transfection RNAs and proteins were extracted from HeLa cells and analysed by Northern and Western methods, respectively. We detected a mild increase at the mRNA level and quite a significant increase at the protein level (Fig. 4A and 4B).}
{Further, this effect was shown to be specific, as mismatched control anti-miR122 (mmanti-miR122) did not result in appreciable effects on miR-122.Consistent with miR-122 underexpression, gene targets of miR-122 were shown to be derepressed in anti-miR122-treated mice relative to mismatched controls (Fig. 4d)}
{Further, this effect was shown to be specific, as mismatched control anti-miR122 (mmanti-miR122) did not result in appreciable effects on miR-122.Consistent with miR-122 underexpression, gene targets of miR-122 were shown to be derepressed in anti-miR122-treated mice relative to mismatched controls (Fig. 4d)}
{Further, this effect was shown to be specific, as mismatched control anti-miR122 (mmanti-miR122) did not result in appreciable effects on miR-122.Consistent with miR-122 underexpression, gene targets of miR-122 were shown to be derepressed in anti-miR122-treated mice relative to mismatched controls (Fig. 4d)}
{Further, this effect was shown to be specific, as mismatched control anti-miR122 (mmanti-miR122) did not result in appreciable effects on miR-122.Consistent with miR-122 underexpression, gene targets of miR-122 were shown to be derepressed in anti-miR122-treated mice relative to mismatched controls (Fig. 4d)}
{Further, this effect was shown to be specific, as mismatched control anti-miR122 (mmanti-miR122) did not result in appreciable effects on miR-122.Consistent with miR-122 underexpression, gene targets of miR-122 were shown to be derepressed in anti-miR122-treated mice relative to mismatched controls (Fig. 4d)}
{Further, this effect was shown to be specific, as mismatched control anti-miR122 (mmanti-miR122) did not result in appreciable effects on miR-122.Consistent with miR-122 underexpression, gene targets of miR-122 were shown to be derepressed in anti-miR122-treated mice relative to mismatched controls (Fig. 4d)}
{As shown in Fig. 5E mir-155 inhibits the expression of endogenous TP53INP1.}{As expected, cells transfected with anti-miR-155 reexpressed TP53INP1 (Fig. 5F) and showed a significant increase in apoptosis (Fig. 5G). Altogether, these results demonstrate that TP53INP1 is a target of miR-155.}
{As shown in Fig. 3A, GFP fluorescence was markedly reduced in cells transfected with miR-155-FADD-target sequence, a result subsequently confirmed by luciferase assay (Fig. 3B) and Western blotting (Fig. 3C).}
{Transfection with Pre-miR-134 downregulated the endogenous protein levels of LRH1 (Fig. 5D, lanes 2 and 4), G_o (lanes 10 and 12), and Nanog (lanes 14 and 16) in both the presence and the absence of RA.}
{Transfection with Pre-miR-134 downregulated the endogenous protein levels of LRH1 (Fig. 5D, lanes 2 and 4), G_o (lanes 10 and 12), and Nanog (lanes 14 and 16) in both the presence and the absence of RA.}
{Transfection with Pre-miR-134 downregulated the endogenous protein levels of LRH1 (Fig. 5D, lanes 2 and 4), G_o (lanes 10 and 12), and Nanog (lanes 14 and 16) in both the presence and the absence of RA.}
{Atrophin protein levels were detectably higher in miR-8 mutant brain (Figure 5A) and in body wall but not in gut tissue (not shown).}
{Atrophin protein levels were detectably higher in miR-8 mutant brain (Figure 5A) and in body wall but not in gut tissue (not shown).}
{Atrophin protein levels were detectably higher in miR-8 mutant brain (Figure 5A) and in body wall but not in gut tissue (not shown).}
{Atrophin protein levels were detectably higher in miR-8 mutant brain (Figure 5A) and in body wall but not in gut tissue (not shown).}
{As expected, introduction of pre-let-7g or pre-let-7d caused down-regulation of IMP-1 protein expression in A549 cells, which express low endogenous levels of let-7 (ref. 3; Fig. 2B and data not shown).}{To inhibit let-7, we designed an antisense oligonucleotides targeting let-7 that contained eight locked nucleic acid (LNA) nucleotides at positions shared by all nine let-7 family members (Supplementary Fig. S1). When let-7 high-expressing HeLa cells were transfected with the LNA-let-7 inhibitor, a substantial up-regulation of IMP-1 was detected (Fig. 2B).}
{Western blot analysis shows that both miR-221 and miR-222 greatly reduce the levels of p57 and p27 proteins (Fig. 3C).}{Cotransfection of miR-221 and miR-222 with the corresponding anti-miRNA oligonucleotides reverses the effect on the protein levels (compare lanes 4 and 8 with lanes 3 and 7, respectively). Our findings show that p57 and p27 mRNAs are both direct targets of miR-221/miR-222.}
{Western blot analysis shows that both miR-221 and miR-222 greatly reduce the levels of p57 and p27 proteins (Fig. 3C).}{Cotransfection of miR-221 and miR-222 with the corresponding anti-miRNA oligonucleotides reverses the effect on the protein levels (compare lanes 4 and 8 with lanes 3 and 7, respectively). Our findings show that p57 and p27 mRNAs are both direct targets of miR-221/miR-222.}
{Western blot analysis shows that both miR-221 and miR-222 greatly reduce the levels of p57 and p27 proteins (Fig. 3C).}{Cotransfection of miR-221 and miR-222 with the corresponding anti-miRNA oligonucleotides reverses the effect on the protein levels (compare lanes 4 and 8 with lanes 3 and 7, respectively). Our findings show that p57 and p27 mRNAs are both direct targets of miR-221/miR-222.}
{Western blot analysis shows that both miR-221 and miR-222 greatly reduce the levels of p57 and p27 proteins (Fig. 3C).}{Cotransfection of miR-221 and miR-222 with the corresponding anti-miRNA oligonucleotides reverses the effect on the protein levels (compare lanes 4 and 8 with lanes 3 and 7, respectively). Our findings show that p57 and p27 mRNAs are both direct targets of miR-221/miR-222.}
{Western blot analysis shows that both miR-221 and miR-222 greatly reduce the levels of p57 and p27 proteins (Fig. 3C).}{Cotransfection of miR-221 and miR-222 with the corresponding anti-miRNA oligonucleotides reverses the effect on the protein levels (compare lanes 4 and 8 with lanes 3 and 7, respectively). Our findings show that p57 and p27 mRNAs are both direct targets of miR-221/miR-222.}
{Western blot analysis shows that both miR-221 and miR-222 greatly reduce the levels of p57 and p27 proteins (Fig. 3C).}{Cotransfection of miR-221 and miR-222 with the corresponding anti-miRNA oligonucleotides reverses the effect on the protein levels (compare lanes 4 and 8 with lanes 3 and 7, respectively). Our findings show that p57 and p27 mRNAs are both direct targets of miR-221/miR-222.}
{We confirmed these results in human neuroblastoma SK-N-SH cells, demonstrating that miR-29a/b-1 could suppress endogenous BACE1 expression by _50% upon transient transfection (Fig. 3D).}
{We confirmed these results in human neuroblastoma SK-N-SH cells, demonstrating that miR-29a/b-1 could suppress endogenous BACE1 expression by _50% upon transient transfection (Fig. 3D).}
{To determine whether miR-322 regulates galectin-3 expression, BT549/vector cells were transfected with an antisense 2'-O-methyl oligoribonucleotide targeted against miR-322 or, as a control,with a scrambled 2'-O-methyl oligoribonucleotide. We found that anti-miR-322, and not the scrambled oligo, upregulates galectin-3 expression at the mRNA(Figure S3A, left) and protein levels (Figure S3A, right).}
{In support of these results, we observed a clear reduction in the level of the endogenous HOXD10 protein in miR-10b-overexpressing cells (tenfold and threefold reduction in HMECs and SUM149 cells, respectively, Fig. 5d).}
{We observed that ectopic expression of miR-24 led to a 50% decrease of the hALK4 mRNA level in HEK293 cells (Figure 2A). Consistently, the endogenous hALK4 protein level was also apparently reduced by miR-24 (Figure 2B).}
{We observed that ectopic expression of miR-24 led to a 50% decrease of the hALK4 mRNA level in HEK293 cells (Figure 2A). Consistently, the endogenous hALK4 protein level was also apparently reduced by miR-24 (Figure 2B).}
{SMAD1 protein was upregulated by the inhibition of miR-26a, because absolute levels of SMAD1 protein were higher in cells treated with the 26a antisense 2_-O-methyl RNA than in control cells (Fig. 3A).}
{Transient transfection of the premiRNA of hsa-miR-199a into Type I cells led to a significant decrease in IKKb expression, whereas transient transfection of the anti-miRNA of hsamiR199a into Type II cells resulted in the expression of IKKb (Figure 6a).}
{Transient transfection of the premiRNA of hsa-miR-199a into Type I cells led to a significant decrease in IKKb expression, whereas transient transfection of the anti-miRNA of hsamiR199a into Type II cells resulted in the expression of IKKb (Figure 6a).}
{Transient transfection of the premiRNA of hsa-miR-199a into Type I cells led to a significant decrease in IKKb expression, whereas transient transfection of the anti-miRNA of hsamiR199a into Type II cells resulted in the expression of IKKb (Figure 6a).}
{To find out whether p27 was an actual target of miR-221 and/or miR-222 in melanoma cells, we analyzed the levels of p27 protein and mRNA in miR- or PLZFtransduced versus control empty vector-transduced Me1402/R melanoma cell line. The level of p27 was increased by PLZF overexpression (Fig. 6A). On the contrary, the amount of the p27 protein was strongly decreased after miR transduction, without affecting the accumulation of p27 mRNA (Fig. 6A) and confirming the typical features of a miRNA target gene.}{In addition, we observed a strong up-regulation of p27 when the endogenously expressed miR-221 and miR-222 were inhibited by antagomir in Me665/1 metastatic melanoma cell line (Fig. 6B).}
{To find out whether p27 was an actual target of miR-221 and/or miR-222 in melanoma cells, we analyzed the levels of p27 protein and mRNA in miR- or PLZFtransduced versus control empty vector-transduced Me1402/R melanoma cell line. The level of p27 was increased by PLZF overexpression (Fig. 6A). On the contrary, the amount of the p27 protein was strongly decreased after miR transduction, without affecting the accumulation of p27 mRNA (Fig. 6A) and confirming the typical features of a miRNA target gene.}{In addition, we observed a strong up-regulation of p27 when the endogenously expressed miR-221 and miR-222 were inhibited by antagomir in Me665/1 metastatic melanoma cell line (Fig. 6B).}
{We found that EFNA3 protein staining was strongly diminished in miR-210 overexpressing cells (Fig. 4C) in the absence of EFNA3 mRNA regulation (Fig. S8C).}
{Transfection of let-7 duplex to H1299 reduced the HMGA2 mRNA and protein(Fig. 2B; Supplementary Fig. S2D).}{Inhibition of let-7 in HeLa cells induced HMGA2 mRNA and protein (Fig. 2C).}
{Transfection of let-7 duplex to H1299 reduced the HMGA2 mRNA and protein(Fig. 2B; Supplementary Fig. S2D).}{Inhibition of let-7 in HeLa cells induced HMGA2 mRNA and protein (Fig. 2C).}
{Transfection of let-7 duplex to H1299 reduced the HMGA2 mRNA and protein(Fig. 2B; Supplementary Fig. S2D).}{Inhibition of let-7 in HeLa cells induced HMGA2 mRNA and protein (Fig. 2C).}
{Transfection of let-7 duplex to H1299 reduced the HMGA2 mRNA and protein(Fig. 2B; Supplementary Fig. S2D).}{Inhibition of let-7 in HeLa cells induced HMGA2 mRNA and protein (Fig. 2C).}
{Transfection of let-7 duplex to H1299 reduced the HMGA2 mRNA and protein(Fig. 2B; Supplementary Fig. S2D).}{Inhibition of let-7 in HeLa cells induced HMGA2 mRNA and protein (Fig. 2C).}
{Transfection of let-7 duplex to H1299 reduced the HMGA2 mRNA and protein(Fig. 2B; Supplementary Fig. S2D).}{Inhibition of let-7 in HeLa cells induced HMGA2 mRNA and protein (Fig. 2C).}
{Transfection of let-7 duplex to H1299 reduced the HMGA2 mRNA and protein(Fig. 2B; Supplementary Fig. S2D).}{Inhibition of let-7 in HeLa cells induced HMGA2 mRNA and protein (Fig. 2C).}
{Transfection of let-7 duplex to H1299 reduced the HMGA2 mRNA and protein(Fig. 2B; Supplementary Fig. S2D).}{Inhibition of let-7 in HeLa cells induced HMGA2 mRNA and protein (Fig. 2C).}
{Transfection of let-7 duplex to H1299 reduced the HMGA2 mRNA and protein(Fig. 2B; Supplementary Fig. S2D).}{Inhibition of let-7 in HeLa cells induced HMGA2 mRNA and protein (Fig. 2C).}
{Transfection of let-7 duplex to H1299 reduced the HMGA2 mRNA and protein(Fig. 2B; Supplementary Fig. S2D).}{Inhibition of let-7 in HeLa cells induced HMGA2 mRNA and protein (Fig. 2C).}
{Transfection of let-7 duplex to H1299 reduced the HMGA2 mRNA and protein(Fig. 2B; Supplementary Fig. S2D).}{Inhibition of let-7 in HeLa cells induced HMGA2 mRNA and protein (Fig. 2C).}
{Transfection of let-7 duplex to H1299 reduced the HMGA2 mRNA and protein(Fig. 2B; Supplementary Fig. S2D).}{Inhibition of let-7 in HeLa cells induced HMGA2 mRNA and protein (Fig. 2C).}
{In concordance with the microarray results, a significant induction of c-Maf mRNAwas detected in bicm2/m2 Th2 cells, and the levels of c-Maf protein were correspondingly increased (Fig. 4,C and D).}
{In addition, c-myc protein expression was induced following Wy-14,643 treatment as assessed by Western blot analysis Fig. 3B). It is now recognized that miRNAs can cause mRNA egradation, and let-7C has been demonstrated to degrade RNA via partial base paring to its targets (3, 53, 57).}
{Introduction of miR-124a2 precursor decreased Foxa2 protein levels and, conversely, miR-124a2 inhibitor increased Foxa2 protein amounts (Fig. 2A).}{Introduction of miR-124a2 precursor decreased Foxa2 protein levels and, conversely, miR-124a2 inhibitor increased Foxa2 protein amounts (Fig. 2A).}
{Whereas the ectopic expression of miRNAs caused a decrease by 70% of the t-trkC isoform (Fig. 4B), LNA-directed miRNA depletion in RA-treated SK-N-BE cells (Fig. 4C) showed a partial recovery of the truncated isoform (_3-fold).}{Whereas the ectopic expression of miRNAs caused a decrease by 70% of the t-trkC isoform (Fig. 4B), LNA-directed miRNA depletion in RA-treated SK-N-BE cells (Fig. 4C) showed a partial recovery of the truncated isoform (_3-fold).}
{Whereas the ectopic expression of miRNAs caused a decrease by 70% of the t-trkC isoform (Fig. 4B), LNA-directed miRNA depletion in RA-treated SK-N-BE cells (Fig. 4C) showed a partial recovery of the truncated isoform (_3-fold).}{Whereas the ectopic expression of miRNAs caused a decrease by 70% of the t-trkC isoform (Fig. 4B), LNA-directed miRNA depletion in RA-treated SK-N-BE cells (Fig. 4C) showed a partial recovery of the truncated isoform (_3-fold).}
{Therefore, by Western blot analysis we examined the expression of ERK5 protein after transfection of the miR-143 precursor into the cells (Fig. 6B). The results indicated that the level of the protein in DLD-1 cells was significantly reduced by the incorporation of miR-143 precursor miRNAs.}
{Expression of miR-132 significantly reduced RFX4 protein abundance relative to miR-1 or miR-219 (Figures 6C and 6D). Likewise, cultures cotransfected with miR-219 showed significantly lower levels of SCOP protein compared with those expressing miR-1 or miR-132 (Figures 6C and 6D). Thus, our data indicate the RFX4 and SCOP are in vitro targets of miR-132 and miR-219, respectively, suggesting the possibility that they may also be in vivo targets of these two miRNAs within the SCN.}
{Expression of miR-132 significantly reduced RFX4 protein abundance relative to miR-1 or miR-219 (Figures 6C and 6D). Likewise, cultures cotransfected with miR-219 showed significantly lower levels of SCOP protein compared with those expressing miR-1 or miR-132 (Figures 6C and 6D). Thus, our data indicate the RFX4 and SCOP are in vitro targets of miR-132 and miR-219, respectively, suggesting the possibility that they may also be in vivo targets of these two miRNAs within the SCN.}
{A Western blot performed on the same cells (Fig. 2B) shows that the p27Kip1 protein was clearly reduced (50%) in both LNCaP and 22Rv1 cells expressing the single miRNAs or a combination of both, as compared with cells transfected with the empty vector.}{PC3 cells transfected with anti-221 and anti-222 LNA showed a 2-fold decrease in miR-221 and miR-222 levels when compared with control cells transfected with LNA against a microRNA not expressed in these cells (Fig. 2C).}
{A Western blot performed on the same cells (Fig. 2B) shows that the p27Kip1 protein was clearly reduced (50%) in both LNCaP and 22Rv1 cells expressing the single miRNAs or a combination of both, as compared with cells transfected with the empty vector.}{PC3 cells transfected with anti-221 and anti-222 LNA showed a 2-fold decrease in miR-221 and miR-222 levels when compared with control cells transfected with LNA against a microRNA not expressed in these cells (Fig. 2C).}
{A Western blot performed on the same cells (Fig. 2B) shows that the p27Kip1 protein was clearly reduced (50%) in both LNCaP and 22Rv1 cells expressing the single miRNAs or a combination of both, as compared with cells transfected with the empty vector.}{PC3 cells transfected with anti-221 and anti-222 LNA showed a 2-fold decrease in miR-221 and miR-222 levels when compared with control cells transfected with LNA against a microRNA not expressed in these cells (Fig. 2C).}
{A Western blot performed on the same cells (Fig. 2B) shows that the p27Kip1 protein was clearly reduced (50%) in both LNCaP and 22Rv1 cells expressing the single miRNAs or a combination of both, as compared with cells transfected with the empty vector.}{PC3 cells transfected with anti-221 and anti-222 LNA showed a 2-fold decrease in miR-221 and miR-222 levels when compared with control cells transfected with LNA against a microRNA not expressed in these cells (Fig. 2C).}
{A Western blot performed on the same cells (Fig. 2B) shows that the p27Kip1 protein was clearly reduced (50%) in both LNCaP and 22Rv1 cells expressing the single miRNAs or a combination of both, as compared with cells transfected with the empty vector.}{PC3 cells transfected with anti-221 and anti-222 LNA showed a 2-fold decrease in miR-221 and miR-222 levels when compared with control cells transfected with LNA against a microRNA not expressed in these cells (Fig. 2C).}
{A Western blot performed on the same cells (Fig. 2B) shows that the p27Kip1 protein was clearly reduced (50%) in both LNCaP and 22Rv1 cells expressing the single miRNAs or a combination of both, as compared with cells transfected with the empty vector.}{PC3 cells transfected with anti-221 and anti-222 LNA showed a 2-fold decrease in miR-221 and miR-222 levels when compared with control cells transfected with LNA against a microRNA not expressed in these cells (Fig. 2C).}
{Western blot analysis for Mylip and Rbp1-like showed lower protein levels in those tumors that overexpressed the four miRNAs compared with negative tumors (Fig. 5C). }
{Western blot analysis for Mylip and Rbp1-like showed lower protein levels in those tumors that overexpressed the four miRNAs compared with negative tumors (Fig. 5C). }
{Western blot analysis for Mylip and Rbp1-like showed lower protein levels in those tumors that overexpressed the four miRNAs compared with negative tumors (Fig. 5C). }
{Western blot analysis for Mylip and Rbp1-like showed lower protein levels in those tumors that overexpressed the four miRNAs compared with negative tumors (Fig. 5C). }
{Western blot analysis for Mylip and Rbp1-like showed lower protein levels in those tumors that overexpressed the four miRNAs compared with negative tumors (Fig. 5C). }
{Western blot analysis for Mylip and Rbp1-like showed lower protein levels in those tumors that overexpressed the four miRNAs compared with negative tumors (Fig. 5C). }
{Western blot analysis for Mylip and Rbp1-like showed lower protein levels in those tumors that overexpressed the four miRNAs compared with negative tumors (Fig. 5C). }
{Western blot analysis for Mylip and Rbp1-like showed lower protein levels in those tumors that overexpressed the four miRNAs compared with negative tumors (Fig. 5C). }
{HMGA2 expression was substantially reduced on introduction of the let-7 miRNAs (Fig. 2B). Introduction of exogenous let-7a, let-7c, or let-7g caused degradation of HMGA2 mRNA (Fig. 2C).}{Both a mixture of inhibitors specific for let-7a, let-7c, and let-7g and the let-7g inhibitor alone caused induction of HMGA2 protein expression, indicating that it is let-7 expression that prevents HMGA2 expression in these cells.}
{HMGA2 expression was substantially reduced on introduction of the let-7 miRNAs (Fig. 2B). Introduction of exogenous let-7a, let-7c, or let-7g caused degradation of HMGA2 mRNA (Fig. 2C).}{Both a mixture of inhibitors specific for let-7a, let-7c, and let-7g and the let-7g inhibitor alone caused induction of HMGA2 protein expression, indicating that it is let-7 expression that prevents HMGA2 expression in these cells.}
{HMGA2 expression was substantially reduced on introduction of the let-7 miRNAs (Fig. 2B). Introduction of exogenous let-7a, let-7c, or let-7g caused degradation of HMGA2 mRNA (Fig. 2C).}{Both a mixture of inhibitors specific for let-7a, let-7c, and let-7g and the let-7g inhibitor alone caused induction of HMGA2 protein expression, indicating that it is let-7 expression that prevents HMGA2 expression in these cells.}
{HMGA2 expression was substantially reduced on introduction of the let-7 miRNAs (Fig. 2B). Introduction of exogenous let-7a, let-7c, or let-7g caused degradation of HMGA2 mRNA (Fig. 2C).}{Both a mixture of inhibitors specific for let-7a, let-7c, and let-7g and the let-7g inhibitor alone caused induction of HMGA2 protein expression, indicating that it is let-7 expression that prevents HMGA2 expression in these cells.}
{HMGA2 expression was substantially reduced on introduction of the let-7 miRNAs (Fig. 2B). Introduction of exogenous let-7a, let-7c, or let-7g caused degradation of HMGA2 mRNA (Fig. 2C).}{Both a mixture of inhibitors specific for let-7a, let-7c, and let-7g and the let-7g inhibitor alone caused induction of HMGA2 protein expression, indicating that it is let-7 expression that prevents HMGA2 expression in these cells.}
{HMGA2 expression was substantially reduced on introduction of the let-7 miRNAs (Fig. 2B). Introduction of exogenous let-7a, let-7c, or let-7g caused degradation of HMGA2 mRNA (Fig. 2C).}{Both a mixture of inhibitors specific for let-7a, let-7c, and let-7g and the let-7g inhibitor alone caused induction of HMGA2 protein expression, indicating that it is let-7 expression that prevents HMGA2 expression in these cells.}
{HMGA2 expression was substantially reduced on introduction of the let-7 miRNAs (Fig. 2B). Introduction of exogenous let-7a, let-7c, or let-7g caused degradation of HMGA2 mRNA (Fig. 2C).}{Both a mixture of inhibitors specific for let-7a, let-7c, and let-7g and the let-7g inhibitor alone caused induction of HMGA2 protein expression, indicating that it is let-7 expression that prevents HMGA2 expression in these cells.}
{HMGA2 expression was substantially reduced on introduction of the let-7 miRNAs (Fig. 2B). Introduction of exogenous let-7a, let-7c, or let-7g caused degradation of HMGA2 mRNA (Fig. 2C).}{Both a mixture of inhibitors specific for let-7a, let-7c, and let-7g and the let-7g inhibitor alone caused induction of HMGA2 protein expression, indicating that it is let-7 expression that prevents HMGA2 expression in these cells.}
{HMGA2 expression was substantially reduced on introduction of the let-7 miRNAs (Fig. 2B). Introduction of exogenous let-7a, let-7c, or let-7g caused degradation of HMGA2 mRNA (Fig. 2C).}{Both a mixture of inhibitors specific for let-7a, let-7c, and let-7g and the let-7g inhibitor alone caused induction of HMGA2 protein expression, indicating that it is let-7 expression that prevents HMGA2 expression in these cells.}
{HMGA2 expression was substantially reduced on introduction of the let-7 miRNAs (Fig. 2B). Introduction of exogenous let-7a, let-7c, or let-7g caused degradation of HMGA2 mRNA (Fig. 2C).}{Both a mixture of inhibitors specific for let-7a, let-7c, and let-7g and the let-7g inhibitor alone caused induction of HMGA2 protein expression, indicating that it is let-7 expression that prevents HMGA2 expression in these cells.}
{HMGA2 expression was substantially reduced on introduction of the let-7 miRNAs (Fig. 2B). Introduction of exogenous let-7a, let-7c, or let-7g caused degradation of HMGA2 mRNA (Fig. 2C).}{Both a mixture of inhibitors specific for let-7a, let-7c, and let-7g and the let-7g inhibitor alone caused induction of HMGA2 protein expression, indicating that it is let-7 expression that prevents HMGA2 expression in these cells.}
{HMGA2 expression was substantially reduced on introduction of the let-7 miRNAs (Fig. 2B). Introduction of exogenous let-7a, let-7c, or let-7g caused degradation of HMGA2 mRNA (Fig. 2C).}{Both a mixture of inhibitors specific for let-7a, let-7c, and let-7g and the let-7g inhibitor alone caused induction of HMGA2 protein expression, indicating that it is let-7 expression that prevents HMGA2 expression in these cells.}
{HMGA2 expression was substantially reduced on introduction of the let-7 miRNAs (Fig. 2B). Introduction of exogenous let-7a, let-7c, or let-7g caused degradation of HMGA2 mRNA (Fig. 2C).}{Both a mixture of inhibitors specific for let-7a, let-7c, and let-7g and the let-7g inhibitor alone caused induction of HMGA2 protein expression, indicating that it is let-7 expression that prevents HMGA2 expression in these cells.}
{HMGA2 expression was substantially reduced on introduction of the let-7 miRNAs (Fig. 2B). Introduction of exogenous let-7a, let-7c, or let-7g caused degradation of HMGA2 mRNA (Fig. 2C).}{Both a mixture of inhibitors specific for let-7a, let-7c, and let-7g and the let-7g inhibitor alone caused induction of HMGA2 protein expression, indicating that it is let-7 expression that prevents HMGA2 expression in these cells.}
{HMGA2 expression was substantially reduced on introduction of the let-7 miRNAs (Fig. 2B). Introduction of exogenous let-7a, let-7c, or let-7g caused degradation of HMGA2 mRNA (Fig. 2C).}{Both a mixture of inhibitors specific for let-7a, let-7c, and let-7g and the let-7g inhibitor alone caused induction of HMGA2 protein expression, indicating that it is let-7 expression that prevents HMGA2 expression in these cells.}
{HMGA2 expression was substantially reduced on introduction of the let-7 miRNAs (Fig. 2B). Introduction of exogenous let-7a, let-7c, or let-7g caused degradation of HMGA2 mRNA (Fig. 2C).}{Both a mixture of inhibitors specific for let-7a, let-7c, and let-7g and the let-7g inhibitor alone caused induction of HMGA2 protein expression, indicating that it is let-7 expression that prevents HMGA2 expression in these cells.}
{HMGA2 expression was substantially reduced on introduction of the let-7 miRNAs (Fig. 2B). Introduction of exogenous let-7a, let-7c, or let-7g caused degradation of HMGA2 mRNA (Fig. 2C).}{Both a mixture of inhibitors specific for let-7a, let-7c, and let-7g and the let-7g inhibitor alone caused induction of HMGA2 protein expression, indicating that it is let-7 expression that prevents HMGA2 expression in these cells.}
{HMGA2 expression was substantially reduced on introduction of the let-7 miRNAs (Fig. 2B). Introduction of exogenous let-7a, let-7c, or let-7g caused degradation of HMGA2 mRNA (Fig. 2C).}{Both a mixture of inhibitors specific for let-7a, let-7c, and let-7g and the let-7g inhibitor alone caused induction of HMGA2 protein expression, indicating that it is let-7 expression that prevents HMGA2 expression in these cells.}
{HMGA2 expression was substantially reduced on introduction of the let-7 miRNAs (Fig. 2B). Introduction of exogenous let-7a, let-7c, or let-7g caused degradation of HMGA2 mRNA (Fig. 2C).}{Both a mixture of inhibitors specific for let-7a, let-7c, and let-7g and the let-7g inhibitor alone caused induction of HMGA2 protein expression, indicating that it is let-7 expression that prevents HMGA2 expression in these cells.}
{HMGA2 expression was substantially reduced on introduction of the let-7 miRNAs (Fig. 2B). Introduction of exogenous let-7a, let-7c, or let-7g caused degradation of HMGA2 mRNA (Fig. 2C).}{Both a mixture of inhibitors specific for let-7a, let-7c, and let-7g and the let-7g inhibitor alone caused induction of HMGA2 protein expression, indicating that it is let-7 expression that prevents HMGA2 expression in these cells.}
{HMGA2 expression was substantially reduced on introduction of the let-7 miRNAs (Fig. 2B). Introduction of exogenous let-7a, let-7c, or let-7g caused degradation of HMGA2 mRNA (Fig. 2C).}{Both a mixture of inhibitors specific for let-7a, let-7c, and let-7g and the let-7g inhibitor alone caused induction of HMGA2 protein expression, indicating that it is let-7 expression that prevents HMGA2 expression in these cells.}
{To confirm this prediction, a 29-O-methylmodified oligonucleotides inhibitor of miR-26a was transfected into primary hippocampal neurons, and the amount of MAP2 protein was measured by immunoblotting the sample. Relative to controls (see Materials and Methods), the hippocampal neurons treated with the inhibitor showed significantly increased MAP2 protein immunoreactivity (Fig. 5).}
{Importantly, blocking endogenous miR-200b by transfection with a specific LNA-modified inhibitor resulted in an increase in the level of endogenous ZFHX1B (Fig. 3D), thereby supporting the notion that miR-200b regulates ZFHX1B in vivo.}{As evident from Figure 3D,E, miR-200b markedly reduced ZFHX1B protein and mRNA levels compared to mock transfected cells. In contrast, none of the control miRNAs repressed ZFHX1B.}
{Consistent with the hypothesis that AML1 translation is negatively regulated by miRNA 17-5p-20a-106a, a 50-60% reduction in AML1 protein level was observed in cells transfected with these miRNAs compared to those transfected with scrambled control oligonucleotides (see Supplementary Information,Fig. S3a) and conversely, no fluctuation in AML1 mRNA levels was observed (see Supplementary Information, Fig. S3b).}{When antimiRNAs were transfected, the level of endogenous miRNAs 17-5p, 20a and 106a declined (see Supplementary Information, Fig. S2b) coupled with increased expression of AML1 protein, whereas the mRNA abundance was unaffected (see Supplementary Information, Fig. S3c, d).}
{Consistent with the hypothesis that AML1 translation is negatively regulated by miRNA 17-5p-20a-106a, a 50-60% reduction in AML1 protein level was observed in cells transfected with these miRNAs compared to those transfected with scrambled control oligonucleotides (see Supplementary Information,Fig. S3a) and conversely, no fluctuation in AML1 mRNA levels was observed (see Supplementary Information, Fig. S3b).}{When antimiRNAs were transfected, the level of endogenous miRNAs 17-5p, 20a and 106a declined (see Supplementary Information, Fig. S2b) coupled with increased expression of AML1 protein, whereas the mRNA abundance was unaffected (see Supplementary Information, Fig. S3c, d).}
{Consistent with the hypothesis that AML1 translation is negatively regulated by miRNA 17-5p-20a-106a, a 50-60% reduction in AML1 protein level was observed in cells transfected with these miRNAs compared to those transfected with scrambled control oligonucleotides (see Supplementary Information,Fig. S3a) and conversely, no fluctuation in AML1 mRNA levels was observed (see Supplementary Information, Fig. S3b).}{When antimiRNAs were transfected, the level of endogenous miRNAs 17-5p, 20a and 106a declined (see Supplementary Information, Fig. S2b) coupled with increased expression of AML1 protein, whereas the mRNA abundance was unaffected (see Supplementary Information, Fig. S3c, d).}
{Transfection of miR-122a in HEP3B and SNU449 caused a reduction in CCNG1 protein level of 55% and 25%, respectively.Cells transfected with the negative control did not exhibit any change in cyclin G1 levels (Fig. 3B).}
{5-aza-CdR significantly reduced the expression of MAP3K8 as well as that of the proliferating cell nuclear antigen, a marker of cell proliferation, in both Mz-1 and Mz-IL-6 cells. These changes were similar to those observed with miR-370 overexpression (Figure 6b).}
{By immunostaining, we found the endogenous p27Kip1 protein level to be 2.5-fold lower in HeLa cells stably expressing miR-221, compared with control cells (Figure 2D).}{Interestingly, when p27Kip1 levels were examined by immunostaining, only the cells containing miR-221&222 (U87 and MDA-MB-231) showed increased levels of the cell cycle inhibitor p27Kip1 after administration of the antagomiRs (Figure 5E).}
{In fact, the overexpression of miR-29 inhibited cav2 protein expression as verified by Western blotting (supplemental Fig. S1B).}
{In fact, the overexpression of miR-29 inhibited cav2 protein expression as verified by Western blotting (supplemental Fig. S1B).}
{In fact, the overexpression of miR-29 inhibited cav2 protein expression as verified by Western blotting (supplemental Fig. S1B).}
{To inhibit all three miRNA34 members, we cotransfected Wi38 cells with antisense locked nucleic acid (LNA) oligonucleotides (miRNA34as) targeting all three isoforms. Inhibition of miRNA34 in Wi38 cells led to an increase in BCL2 levels within 36 hr (Figure 4C). }
{We transfected cells with the let-7i precursor or the let-7i antisense 2-methoxy oligonucleotides for 12 h and then measured TLR4 protein expression in cells by quantitative Western blotting. We found a dose-dependent increase of TLR4 protein content in cultured cholangiocytes after treatment with the let-7i antisense 2-methoxy oligonucleotides (Fig. 2A).}{In contrast, overexpression of let-7i with the precursor decreased TLR4 protein content in a dose-dependent manner (Fig. 2A).}
{Overexpression of the miR-24 mimic led to down-regulation of DHFR protein (Fig. 1B).}
{Consistent with the microarray analysis, we found that protein levels of both CDK6 and CDC25A decrease in cells transfected with pre-let-7 compared with cells transfected with a control pre-miRNA (Fig. 5A).}
{Consistent with the microarray analysis, we found that protein levels of both CDK6 and CDC25A decrease in cells transfected with pre-let-7 compared with cells transfected with a control pre-miRNA (Fig. 5A).}
{Western Blot demonstrated decreased Bcl-2 protein level in miR-15b or miR-16 precursor-transfected cells compared to precursor control-transfected cells. In contrast, RT-PCR did not show any difference in the mRNA level (Fig. 3c).}
{Western Blot demonstrated decreased Bcl-2 protein level in miR-15b or miR-16 precursor-transfected cells compared to precursor control-transfected cells. In contrast, RT-PCR did not show any difference in the mRNA level (Fig. 3c).}
{Next, we transfected the C/EBPa-positive cell line HL60 with miR-124a or the negative control oligonucleotides. After 48 h, C/EBPa protein decreased substantially, supporting our hypothesis that miR-124a negatively regulates C/EBPa (Fig. 5B).}
{Aicda155/_ showed a similar expression pattern, but in all cases the amounts were 2- to 3-fold higher than in Aicda+/_ controls, as determined by immunoblot (Figures 1A and 1B). Similar effects were also found in miR-155-deficient B cells (Figures 1C and 1D). We conclude that miR-155 regulates the amount of AID protein in stimulated B cells.}
{Transfection of either AMO-1 or AMO-133 at 100 nM did not cause significant changes of HCN2 protein level (Fig. 2D). Co-transfection of AMO-1 and AMO-133, however, was able to increase the level by approximately 87±6.5% (p<0.05).}{Transfection of miR-1 or miR-133 alone produced remarkable depression of HCN2 (Fig. 2D), but only miR-1 not miR-133 decreased HCN4 level (Fig. 2E).}
{Transfection of either AMO-1 or AMO-133 at 100 nM did not cause significant changes of HCN2 protein level (Fig. 2D). Co-transfection of AMO-1 and AMO-133, however, was able to increase the level by approximately 87 ± 6.5% (p<0.05).}{Transfection of miR-1 or miR-133 alone produced remarkable depression of HCN2 (Fig. 2D), but only miR-1 not miR-133 decreased HCN4 level (Fig. 2E).}
{By comparison, application of AMO-1 alone increased HCN4 protein level by 46±3.7% and AMO-133 did not produce any effects (Fig. 2E).}{Transfection of miR-1 or miR-133 alone produced remarkable depression of HCN2 (Fig. 2D), but only miR-1 not miR-133 decreased HCN4 level (Fig. 2E).}
{Computational target gene prediction suggested that both miR-133a and miR-133b are targeting transcript of pyruvate kinase type M2 (PKM2), a potential oncogene in solid cancers. In tongue SCC cell lines, PKM2 expression was reduced in response to miR-133a and miR-133b precursors transfection.}
{Computational target gene prediction suggested that both miR-133a and miR-133b are targeting transcript of pyruvate kinase type M2 (PKM2), a potential oncogene in solid cancers. In tongue SCC cell lines, PKM2 expression was reduced in response to miR-133a and miR-133b precursors transfection.}
{Investigating further, we performed immunoblot for IRS-1 andIR S-2, which contain target sites for miR-7 in their 3-UTRs (Fig. 2B). pre-miR-7 transfection of glioma cells decreasede xpression of both IRS-1 andI RS-2 protein (Fig. 2C).}
{Investigating further, we performed immunoblot for IRS-1 andIR S-2, which contain target sites for miR-7 in their 3-UTRs (Fig. 2B). pre-miR-7 transfection of glioma cells decreasede xpression of both IRS-1 andI RS-2 protein (Fig. 2C).}
{Investigating further, we performed immunoblot for IRS-1 andIR S-2, which contain target sites for miR-7 in their 3-UTRs (Fig. 2B). pre-miR-7 transfection of glioma cells decreasede xpression of both IRS-1 andI RS-2 protein (Fig. 2C).}
{We tested the efficiency of our pre-miR-7 transfection with a miR-7 activity reporter, a plasmidcontaining three fully complementary miR-7 target sites as a 3-UTR for the luciferase gene. Transfection with pre-miR-7 ablatedreporter activity (P < 0.001; Fig. 1B), indicating high transfection efficiency andef ficacy. Immunoblot showed profoundly decreased EGFR protein (Fig. 1C).}
{We tested the efficiency of our pre-miR-7 transfection with a miR-7 activity reporter, a plasmidcontaining three fully complementary miR-7 target sites as a 3-UTR for the luciferase gene. Transfection with pre-miR-7 ablatedreporter activity (P < 0.001; Fig. 1B), indicating high transfection efficiency andef ficacy. Immunoblot showed profoundly decreased EGFR protein (Fig. 1C).}
{We tested the efficiency of our pre-miR-7 transfection with a miR-7 activity reporter, a plasmidcontaining three fully complementary miR-7 target sites as a 3-UTR for the luciferase gene. Transfection with pre-miR-7 ablatedreporter activity (P < 0.001; Fig. 1B), indicating high transfection efficiency andef ficacy. Immunoblot showed profoundly decreased EGFR protein (Fig. 1C).}
{These cells were transduced with retroviral vectors expressing miR-126, miR-145, or let-7a alone, let-7a with either miR-126 or miR-145, or the triple expression plasmid. Although the transduction efficiency of the single and double vectors was typically ~90% or greater, the triple vector efficiency was ~40-50%.}
{These cells were transduced with retroviral vectors expressing miR-126, miR-145, or let-7a alone, let-7a with either miR-126 or miR-145, or the triple expression plasmid. Although the transduction efficiency of the single and double vectors was typically ~90% or greater, the triple vector efficiency was ~40-50%.}
{These cells were transduced with retroviral vectors expressing miR-126, miR-145, or let-7a alone, let-7a with either miR-126 or miR-145, or the triple expression plasmid. Although the transduction efficiency of the single and double vectors was typically ~90% or greater, the triple vector efficiency was ~40-50%.}
{As predicted, miR-141 had the strongest inhibitory effect on TGFb2 and miR-200c on ZEB1 expression, as shown by RNA and protein levels.}
{Intraocular injection of pre-miR-31 caused reductionsin retinal HIF-1? and PDGF-B (Figure 4b and c).}
{Intraocular injection of pre-miR-31 caused reductionsin retinal HIF-1? and PDGF-B (Figure 4b and c).}
{Injection of pre-miR-31 or -150,but not pre-miR-184, caused a significant reduction in VEGF levels in ischemic retina (Figure 4a).}
{Injection of pre-miR-150 caused reductions in PDGF-B and VEGF (Figure 4c and d).}
{We first performed a western blot analysis on the total protein extracts from IMR32 and LA-N-5 cells transfected with miR-34a 48 h after transfection (Figure 3a, left panel). Quantification of the MYCN mmunobands on the western blot demonstrated that iR-34a caused an 80 or 95% reduction of MYCN rotein in both IMR32 and LA-N-5 cells, respectively, fter normalization by glyceraldehyde 3-phosphate  hydrogenase (GAPDH) (Figure 3a, right panel).}
{We first performed a western blot analysis on the total protein extracts from IMR32 and LA-N-5 cells transfected with miR-34a 48 h after transfection (Figure 3a, left panel). Quantification of the MYCN mmunobands on the western blot demonstrated that iR-34a caused an 80 or 95% reduction of MYCN rotein in both IMR32 and LA-N-5 cells, respectively, fter normalization by glyceraldehyde 3-phosphate  hydrogenase (GAPDH) (Figure 3a, right panel).}
{Using Western blot analysis we confirmed down-regulation of endogenous SPRY2 (52±4%, n=4 averaged ± SD) upon over-expression of miR-21 for 48 hr (Fig. 2e).}
{In both assays, we observed increased abundance of endogenous UNC-29 and UNC-63 subunits in mir-1 mutants, whereas the abundance of a control subunit (UNC-38) was unaltered.}
{In both assays, we observed increased abundance of endogenous UNC-29 and UNC-63 subunits in mir-1 mutants, whereas the abundance of a control subunit (UNC-38) was unaltered.}
{In both assays, we observed increased abundance of endogenous UNC-29 and UNC-63 subunits in mir-1 mutants, whereas the abundance of a control subunit (UNC-38) was unaltered.}
{In both assays, we observed increased abundance of endogenous UNC-29 and UNC-63 subunits in mir-1 mutants, whereas the abundance of a control subunit (UNC-38) was unaltered.}
{In both assays, we observed increased abundance of endogenous UNC-29 and UNC-63 subunits in mir-1 mutants, whereas the abundance of a control subunit (UNC-38) was unaltered.}
{In both assays, we observed increased abundance of endogenous UNC-29 and UNC-63 subunits in mir-1 mutants, whereas the abundance of a control subunit (UNC-38) was unaltered.}
{The increased levels of miRNA-98 were validated by miRVana qRT-PCRô as before. These treatments resulted in diminishment in protein levels and mRNA expression of HMGA2 in control the normoxic SCC-4 cell line as well as cell lines exposed to hypoxia (Figure 9a,b);}{Anti-miR98ô transfection had a significant effect on enhancing HMGA2 expression when cells lines were grown during normoxia (*p ? 0.05) but were ineffective under hypoxic conditions.}
{The increased levels of miRNA-98 were validated by miRVana qRT-PCRô as before. These treatments resulted in diminishment in protein levels and mRNA expression of HMGA2 in control the normoxic SCC-4 cell line as well as cell lines exposed to hypoxia (Figure 9a,b);}{Anti-miR98ô transfection had a significant effect on enhancing HMGA2 expression when cells lines were grown during normoxia (*p ? 0.05) but were ineffective under hypoxic conditions.}
{The increased levels of miRNA-98 were validated by miRVana qRT-PCRô as before. These treatments resulted in diminishment in protein levels and mRNA expression of HMGA2 in control the normoxic SCC-4 cell line as well as cell lines exposed to hypoxia (Figure 9a,b);}{Anti-miR98ô transfection had a significant effect on enhancing HMGA2 expression when cells lines were grown during normoxia (*p ? 0.05) but were ineffective under hypoxic conditions.}
{The increased levels of miRNA-98 were validated by miRVana qRT-PCRô as before. These treatments resulted in diminishment in protein levels and mRNA expression of HMGA2 in control the normoxic SCC-4 cell line as well as cell lines exposed to hypoxia (Figure 9a,b);}{Anti-miR98ô transfection had a significant effect on enhancing HMGA2 expression when cells lines were grown during normoxia (*p ? 0.05) but were ineffective under hypoxic conditions.}
{The increased levels of miRNA-98 were validated by miRVana qRT-PCRô as before. These treatments resulted in diminishment in protein levels and mRNA expression of HMGA2 in control the normoxic SCC-4 cell line as well as cell lines exposed to hypoxia (Figure 9a,b);}{Anti-miR98ô transfection had a significant effect on enhancing HMGA2 expression when cells lines were grown during normoxia (*p ? 0.05) but were ineffective under hypoxic conditions.}
{The increased levels of miRNA-98 were validated by miRVana qRT-PCRô as before. These treatments resulted in diminishment in protein levels and mRNA expression of HMGA2 in control the normoxic SCC-4 cell line as well as cell lines exposed to hypoxia (Figure 9a,b);}{Anti-miR98ô transfection had a significant effect on enhancing HMGA2 expression when cells lines were grown during normoxia (*p ? 0.05) but were ineffective under hypoxic conditions.}
{The increased levels of miRNA-98 were validated by miRVana qRT-PCRô as before. These treatments resulted in diminishment in protein levels and mRNA expression of HMGA2 in control the normoxic SCC-4 cell line as well as cell lines exposed to hypoxia (Figure 9a,b);}{Anti-miR98ô transfection had a significant effect on enhancing HMGA2 expression when cells lines were grown during normoxia (*p ? 0.05) but were ineffective under hypoxic conditions.}
{PS-206 induced a dose-dependent decrease in ER_, reducing it by approximately 70% at 20 _g of vector in both cell lines.}
{PS-206 induced a dose-dependent decrease in ER_, reducing it by approximately 70% at 20 _g of vector in both cell lines.}
{In P19 cells induced to differentiate, the presence of the complementary oligonucleotides impaired Over a period of 4 to 5 days following induction with retinoic acid, the cellular concentration of miR-125b and, to a lesser extent, miR-125a increases significantly, while Lin-28 protein and mRNA levels decline markedly (Fig. 8) (48, 53).the ability of endogenous miR-125b to repress Lin-28 protein synthesis (Fig. 10B).}{The increased concentration of miR-125b in these cells resulted in a marked reduction in Lin-28 protein synthesis compared to that in P19 cells transfected with a control plasmid that did not encode the miRNA (Fig. 9).}
{In P19 cells induced to differentiate, the presence of the complementary oligonucleotides impaired Over a period of 4 to 5 days following induction with retinoic acid, the cellular concentration of miR-125b and, to a lesser extent, miR-125a increases significantly, while Lin-28 protein and mRNA levels decline markedly (Fig. 8) (48, 53).the ability of endogenous miR-125b to repress Lin-28 protein synthesis (Fig. 10B).}{The increased concentration of miR-125b in these cells resulted in a marked reduction in Lin-28 protein synthesis compared to that in P19 cells transfected with a control plasmid that did not encode the miRNA (Fig. 9).}
{The presence of an LCE suggested that lin-28 expression is regulated by lin-4. To test this possibility, we crossed the lin-28:GFP transgenic array into a lin-4(e912) mutant background to examine the expression of the fusion in the absence of lin-4 activity. Unlike in a wild-type background, lin-28:GFP was expressed in late larval stages and adults of lin-4(e912) animals (Figure4A; Table 1). L1 larvae of lin-4(e912) transgenic animals fluoresce no more intensely than L1s of lin-4(1) transgenic animals (data not shown) and older lin-4(2);lin-28:GFP larvae display relatively undiminished fluorescence compared to the L1 (Table 1; compare Figure 3 [L4] to Figure 4A).}
{As results, the level of HOXB8 in cells that expressed miR-196 was decreased compared with that in cells that expressed mutant miR-196. In addition, the level of HOXB8 mRNA was also decreased in the presence of exogenous miRNA. These results suggest that miR-196 can regulate expression of HOXB8 at a post-transcriptional level.}{As results, the level of HOXB8 in cells that expressed miR-196 was decreased compared with that in cells that expressed mutant miR-196. In addition, the level of HOXB8 mRNA was also decreased in the presence of exogenous miRNA. These results suggest that miR-196 can regulate expression of HOXB8 at a post-transcriptional level.}
{By proteomics analysis, we identified proteins whose intensity was reduced 4-fold or more in the pRS15/16 group with respect to the pRS-E group. We isolated 27 different proteins (Table 3 and SI Table 9). Interestingly, BCL2, which we had already shown as a target of miR-15a/16-1 (22), and WT1, another predicted target of these miRNAs, were identified. The targeted proteins have a variety of biological functions and can be grouped into four groups. The first group includes proteins that play a role in regulation of cell growth and cell cycle (Ruvbl1, Anxa2, Rcn1, Cct7, Sugt1, Cdc2, Psf1), another category is formed by antiapoptotic proteins (Grp78, Bcl2,Pdia2), and proteins involved in human tumorigenesis, either as oncogenes, or as tumor-suppressor genes (Wt1, MageB3, Rab9B).}
{By proteomics analysis, we identified proteins whose intensity was reduced 4-fold or more in the pRS15/16 group with respect to the pRS-E group. We isolated 27 different proteins (Table 3 and SI Table 9). Interestingly, BCL2, which we had already shown as a target of miR-15a/16-1 (22), and WT1, another predicted target of these miRNAs, were identified. The targeted proteins have a variety of biological functions and can be grouped into four groups. The first group includes proteins that play a role in regulation of cell growth and cell cycle (Ruvbl1, Anxa2, Rcn1, Cct7, Sugt1, Cdc2, Psf1), another category is formed by antiapoptotic proteins (Grp78, Bcl2,Pdia2), and proteins involved in human tumorigenesis, either as oncogenes, or as tumor-suppressor genes (Wt1, MageB3, Rab9B).}
By proteomics analysis, we identified proteins whose intensity was reduced 4-fold or more in the pRS15/16 group with respect to the pRS-E group. We isolated 27 different proteins (Table 3 and SI Table 9). Interestingly, BCL2, which we had already shown as a target of miR-15a/16-1 (22), and WT1, another predicted target of these miRNAs, were identified. The targeted proteins have a variety of biological functions and can be grouped into four groups. The first group includes proteins that play a role in regulation of cell growth and cell cycle (Ruvbl1, Anxa2, Rcn1, Cct7, Sugt1, Cdc2, Psf1), another category is formed by antiapoptotic proteins (Grp78, Bcl2,Pdia2), and proteins involved in human tumorigenesis, either as oncogenes, or as tumor-suppressor genes (Wt1, MageB3, Rab9B).
By proteomics analysis, we identified proteins whose intensity was reduced 4-fold or more in the pRS15/16 group with respect to the pRS-E group. We isolated 27 different proteins (Table 3 and SI Table 9). Interestingly, BCL2, which we had already shown as a target of miR-15a/16-1 (22), and WT1, another predicted target of these miRNAs, were identified. The targeted proteins have a variety of biological functions and can be grouped into four groups. The first group includes proteins that play a role in regulation of cell growth and cell cycle (Ruvbl1, Anxa2, Rcn1, Cct7, Sugt1, Cdc2, Psf1), another category is formed by antiapoptotic proteins (Grp78, Bcl2,Pdia2), and proteins involved in human tumorigenesis, either as oncogenes, or as tumor-suppressor genes (Wt1, MageB3, Rab9B).
By proteomics analysis, we identified proteins whose intensity was reduced 4-fold or more in the pRS15/16 group with respect to the pRS-E group. We isolated 27 different proteins (Table 3 and SI Table 9). Interestingly, BCL2, which we had already shown as a target of miR-15a/16-1 (22), and WT1, another predicted target of these miRNAs, were identified. The targeted proteins have a variety of biological functions and can be grouped into four groups. The first group includes proteins that play a role in regulation of cell growth and cell cycle (Ruvbl1, Anxa2, Rcn1, Cct7, Sugt1, Cdc2, Psf1), another category is formed by antiapoptotic proteins (Grp78, Bcl2,Pdia2), and proteins involved in human tumorigenesis, either as oncogenes, or as tumor-suppressor genes (Wt1, MageB3, Rab9B).
By proteomics analysis, we identified proteins whose intensity was reduced 4-fold or more in the pRS15/16 group with respect to the pRS-E group. We isolated 27 different proteins (Table 3 and SI Table 9). Interestingly, BCL2, which we had already shown as a target of miR-15a/16-1 (22), and WT1, another predicted target of these miRNAs, were identified. The targeted proteins have a variety of biological functions and can be grouped into four groups. The first group includes proteins that play a role in regulation of cell growth and cell cycle (Ruvbl1, Anxa2, Rcn1, Cct7, Sugt1, Cdc2, Psf1), another category is formed by antiapoptotic proteins (Grp78, Bcl2,Pdia2), and proteins involved in human tumorigenesis, either as oncogenes, or as tumor-suppressor genes (Wt1, MageB3, Rab9B).
By proteomics analysis, we identified proteins whose intensity was reduced 4-fold or more in the pRS15/16 group with respect to the pRS-E group. We isolated 27 different proteins (Table 3 and SI Table 9). Interestingly, BCL2, which we had already shown as a target of miR-15a/16-1 (22), and WT1, another predicted target of these miRNAs, were identified. The targeted proteins have a variety of biological functions and can be grouped into four groups. The first group includes proteins that play a role in regulation of cell growth and cell cycle (Ruvbl1, Anxa2, Rcn1, Cct7, Sugt1, Cdc2, Psf1), another category is formed by antiapoptotic proteins (Grp78, Bcl2,Pdia2), and proteins involved in human tumorigenesis, either as oncogenes, or as tumor-suppressor genes (Wt1, MageB3, Rab9B).
By proteomics analysis, we identified proteins whose intensity was reduced 4-fold or more in the pRS15/16 group with respect to the pRS-E group. We isolated 27 different proteins (Table 3 and SI Table 9). Interestingly, BCL2, which we had already shown as a target of miR-15a/16-1 (22), and WT1, another predicted target of these miRNAs, were identified. The targeted proteins have a variety of biological functions and can be grouped into four groups. The first group includes proteins that play a role in regulation of cell growth and cell cycle (Ruvbl1, Anxa2, Rcn1, Cct7, Sugt1, Cdc2, Psf1), another category is formed by antiapoptotic proteins (Grp78, Bcl2,Pdia2), and proteins involved in human tumorigenesis, either as oncogenes, or as tumor-suppressor genes (Wt1, MageB3, Rab9B).
{The expression of these genes was studied by immunoblotting in MIN6 and N2A neuroblastoma cells (devoid of miR-375) that were infected with either Ad-375 or Ad-eGFP (Fig. 4a). Expression of miR-375 in N2A cells led to reduced protein levels of Mtpn and Vti1a, whereas expression of the other genes was unaffected.}{Furthermore, transfection of 2 0-Ome- 375 increased protein levels of Mtpn but not Vti1a in MIN6 cells (Fig. 4b; data not shown).}
{By proteomics analysis, we identified proteins whose intensity was reduced 4-fold or more in the pRS15/16 group with respect to the pRS-E group. We isolated 27 different proteins (Table 3 and SI Table 9). Interestingly, BCL2, which we had already shown as a target of miR-15a/16-1 (22), and WT1, another predicted target of these miRNAs, were identified. The targeted proteins have a variety of biological functions and can be grouped into four groups. The first group includes proteins that play a role in regulation of cell growth and cell cycle (Ruvbl1, Anxa2, Rcn1, Cct7, Sugt1, Cdc2, Psf1), another category is formed by antiapoptotic proteins (Grp78, Bcl2,Pdia2), and proteins involved in human tumorigenesis, either as oncogenes, or as tumor-suppressor genes (Wt1, MageB3, Rab9B).}
{By proteomics analysis, we identified proteins whose intensity was reduced 4-fold or more in the pRS15/16 group with respect to the pRS-E group. We isolated 27 different proteins (Table 3 and SI Table 9). Interestingly, BCL2, which we had already shown as a target of miR-15a/16-1 (22), and WT1, another predicted target of these miRNAs, were identified. The targeted proteins have a variety of biological functions and can be grouped into four groups. The first group includes proteins that play a role in regulation of cell growth and cell cycle (Ruvbl1, Anxa2, Rcn1, Cct7, Sugt1, Cdc2, Psf1), another category is formed by antiapoptotic proteins (Grp78, Bcl2,Pdia2), and proteins involved in human tumorigenesis, either as oncogenes, or as tumor-suppressor genes (Wt1, MageB3, Rab9B).}
{By proteomics analysis, we identified proteins whose intensity was reduced 4-fold or more in the pRS15/16 group with respect to the pRS-E group. We isolated 27 different proteins (Table 3 and SI Table 9). Interestingly, BCL2, which we had already shown as a target of miR-15a/16-1 (22), and WT1, another predicted target of these miRNAs, were identified. The targeted proteins have a variety of biological functions and can be grouped into four groups. The first group includes proteins that play a role in regulation of cell growth and cell cycle (Ruvbl1, Anxa2, Rcn1, Cct7, Sugt1, Cdc2, Psf1), another category is formed by antiapoptotic proteins (Grp78, Bcl2,Pdia2), and proteins involved in human tumorigenesis, either as oncogenes, or as tumor-suppressor genes (Wt1, MageB3, Rab9B).}
{By proteomics analysis, we identified proteins whose intensity was reduced 4-fold or more in the pRS15/16 group with respect to the pRS-E group. We isolated 27 different proteins (Table 3 and SI Table 9). Interestingly, BCL2, which we had already shown as a target of miR-15a/16-1 (22), and WT1, another predicted target of these miRNAs, were identified. The targeted proteins have a variety of biological functions and can be grouped into four groups. The first group includes proteins that play a role in regulation of cell growth and cell cycle (Ruvbl1, Anxa2, Rcn1, Cct7, Sugt1, Cdc2, Psf1), another category is formed by antiapoptotic proteins (Grp78, Bcl2,Pdia2), and proteins involved in human tumorigenesis, either as oncogenes, or as tumor-suppressor genes (Wt1, MageB3, Rab9B).}
{By proteomics analysis, we identified proteins whose intensity was reduced 4-fold or more in the pRS15/16 group with respect to the pRS-E group. We isolated 27 different proteins (Table 3 and SI Table 9). Interestingly, BCL2, which we had already shown as a target of miR-15a/16-1 (22), and WT1, another predicted target of these miRNAs, were identified. The targeted proteins have a variety of biological functions and can be grouped into four groups. The first group includes proteins that play a role in regulation of cell growth and cell cycle (Ruvbl1, Anxa2, Rcn1, Cct7, Sugt1, Cdc2, Psf1), another category is formed by antiapoptotic proteins (Grp78, Bcl2,Pdia2), and proteins involved in human tumorigenesis, either as oncogenes, or as tumor-suppressor genes (Wt1, MageB3, Rab9B).}
{First, we transfected neuroblastoma N2A cells with short interfering RNA (siRNA) duplexes that are homologous in sequence to miR-124 (si-124) or let-7b (si-let-7b). We observed in both cases a decrease in endogenous Mtpn expression by western blotting (Fig. 4a).}
{First, we transfected neuroblastoma N2A cells with short interfering RNA (siRNA) duplexes that are homologous in sequence to miR-124 (si-124) or let-7b (si-let-7b). We observed in both cases a decrease in endogenous Mtpn expression by western blotting (Fig. 4a).}
{[assessed from the table in the paper]}
{In the presence of a miR-29 miRNA mix, the p85Œ± protein level decreased, as determined by western blotting (Fig. 4a)}
{The CDC42 protein level fell in HeLa cells that were transfected with miR-29 miRNAs, as determined by western blotting}
{The CDC42 protein level fell in HeLa cells that were transfected with miR-29 miRNAs, as determined by western blotting}
{Under these conditions, Cdc34 protein levels decreased with pre-let-7b transfection as compared to samples transfected with a negative control (Figure 2B). By 24 hours post-transfection, Cdc34 protein levels had dropped significantly and the levels continued to decline through 24 hours of restimulation (Figure 2B)}
{Overexpression of miR-128 in T98G cells resulted in down-regulation of E2F3a at posttranscriptional level as assessed by western blot (Fig. 3c).}
{Similar results were obtained using the other two miRNAs as the introduced silent mutations rescued the corresponding Nanog-,Oct4- and Sox2-CDS constructs from downregulation by pre-miR-470 and pre-miR-134 (Fig. 2b, c).}
{We transfected mouse ESCs with precursor miRNA (pre-miRNA) oligomers (see Methods) separately for each miRNA and measured the effect on the predicted corresponding endogenous target using quantitative PCR and western blotting. Each miRNA had only a limited effect on the mRNA of the endogenous target but affected the corresponding protein more substantially (Fig. 1a).}
{We transfected mouse ESCs with precursor miRNA (pre-miRNA) oligomers (see Methods) separately for each miRNA and measured the effect on the predicted corresponding endogenous target using quantitative PCR and western blotting. Each miRNA had only a limited effect on the mRNA of the endogenous target but affected the corresponding protein more substantially (Fig. 1a).}
{We transfected mouse ESCs with precursor miRNA (pre-miRNA) oligomers (see Methods) separately for each miRNA and measured the effect on the predicted corresponding endogenous target using quantitative PCR and western blotting. Each miRNA had only a limited effect on the mRNA of the endogenous target but affected the corresponding protein more substantially (Fig. 1a).}
{As shown in Figure 3, Western blot analysis revealed that transfection of miR-34 mimics downregulated target gene Bcl-2 expression at the protein level,}
{As shown in Figure 3, Western blot analysis revealed that transfection of miR-34 mimics downregulated target gene Bcl-2 expression at the protein level,}
{As shown in Figure 3, Western blot analysis revealed that transfection of miR-34 mimics downregulated target gene Bcl-2 expression at the protein level,}
{Immunofluorescence confirmed an inverse correlation between hsa-mir-34b transfection and c-Myc expression(Figure 5c).}
{Using prediction algorithms, we identified 30-untranslated regions of IGF-1 gene are the target of miR-1 (Fig. 4B). Moreover, overexpression of specific miR-1 mimics, but not mutant miR-1, blocked the capacity of IGF-1 to prevent glucose-induced apoptosis (Fig. 1B), mitochondrial dysfunction (Fig. 2B) and cytochrome-c release (Fig. 3B).}
{In AGS or SGC7901 cells, RECK can be regulated negatively by miR-21 at protein level (Figure 7c; P<0.05).}
{Immunoblotting and RT-PCR analyses revealed that RhoA protein but not RhoA mRNA was considerably decreased in miR-155-transfected cells (Fig. 5B).}
{Immunoblotting and RT-PCR analyses revealed that RhoA protein but not RhoA mRNA was considerably decreased in miR-155-transfected cells (Fig. 5B).}
{Immunoblotting and RT-PCR analyses revealed that RhoA protein but not RhoA mRNA was considerably decreased in miR-155-transfected cells (Fig. 5B).}
{As indicated, in the cells transfected with miR-146a, the expression of IRAK-1, a confirmed miR-146 target,was decreased compared with that in negative mimic transfected cells (Figure 2B).}
{Transient transfection of increasing amounts of miR-221 and/or miR-222 in MCF-7 cells reduced ERŒ±expression in a dose-dependent manner (Fig. 2B). Furthermore,stably miR-221- and miR-222-transfected MCF-7 and T47D cells decreased the protein but not mRNA levels of ERŒ±(Fig. 2C).}
{Transient transfection of increasing amounts of miR-221 and/or miR-222 in MCF-7 cells reduced ERŒ±expression in a dose-dependent manner (Fig. 2B). Furthermore,stably miR-221- and miR-222-transfected MCF-7 and T47D cells decreased the protein but not mRNA levels of ERŒ±(Fig. 2C).}
{Expression of miR-133 down-regulated endogenous Runx2 protein (Fig. 3C),with no effect on mRNA levels(Fig. 3D)}
{Similar results were obtained in the Ramos Burkitt lymphoma cell line (not depicted) or when protein levels were analyzed by Western blotting (52 ¬±8%; Fig. 2 D ).}
{Similar results were obtained in the Ramos Burkitt lymphoma cell line (not depicted) or when protein levels were analyzed by Western blotting (52 ¬±8%; Fig. 2 D ).}
{By Western blot analysis, procaspase-3 protein was down-regulated in both A431 and HepG2 cells after transfection with let-7a precursor while there was no effect for the control transfection. }
{we transfected different human cell lines (HEK293T, HeLa and MCF7 cells) with either each individual miRNA (miR-19a, miR-101 and miR-130a) or all of them combined. We observed a marked decrease in ATXN1 levels on cotransfection of all three miRNAs; transfection of 120 pmol of each miRNA individually also decreased ATXN1 levels (Fig. 1a,b and Supplementary Fig. 3 online).}
{we transfected different human cell lines (HEK293T, HeLa and MCF7 cells) with either each individual miRNA (miR-19a, miR-101 and miR-130a) or all of them combined. We observed a marked decrease in ATXN1 levels on cotransfection of all three miRNAs; transfection of 120 pmol of each miRNA individually also decreased ATXN1 levels (Fig. 1a,b and Supplementary Fig. 3 online).}
{we transfected different human cell lines (HEK293T, HeLa and MCF7 cells) with either each individual miRNA (miR-19a, miR-101 and miR-130a) or all of them combined. We observed a marked decrease in ATXN1 levels on cotransfection of all three miRNAs; transfection of 120 pmol of each miRNA individually also decreased ATXN1 levels (Fig. 1a,b and Supplementary Fig. 3 online).}
{As expected, ectopic expression of miR-324-5p caused a reduction of Gli1 protein levels (Figure 3E).}
{Further evidence of Smo repression through miRNA activity was provided by the reduction of Smo mRNA and protein levels following overexpression of miR-125b, miR-326 and miR-324-5p in Daoy cells (Figure 2E and F).}
{Further evidence of Smo repression through miRNA activity was provided by the reduction of Smo mRNA and protein levels following overexpression of miR-125b, miR-326 and miR-324-5p in Daoy cells (Figure 2E and F).}
{Further evidence of Smo repression through miRNA activity was provided by the reduction of Smo mRNA and protein levels following overexpression of miR-125b, miR-326 and miR-324-5p in Daoy cells (Figure 2E and F).}
{We found a significant increase of BIM protein levels at 48 h after treatment with anti-miR-19s compared to scrambled oligonucleotides (Fig. S5).}
{We found a significant increase of BIM protein levels at 48 h after treatment with anti-miR-19s compared to scrambled oligonucleotides (Fig. S5).}
{A highly significant decrease in the expression of endogenous GRN was observed in M17 cells treated with miR-659 (P<0.001)(Fig. 4A and B)}
{Exogenous miR-377 added to normal mesangial cells led to reduced PAK1 and SOD2, but not PPM1A protein quantities (Fig. 2B).}
{In hESCs transfected with anti-miR-302, mature miR-302a levels were decreased due to the fact that the anti-miR inhibitors bind irreversibly to the miRNA, causing them to be unavailable both functionally and for the analysis (Fig. 7A). Under these conditions, the cyclin D1 protein level was increased, but there was no change in the cyclin D1 mRNA level.}
{Indeed, Western blot analysis revealed 50% reduction in the level of p27Kip1 in MCF-7 cells compared with OHTR cells (Fig. 3A). Furthermore,the level of p27Kip1 was reduced by 50 and 28% in miR-221/222-expressing clone 9 and the G418-selected pool, respectively, compared with the vector-transfected cells (Fig. 3A).}
{Indeed, Western blot analysis revealed 50% reduction in the level of p27Kip1 in MCF-7 cells compared with OHTR cells (Fig. 3A). Furthermore,the level of p27Kip1 was reduced by 50 and 28% in miR-221/222-expressing clone 9 and the G418-selected pool, respectively, compared with the vector-transfected cells (Fig. 3A).}
{Figure 4, miR-16 and miR-424 markedly reduced CCND3, CCNE1 and CDK6 protein levels compared with mock transfected cells (Figure 4A, C and E)}
{Figure 4, miR-16 and miR-424 markedly reduced CCND3, CCNE1 and CDK6 protein levels compared with mock transfected cells (Figure 4A, C and E)}
{Figure 4, miR-16 and miR-424 markedly reduced CCND3, CCNE1 and CDK6 protein levels compared with mock transfected cells (Figure 4A, C and E)}
{Figure 4, miR-16 and miR-424 markedly reduced CCND3, CCNE1 and CDK6 protein levels compared with mock transfected cells (Figure 4A, C and E)}
{Figure 4, miR-16 and miR-424 markedly reduced CCND3, CCNE1 and CDK6 protein levels compared with mock transfected cells (Figure 4A, C and E)}
{Figure 4, miR-16 and miR-424 markedly reduced CCND3, CCNE1 and CDK6 protein levels compared with mock transfected cells (Figure 4A, C and E)}
{Western blot analysis revealed that miR-16 and miR-424 markedly reduced CCND1 protein levels compared to Luc-siRNA and mock transfected cells (Figure 2F)}
{Western blot analysis revealed that miR-16 and miR-424 markedly reduced CCND1 protein levels compared to Luc-siRNA and mock transfected cells (Figure 2F)}
{Western blot analysis revealed that miR-16 and miR-424 markedly reduced CCND1 protein levels compared to Luc-siRNA and mock transfected cells (Figure 2F)}
{This analysis showed that let-7a, c, and d induced a nearly identical reduction in Dicer expression, suggesting that other let-7 family members could also downregulate Dicer}
{Upon transfection of the let-7d precursor molecule, which was a double-stranded RNA designed to mimic mature miRNA, the level of Dicer protein was reduced when compared with transfections of mock or negative control Calu6 cells (Figure 3A).}
{Cells were transfected with miR-122_D, miR-NC, and Bcl-w_siR. The Bcl-w protein level was significantly reduced in cells transfected with miR-122 duplex (47% and 53%) and Bcl-w siRNA (76% and 78% in Hep3B and HepG2, respectively) relative to miR-NC (Fig. 3B).}
{Over-expression of mir-520c had a similar effect on APP levels as mir-106a (Figure 2B and 2D).}
{We observed that mir-106a over-expression significantly decreased APP levels (Figure 2A)}
{As shown in figure 3A, comparing to the precursor negative control oligo (pre-ctrl oligo, lane 1), miR-150 (lane 2) downregulated endogenous c-Myb protein to 22%, 24%, 45%, and 52%, respectively}
{As shown in figure 3A, comparing to the precursor negative control oligo (pre-ctrl oligo, lane 1), miR-150 (lane 2) downregulated endogenous c-Myb protein to 22%, 24%, 45%, and 52%, respectively}
{As shown in Figure 3B, miR-126 substantially reduced p85Œ≤ protein (~3-fold) with no apparent degradation of the transcript, thereby indicating a translational repression of the p85b mRNA by miR-126.}
{The decrease in the ANXA1 mRNA levels was accompanied by a decrease in ANXA1 protein levels in these four cell lines (Figure 4b).}
{Western blotting from protein extracts of 293T cells revealed a dramatic reduction in the PTPRO protein after pCMVpuro-miR-17-92 transfection, compared with the empty vector(Fig. 4E)}
{There was a significant, ~30% decrease in BDNF protein levels in neuronal cultures overexpressing miR-30a (Fig. 4B)}
{As expected, Western blot analysis revealed a significant down-regulation of HIF-1a protein in BEAS2B cells stably overexpressing miR-17-92. HIF-2a carrying potential target sites was also found to be down-regulated, whereas the expression of HIF-1h did not show any obvious changes (Fig. 2C, left)}
{As shown in Figure 3, let-7f transfection led to a significant decrease in KLK6 and KLK10 protein levels (p-0.05), and this was most apparent between days 3 and 7 post-transfection. Control transfection did not cause a significant decrease in KLK levels}
{As shown in Figure 3, let-7f transfection led to a significant decrease in KLK6 and KLK10 protein levels (p<0.05), and this was most apparent between days 3 and 7 post-transfection. Control transfection did not cause a significant decrease in KLK levels}
{Re-expression of miR 124 in Daoy medulloblastoma cells decreased expression of CDK6 protein (Fig. 2d).}
{Re-expression of miR 124 in Daoy medulloblastoma cells decreased expression of CDK6 protein (Fig. 2d).}
{The data exhibit that Hep G2 cells transfected with hsa-miR-657 show a signif¬≠i¬≠cant reduction in the expression of human soluble IGF2R compared with N.C.(Fig. 3B).}
{Transfection with 50 and 100 nM of pre-miR 126 resulted in a 1000 to 5000-fold increase in miR-126 expression measured by RT-PCR and a decrease in Crk II protein while mRNA remained unchanged (Fig. 3A and B)}
{We observed that miR-20a over-expression, by transfection of 80 nM mature miR-20a, reduces LRF protein by 40% (Figure 1h).}
{We then examined the protein levels of RECK and TIMP3 and found that both proteins were upregulated by miR-21 inhibition in glioma A172 and LN229 cells (Fig. 4D)}
{We then examined the protein levels of RECK and TIMP3 and found that both proteins were upregulated by miR-21 inhibition in glioma A172 and LN229 cells (Fig. 4D)}
{In independent experiments we observed marked reductions of CDK6 transcript (Figure 6A) and CDK6 protein (Figure 6B) in response to miR-124 and miR-137 transfection}
{In independent experiments we observed marked reductions of CDK6 transcript (Figure 6A) and CDK6 protein (Figure 6B) in response to miR-124 and miR-137 transfection}
{Western blot analysis confirmed that CD44 expression was repressed in the cells stably transfected with miR-328 (Fig 3B,left panel)}
{This antiproliferative response is accompanied by a dramatic reduction (of about70%) in ABL1 levels in these tumor lymphocytes in both KARPAS-45 and PEER cell lines (Figure 5 and Figure S4)}
{As expected, p27(Kip1) and p57(Kip2) were downregulated by Ad-miR-221 or Ad-miR-222 but not by unrelated adenovirus Ad-miR-31 (Figure 4E) at protein levels}
{As expected, p27(Kip1) and p57(Kip2) were downregulated by Ad-miR-221 or Ad-miR-222 but not by unrelated adenovirus Ad-miR-31 (Figure 4E) at protein levels}
{As expected, p27(Kip1) and p57(Kip2) were downregulated by Ad-miR-221 or Ad-miR-222 but not by unrelated adenovirus Ad-miR-31 (Figure 4E) at protein levels}
{As expected, p27(Kip1) and p57(Kip2) were downregulated by Ad-miR-221 or Ad-miR-222 but not by unrelated adenovirus Ad-miR-31 (Figure 4E) at protein levels}
{As shown in Figure 3b, the expression of KRAS was significantly abolished by the introduction of pre-miR-143.}
{As shown in Figure 3b, the expression of KRAS was significantly abolished by the introduction of pre-miR-143.}
{Overexpression of miR106b was associated with a decrease in Itch protein levels (Figure 4E),}
{The expression of DHFR protein was analyzed using Western immunoblot analysis and the results are shown in Fig. 1B.Overexpression of miR-192 clearly decreased the expression of DHFR protein (Fig. 1B, lane 4)}
{Similarly, KU812 cells nucleofected with pre-miR-10 showed an increase in expression of hsa-miR-10a and a decrease in HOXA1 mRNA and USF2 mRNA and protein levels (Fig.3A and B)}
{Based on these results we chose to use 20 nM and 100 nM concentrations of miRNA mimics and inhibitors in a larger scale experiment. We demonstrated that both miR-20 and miR-93mimics, transfected at the 100 nM concentration, could significantly reduce STAT3 expression (Fig. S3A)}
{After 48 h, lysates were made and analyzed by Western blot for STAT3 expression. We constructed a dose response graph and demonstrated a significant decrease in STAT3 when ES cells were transfected with 80‚Äì160 nM of miR-93 mimic (Fig. 7C)}
{As shown in Fig. 3B, the level of Mcl-1 protein was significantly decreased in all three cell lines in the presence of mir-320}
{As shown in Fig. 3B, the level of Mcl-1 protein was significantly decreased in all three cell lines in the presence of mir-320}
{Western blot and real-time PCR analysis showed that HOXC8 expression was decreased in miR-196a-overexpressing hASC but increased in anti-miR-196a-transfected hASC(Fig 6C and D, respectively)}
{Transfection of miR-101 led to COX-2 silencing in all three cell lines and was found to be stronger in LS-174T where miR-101 basal levels are low.}
{Using 100 nM of miRs we observed a significant (p<0.01) reduction of GR protein levels for miR-18 (34.7% + 5.1% reduction vs mock transfected cells, n=3) and miR-124a (42.1 + 6.7% reduction vs mock transfected cells, n=3) while miR-22, 328 and miR-524 did not reduce GR protein levels (Figure 1)}
{Using 100 nM of miRs we observed a significant (p<0.01) reduction of GR protein levels for miR-18 (34.7% + 5.1% reduction vs mock transfected cells, n=3) and miR-124a (42.1 + 6.7% reduction vs mock transfected cells, n=3) while miR-22, 328 and miR-524 did not reduce GR protein levels (Figure 1)}
{This conclusion is supported by the data presented in Fig. 2C, which show that miR-27a inhibits the production of Runx1 protein made in HEK293 by a genuine Runx1 cDNA bearing the S3'UTR}
{Fig. 3D shows a representative image of Western blot analyses of total protein extracts from the same tumors already tested for miR-221/222 expression: in all cases assayed, a high p27 expression was measured where miR-221 and miR-222 were kept low by antagomir action, whereas the lack of inhibition of the two microRNAs matched with a low level of p27 expression}
{Fig. 3D shows a representative image of Western blot analyses of total protein extracts from the same tumors already tested for miR-221/222 expression: in all cases assayed, a high p27 expression was measured where miR-221 and miR-222 were kept low by antagomir action, whereas the lack of inhibition of the two microRNAs matched with a low level of p27 expression}
{transfection with the miR-142-3p inhibitor increased the levels of AC9 protein and cAMP in CD4+CD25-T cells, and transfection with miR-142-3p decreased the levels of AC9 protein and cAMP in CD4+CD25-TREG cells (Fig 2B,C).}
{Transfection of primary cardiac fibroblasts with miR-21 precursors had no detectable effect on the Spry1 mRNA level (data not shown), but resulted in strong repression of SPRY1 protein expression (Fig. 3a)}
{miR-338 was over-expressed in SCG neurons by transfection of miR-338 precursor RNA, and COXIV protein levels were evaluated by immunoblot analysis. Overexpression of miR-338 lead to a ~40% decrease in COXIV protein levels in both the soma and distal axons of SCG neurons (Fig. 4E,G)}
{we measured LMO2 mRNA and protein in pcDNA3.1-pri-miR-223/K562 cells by real-time PCR and Western blot analysis. LMO2 mRNA levels in pcDNA3.1-pri-miR-223/K562 cells were much lower than that in pcDNA3.1/K562 cells both before and after hemin induction (Fig.8B)}
{immunostaining and western blot analyses of Lef1 protein levels in the dorsal halves of injected fins demonstrated significantly increased expression of Lef1 (Fig. 3 C and F)}
{Western blot analysis revealed that miR-22 regulates both PPARA and BMP7 protein expression levels in normal and osteoarthritic chondrocytes (Figure 3D) Overexpression of miR-22 inhibited BMP-7 (76%) and PPARA (93%) protein expression in normal chondrocytes}
{Western blot analysis revealed that miR-22 regulates both PPARA and BMP7 protein expression levels in normal and osteoarthritic chondrocytes (Figure 3D) Overexpression of miR-22 inhibited BMP-7 (76%) and PPARA (93%) protein expression in normal chondrocytes}
{Following transfection, the secretion of LIF was significantly decreased in cells transfected with miRNA hsa-mir199a and hsa-mir346 (Fig.4B)}
{Following transfection, the secretion of LIF was significantly decreased in cells transfected with miRNA hsa-mir199a and hsa-mir346 (Fig.4B)}
{To determine whether miR-101 represses EZH2 protein expression, we performed immunoblot analysis using an EZH2-specific antibodyas well as antibodies to other PRC2 members,including EED and SUZ12 (Fig. 1D)}
{To determine whether miR-101 represses EZH2 protein expression, we performed immunoblot analysis using an EZH2-specific antibodyas well as antibodies to other PRC2 members,including EED and SUZ12 (Fig. 1D)}
{Introduction of miR-138 into mouse NIH 3T3 cells decreased endogenous cspg2 mRNA and protein accumulation by approximately 40% (Fig. 5 B and C)}
{Transfection of decidual cells with anti-miR-338 induced a 2.8-fold reduction in miR- 338 expression (Figures 3A and 3B), while increasing PLA2G4B mRNA 3.7-fold (Figure 3C).immunoblotting also demonstrated an increase in PLA2G4B protein expression in anti-miR-338 transfected cells (Figure 3D)}
{The reduction of PMP22 mRNA level is accompanied by a comparable reduction at the protein level as determined by Western blot ( p<0.05, Student‚Äôs t test) (Fig. 5B)}
{Western blot analysis of protein extracts showed that cells transfected with a combination of miR-298 and miR-328 duplexes express lower levels of BACE1, as compared to N2a cells treated with the miR-196 control duplex (Fig. 7A). Inhibition of BACE1 expression was less pronounced when duplexes of either miRNAs were transfected individually into N2a cells}
{Western blot analysis of protein extracts showed that cells transfected with a combination of miR-298 and miR-328 duplexes express lower levels of BACE1, as compared to N2a cells treated with the miR-196 control duplex (Fig. 7A). Inhibition of BACE1 expression was less pronounced when duplexes of either miRNAs were transfected individually into N2a cells}
{Indeed, introduction of Pre-29 caused YY1 protein, but not RNA, to be reduced(figure D)}
{Indeed, introduction of Pre-29 caused YY1 protein, but not RNA, to be reduced(figure D)}
{Indeed, introduction of Pre-29 caused YY1 protein, but not RNA, to be reduced(figure D)}
{1. Target region level evidence:The targeting of KIT by mir-221 was studied in  mir-221 expressing CHO cells. expressing vectors containing human kit cDNA with or without 3'UTR.forty-eight hours after nucleofection, cells were lysed and expression of the cKit was examined by Western blot analysis. In mir-221-positive cells, expression of the cKit from 3'UTR-containing plasmid was decreased, whereas in cells transduced with cDNA it was unaffected (Fig. 4C). These studies confirmed that mir-221 could directly act on 3'UTR of the c-kit mRNA.}
{Also, only pre-miR-9* significantly reduced endogenous CoREST expression in HEK 293 cells (F(2,9)=11.173, p<0.0036) (Fig. 3B,C)}
{This resulted in significant knockdown of endogenous REST protein expression (F(2,9)=7.552, p<0.0119) compared with pre-miR-9* or the pre-miR negative control (Fig.2B,C)}
{Transfection with pre‚ÄìmiR-15a confirmed that miR-15a reduces cyclin E protein levels in SQ20B cells; this decrease occurred both in cells that were untreated or pretreated with the PKCa inhibitor Go6976 (Fig. 4C)}
{The CDC42 protein level fell in HeLa cells that were transfected with miR-29 miRNAs, as determined by western blotting}
{The CDC42 protein level fell in HeLa cells that were transfected with miR-29 miRNAs, as determined by western blotting}
{In the presence of a miR-29 miRNA mix, the p85Œ± protein level decreased, as determined by western blotting (Fig. 4a)}
{The CDC42 protein level fell in HeLa cells that were transfected with miR-29 miRNAs, as determined by western blotting}
{The CDC42 protein level fell in HeLa cells that were transfected with miR-29 miRNAs, as determined by western blotting}
{In the presence of a miR-29 miRNA mix, the p85Œ± protein level decreased, as determined by western blotting (Fig. 4a)}
{Western blotting of these cell lysates revealed that c-Myb protein expression was downregulated to 30% of baseline following transduction with miR-150, but not with the miR-negative control or miR195 (Fig. 4C).}
{Western blot analysis showed significant reduction in MET (~50 and 65% in clones 1 and 2, respectively) in both miR-1-expressing clones (Fig. 5, B and C).}
{Western blot analysis showed significant reduction in MET (~50 and 65% in clones 1 and 2, respectively) in both miR-1-expressing clones (Fig. 5, B and C).}
{Western blot analysis of A549 cell extracts showed a significant decrease in the endogenous FoxP1 level in miR-1-expressing cells (Fig. 5, B andC).}
{Western blot analysis revealed a dramatic decrease in HDAC4 following miR-1 expression (Fig. 5, B and C).}
{Significant reduction in endogenous Pim-1 protein (Fig. 5, B andC) andRNAlevels (Fig.5G) in miR-1-expressing cells indicates its negative regulation by miR-1 in A549 cells.}
{Of the candidates examined, only miR-15a was able to effect a significant reduction in K562 c-Myb protein content compared with controls (Figure 3A)}
{Of the candidates examined, only miR-15a was able to effect a significant reduction in K562 c-Myb protein content compared with controls (Figure 3A)}
{while IRS1 protein level decreased dose-dependently by psilencer-mir-126 transfection(Fig. 3E and F}
{In addition, the expression levels of proteins involved in cell cycle control and apoptosis such as CDK6, Cyclin D1, E2F3, E2F1, and BCL2 were decreased in PC3 cells as a result of ectopic expression of miR-34a, all of which except for E2F1 were identified to be its direct targets}
{In addition, the expression levels of proteins involved in cell cycle control and apoptosis such as CDK6, Cyclin D1, E2F3, E2F1, and BCL2 were decreased in PC3 cells as a result of ectopic expression of miR-34a, all of which except for E2F1 were identified to be its direct targets}
{In addition, the expression levels of proteins involved in cell cycle control and apoptosis such as CDK6, Cyclin D1, E2F3, E2F1, and BCL2 were decreased in PC3 cells as a result of ectopic expression of miR-34a, all of which except for E2F1 were identified to be its direct targets}
{In addition, the expression levels of proteins involved in cell cycle control and apoptosis such as CDK6, Cyclin D1, E2F3, E2F1, and BCL2 were decreased in PC3 cells as a result of ectopic expression of miR-34a, all of which except for E2F1 were identified to be its direct targets}
{after transfection with miR-183, Ezrin expression in 801D cell was almost abolished. In contrast, negative control RNA had little or none effect on Ezrin expression (Fig. 4a).}
{Immunoblotting analysis showed that, at 48 and 72 h,coinciding with the maximum accumulation of miR-21,the RAS-induced downregulation of PDCD4 was almost completely antagonized by the anti-miR-21 LNA, compared with the control cells treated with a scrambled LNA.}
{Differing from PDCD4, PTEN expression was only partially inhibited by RAS induction. Nevertheless,the partial downregulation of PTEN was abrogated by the antagomir-LNA, thus showing that also PTEN is targeted by miR-21, in the RAS-inducible thyroid system (Figure 6c)}
{cell lines were transfected with miR-7, miR-465, or negative control(designated miR-con) miRNA mimic, and Pak1 expression levels were determined by Western blotting. MiR-7 and miR-465 inhibited the levels of Pak1 protein but not Pak2, actin, or vinculin (Fig. 1B;Supplementary Fig. S1B).}
{Immunoblot analysis supported the data obtained with the reporter gene assays for BCL2,WNT3A and CCND1 (Fig. 3b,c and upplementary Fig. 8a‚Äìd online).}
{Immunoblot analysis supported the data obtained with the reporter gene assays for BCL2,WNT3A and CCND1 (Fig. 3b,c and supplementary Fig. 8a‚Äìd online).}
{Immunoblot analysis supported the data obtained with the reporter gene assays for BCL2,WNT3A and CCND1 (Fig. 3b,c and Supplementary Fig. 8a‚Äìd online).}
{Immunoblot analysis supported the data obtained with the reporter gene assays for BCL2,WNT3A and CCND1 (Fig. 3b,c and Supplementary Fig. 8a‚Äìd online).}
{Immunoblot analysis supported the data obtained with the reporter gene assays for BCL2,WNT3A and CCND1 (Fig. 3b,c and Supplementary Fig. 8a‚Äìd online).}
{Immunoblot analysis supported the data obtained with the reporter gene assays for BCL2,WNT3A and CCND1 (Fig. 3b,c and Supplementary Fig. 8a‚Äìd online).}
{Consistent with our prediction, overexpression of miR15a in PCK-CCL was associated with an approximately 50% reduction in the level of Cdc25A protein (Figure 6, D and E).}
{Meis1 protein and constant MEIS1 mRNA levels were observed in MIRN155-transfected MK cells (Fig 5C).}
{CREB3L3 was increased after treatment with P-2 inhibitor}
The immunoblotting showed that the protein expression of LAMB3 was decreased after treatment with P-27‚Äì5p mimic but increased after treatment with P-27‚Äì5p inhibitor (Fig. 3D).
{As shown in Fig. 5C, LNA-anti-miR-21 increased, whereas pre-miR-21 decreased PDCD4 expression in cultured VSMCs.}
{miR-373 and miR-520c both suppressed total CD44 protein compared with untreated positive control or with transfection of an irrelevant, scrambled miRNA (Figure 4b).}
{miR-373 and miR-520c both suppressed total CD44 protein compared with untreated positive control or with transfection of an irrelevant, scrambled miRNA (Figure 4b).}
{overexpression of mir-9a by patched (ptc)-Gal4, that drives expression of mir-9a in a stripe along the anterior‚Äìposterior compartment boundary in the wing pouch (Fig. 3J), reduces the level of dLMO expression according to this pattern (Fig. 3K).}
{each of the 5 shows significantly lower expression (P<0.05) in the presence of elevated mir-218 in vitro(figure 4)}
{each of the 5 shows significantly lower expression (P<0.05) in the presence of elevated mir-218 in vitro(figure 4)}
{each of the 5 shows significantly lower expression (P<0.05) in the presence of elevated mir-218 in vitro(figure 4)}
{each of the 5 shows significantly lower expression (P<0.05) in the presence of elevated mir-218 in vitro(figure 4)}
{each of the 5 shows significantly lower expression (P<0.05) in the presence of elevated mir-218 in vitro(figure 4)}
{Introduction of miR-101b decreased the protein levels significantly (Figures 4b and c).}
{Introduction of miR-101b decreased the protein levels significantly (Figures 4b and c).}
{Real-time PCR and Western blots revealed significantly increased expression of both RXRa mRNA and protein in activated HSCs cotransfected by anti-miR-27a and anti-miR-27b (Fig. 5)}
{Real-time PCR and Western blots revealed significantly increased expression of both RXRa mRNA and protein in activated HSCs cotransfected by anti-miR-27a and anti-miR-27b (Fig. 5)}
{miR-1 overexpression in NRVM decreased Mef2a protein (Fig. 7d). Mef2a mRNA was downregulated by miR-1 overexpression, suggesting that miR-1 decreased Mef2a transcript stability (Fig. 7e).}
{miR-1 overexpression in NRVM decreased Mef2a protein (Fig. 7d). Mef2a mRNA was downregulated by miR-1 overexpression, suggesting that miR-1 decreased Mef2a transcript stability (Fig. 7e).}
{miR-1 overexpression of miR-1 significantly downregulated CaM protein (Fig. 5e). }
{miR-1 overexpression of miR-1 significantly downregulated CaM protein (Fig. 5e). }
{overexpression of miR-134 duplex RNA in hippocampal neurons resulted in a significant decrease in endogenous Pum2 protein levels as assessed by immunocytochemistry (Figure 5D and E).}
{Immunostaining further confirmed that the ectopic expression of miR-145 substantially suppressed c-Myc expression (Fig. S4).}
{a significant down-regulation of endogenous DNMT1, and DNMT3A and 3B protein levels was also observed in all 3 AML cell lines 48 hours after pre-miR-29b transfection(Figure1D).}
{a significant down-regulation of endogenous DNMT1, and DNMT3A and 3B protein levels was also observed in all 3 AML cell lines 48 hours after pre-miR-29b transfection(Figure1D).}
{Western blot analysis revealed that both miR-30e and miR-30c suppressed Ubc9 expression at the protein level (Fig. 2B).}
{Western blot analysis revealed that both miR-30e and miR-30c suppressed Ubc9 expression at the protein level (Fig. 2B).}
{Western blot analysis revealed that both miR-30e and miR-30c suppressed Ubc9 expression at the protein level (Fig. 2B).}
{Western blot analysis revealed that both miR-30e and miR-30c suppressed Ubc9 expression at the protein level (Fig. 2B).}
{miR-205 suppressed the ErbB3 level in MCF-7 cells significantly compared to vector control.}
{Exposure to pre205 resulted in a significant reduction of PKCq mRNA levels accompanied by a pronounced decrease in protein abundance in DU145 and PC-3 cells (Fig. 4B and C).}
{Western blotting showed that IGF-1 protein was reduced by miR-1 as significantly as by IGF-1 siRNA (Fig. 4B and  Supplemental Fig. 3B).}
{RasXT cells also showed lower amounts of TTP protein than epithelial EpRas cells (Fig 2B).}
{This was also confirmed via immunoblotting analysis‚Äîoverexpression of miR-199a* resulted in decreased Smad1 protein levels(Figure 5F).}
{This was also confirmed via immunoblotting analysis‚Äîoverexpression of miR-199a* resulted in decreased Smad1 protein levels(Figure 5F).}
{Results confirmed that miR-34b, not miR-neg,inhibited CREB protein expression, which was found drastically reduced in all myeloid cell lines studied.}
{Western blot was used to reveal that endogenous LMNB1 levels were reduced by miR-23a and miR-23b (Fig. 5B) in HEK293 cells}
{Western blot was used to reveal that endogenous LMNB1 levels were reduced by miR-23a and miR-23b (Fig. 5B) in HEK293 cells}
{Western blot was used to reveal that endogenous LMNB1 levels were reduced by miR-23a and miR-23b (Fig. 5B) in HEK293 cells}
{Western blot was used to reveal that endogenous LMNB1 levels were reduced by miR-23a and miR-23b (Fig. 5B) in HEK293 cells}
{Western blot was used to reveal that endogenous LMNB1 levels were reduced by miR-23a and miR-23b (Fig. 5B) in HEK293 cells}
{Western blot was used to reveal that endogenous LMNB1 levels were reduced by miR-23a and miR-23b (Fig. 5B) in HEK293 cells}
{By Western blot analysis, transfection with miR-16 precursor was found to suppress}
{Over-expression of miR-181a caused a 5-fold reduction of p27  protein levels, while the amount of p27 mRNA was reduced only ~1.5-fold (Fig.7C and D).}
{We observed a dramatic reduction in MYB protein level upon ectopic expression of miR-150 (Figure 5A and Figure S10)}
{ABCG2 protein expression was suppressed more than 70% at 48 h after transfection with a higher dose (1.0 Œºg) of pS-miR-328 plasmid.}
{The down-regulation of endogenous microRNA-21 with anti‚ÄìmicroRNA-21 (Fig. 5A) led to a significant increase in PDCD4 protein (Fig. 5C and D) without any change in PDCD4 mRNA (Fig. 5B).}
{Overexpression of miR-205 (verified by Northern blot in Supplementary Fig. S3 and real-time PCR in Fig. 1A) decreased HER3 protein expression of f44% and 30% in SKBr3 and MCF7, respectively,as evaluated by densitometric analysis (Fig. 1B).}
{Accordingly, LMO2 protein level was markedly lower in miR-223- or siLMO2 transfected cells, as compared with control groups (Figure 3B, right).}
{Examination of endogenous APC protein levels from HEK293T cells stably expressing miR-135b revealed a clear down-regulation of APC protein compared with cells expressing a control construct (Fig. 2A).}
{Interestingly, miR-26a did not show any detectable inhibition to Ezh2 (Fig. 3E), although it efficiently repressed the expression of the reporter construct containing Ezh2 3'-UTR (Fig. 3B).}
{To investigate whether downregulation of endogenous miR-100 can modulate the levels of mTOR, raptor, and IGF-IR proteins, we transfected a specific miR-100 knockdown probe or a control probe into two different human adrenocortical cell lines (H295R and SW-13) and detected a dose-dependent increase in mTOR, raptor, and IGF-IR protein expression only after miR-100‚Äìspecific knockdown (Fig. 2D).}
{To investigate whether downregulation of endogenous miR-100 can modulate the levels of mTOR, raptor, and IGF-IR proteins, we transfected a specific miR-100 knockdown probe or a control probe into two different human adrenocortical cell lines (H295R and SW-13) and detected a dose-dependent increase in mTOR, raptor, and IGF-IR protein expression only after miR-100‚Äìspecific knockdown (Fig. 2D).}
{To investigate whether downregulation of endogenous miR-100 can modulate the levels of mTOR, raptor, and IGF-IR proteins, we transfected a specific miR-100 knockdown probe or a control probe into two different human adrenocortical cell lines (H295R and SW-13) and detected a dose-dependent increase in mTOR, raptor, and IGF-IR protein expression only after miR-100‚Äìspecific knockdown (Fig. 2D).}
{Totally 2 cell cycle related genes, cyclin D1 and p27, were found altered. A strong reduction in the expression of cyclin D1 and increased level of p27 was observed in miR-296 antagomir cells.The results were further confirmed by Western blot.}
{Totally 2 cell cycle related genes, cyclin D1 and p27, were found altered. A strong reduction in the expression of cyclin D1 and increased level of p27 was observed in miR-296 antagomir cells.The results were further confirmed by Western blot.}
{To evaluate whether MDR1 was a genuine target of miR-296, we analyzed the expression correlation of miR-296 and MDR1 in miR-296 antagomir and control cells by real-time PCR (Fig. 4A) and Western blot (Fig. 4B). The results showed that down-regulation of miR-296 significantly decreased expression of MDR1.}
{As shown in Figure 4A and B,the expression of Bcl-2 was decreased and the expression of Bax was increased in response to down-regulation of miR-296. However, relatively equal levels of Bcl-xL and Bak were detected in all ECA109 derived cell lines.}
{As shown in Figure 4A and B,the expression of Bcl-2 was decreased and the expression of Bax was increased in response to down-regulation of miR-296. However, relatively equal levels of Bcl-xL and Bak were detected in all ECA110 derived cell lines.}
{The inhibition of miR-199a-3p in SNU475 cells determined an increase of both mTOR and P-S6 proteins, with no variations in the mRNA level (Fig. 2C).}
{To evaluate the specific effect of miR-33 on ABCA1 expression, we transfected cells with synthetic miR-33 precursor oligonucleotides (pre-miR-33a/b) to increase the intracellular levels of miR-33a and b, respectively. These studies show that ABCA1 expression is repressed by excess miR-33, especially in mouse J774 macrophages and the human IMR-90 fibroblasts (Fig. 2B).}
{To evaluate the specific effect of miR-33 on ABCA1 expression, we transfected cells with synthetic miR-33 precursor oligonucleotides (pre-miR-33a/b) to increase the intracellular levels of miR-33a and b, respectively. These studies show that ABCA1 expression is repressed by excess miR-33, especially in mouse J774 macrophages and the human IMR-90 fibroblasts (Fig. 2B).}
{Expression of endogenous TBP in mRNA as well as protein level was decreased in STHdhQ111/HdhQ111 cells expressing exogenous miR-146a (Fig. 4B and C).}
{Transfection of Par-4-overexpressing cells with the miR-34a antagomir gradually increased BCL2 protein levels at 24 h and 72 h, when compared with negative control scrambled oligonucleotide transfected cells (Figure 9B, bottom panel). In contrast, no significant changes in BCL2 protein levels were detected at 0, 24, or 72 h in the empty vector-transfected cells with or without miR-34a inhibition (Figure 9B, bottom panel).}
{Ectopic expression of miR-215 by transient transfection decreased the expression of both DHFR and TS proteins in both osteosarcoma U-2 OS and colon cancer HCT 116 (wt-p53) cells analyzed by Western immunoblot analysis (Figure 1B).}
{Ectopic expression of miR-215 by transient transfection decreased the expression of both DHFR and TS proteins in both osteosarcoma U-2 OS and colon cancer HCT 116 (wt-p53) cells analyzed by Western immunoblot analysis (Figure 1B).}
{Ectopic expression of miR-215 by transient transfection decreased the expression of both DHFR and TS proteins in both osteosarcoma U-2 OS and colon cancer HCT 116 (wt-p53) cells analyzed by Western immunoblot analysis (Figure 1B).}
{We confirmed that miR-215 down-regulated DTL protein expression by Western immunoblot.}
{In accordance with the changes of mRNA level, the p53 protein level was also substan-tially reduced when miR-1285 was expressed in these cells (Fig. 3B).}
{In accordance with the changes of mRNA level, the p53 protein level was also substan-tially reduced when miR-1285 was expressed in these cells (Fig. 3B).}
{These results were also observed at protein level (Figure 4c).}
{miR-650 precursor transfection led to a significant decrease in ING4 protein level (Fig. 3B, p < 0.05).}
{We observed that in vitro AM-125b reduced miRNA-125b, restored CDKN2A back to homeostatic levels, and markedly reduced both GFAP expression and NHA proliferation (Fig. 5).}
{Antagomir-10b‚Äìtreated mice, which showed abundant, derepressed Hoxd10 protein expression in primary tumors (Fig. 2b).}
{miR-148a precursor significantly decreased the MSK1 protein level in PC3 cells on days 2, 3, 4, and 5 after transfection.}
{miR-148a precursor significantly decreased the MSK1 protein level in PC3 cells on days 2, 3, 4, and 5 after transfection.}
{miR-148a precursor significantly decreased the MSK1 protein level in PC3 cells on days 2, 3, 4, and 5 after transfection.}
{Quantitative analyses by ELISA confirmed that IL-8 and CXCL1 were reduced in miR-17/20‚Äìtransduced breast cancer cell medium (Fig. 2 E and F).}
{Quantitative analyses by ELISA confirmed that IL-8 and CXCL1 were reduced in miR-17/20‚Äìtransduced breast cancer cell medium (Fig. 2 E and F).}
{We found that, interestingly, both IL-10 mRNA and protein levels were higher in the miR-466loverexpressed RAW264.7 cells than that in mock control cells when these cells were stimulated with TLR ligands (Fig. 2B‚ÄìD; Supplemental Fig. 1).}
{After 72 h of miR-34a mimic transfection, Western blot analysis demonstrated that Sirt1 level was diminished by  40% in EPCs compared with that of scramble miRNA mimic (Fig. 3).}
{"To confirm the causative role of miR-15a in the regulation of bcl-2, both gain- and loss-of-miR-15a function were applied transfecting CECs with an miR-15a mimic or inhibitor reagent. After 48 h of treatment, the transfected CEC cultures weresubjected to 16 h OGD, and then the expression of bcl-2 was examined by Western blot. As shown in Figure 6,The miR-15a inhibitor effectively increases (Fig. 6A) whereas the mimic decreases (Fig. 6B)."}
{We examined the differences in miR-155 and CYR61 expression levels between the control and PE placentas by quantitative real time reverse transcriptase-polymerase chain reaction (qRT-PCR) (Figure 1, A). The expression of miR-155 in severe PE placenta is 2.58-fold higher in comparison to the control placentas (P  <0.001). The transcript level of CYR61 was significantly lower in severe PE placenta compared to the control group (Figure 1, A) (P <0.01).}
{"As expected, transfection of mir-375-NC did not show discernible effect on V1 expression. However, Ectopic mir-375 duplexes reducedV1 protein levels in NIT-1 cells by 3-fold, while inhibition of the endogenous mir-375 expression by transfection of 2'-O-Me-375 increased V1 protein levels by 1-fold (Figure 5)."}
{In contrast, overexpression of miR-155 mimics decreased MyD88 protein content in a dose and time-dependent manner (Fig. 2B and C).}
{miR-22 not miR-320 was able to repress Max protein expression (Fig. 3C).}
{Enforced expression of all three miR-34 family members reduced Myc protein abundance; however, miR-34c was the most potent (Fig. S1E).}
{Enforced expression of all three miR-34 family members reduced Myc protein abundance; however, miR-34c was the most potent (Fig. S1E).}
{Enforced expression of all three miR-34 family members reduced Myc protein abundance; however, miR-34c was the most potent (Fig. S1E).}
{The knockdown of DICER1 upon overexpression of miR-125b-2 in K562 cells was validated further by Western blot (Supplemental Fig. S7B).}
{The knockdown of DICER1 upon overexpression of miR-125b-2 in K562 cells was validated further by Western blot (Supplemental Fig. S7B).}
{The results showed that an ectopic increase in either synthesized or vector-expressed miR-22 in MCF-7 led to an  50% reduction in ERa protein levels (Fig. 2E).}
{To determine whether these effects depend specifically on E-cadherin suppression, we employed an expression construct that encodes the entire E-cadherin coding sequence but lacks the 3¬¥ UTR, yielding an mRNA resistant to miRNA-mediated suppression. Ectopic expression of E-cadherin with this construct decreased migration and invasion in miR-9-overexpressing cells but not in control cells, which have a low basal level of miR-9 (Fig. 1e, f).}
{Increased PTEN protein in 16HBE-T cells was detected in an miR-22 (147.6%) inhibition study (P < 0.05) (Fig. 3A and B). The miR-22 over-expression study in 16HBE-T cells showed PTEN protein expression decreased by 48.0% (P < 0.05) (Fig. 3A and C). No change of PTEN mRNA was observed.}
{We transfected the cells with pre-miR-155. This resulted in about 50-60% reduction of FOXO3a proteins (Fig. 2B and data not shown).}
{Transfection of any of the three miRNAs decreased both KLK10 protein expression levels and the rate of cellular proliferation when compared with the untransfected control cells.}
{Transfection of any of the three miRNAs decreased both KLK10 protein expression levels and the rate of cellular proliferation when compared with the untransfected control cells.}
{Transfection of any of the three miRNAs decreased both KLK10 protein expression levels and the rate of cellular proliferation when compared with the untransfected control cells.}
{We overexpressed miR-155 in MCF-7 cells using a vector coexpressing mir-155 and GFP. As shown by immunofluorescent staining, SOCS1 protein was largely reduced in cells with a strong GFP signal and miR-155 overexpression.}
{Western blot analysis using specific antibody against Notch1 showed that the expression level of Notch1 protein was decreased by 24 and 31% in the pre-miR-34a-transfected HeLa and JAR cells, respectively (Figure 2B).}
{The expression of Jagged1 in the HeLa and JAR cells was decreased by 20 and 25%, respectively upon pre-miR-34a transfection (Figure 2C).}
{Since Jak1 was efficiently down-regulated on mRNA as well as on protein level by miR-17 and the closely related miR-20a (Fig. 5A/B/C)}
{We observed a minor down-regulation of the anti-angiogenic proteins CTGF and Tsp1 by overexpression of miR-18a and miR-19a.}
{We observed a minor down-regulation of the anti-angiogenic proteins CTGF and Tsp1 by overexpression of miR-18a and miR-19a.}
{The reduction of p21 mediated by miR-17 was associated with an increased proliferation of pre-miR-17 transfected endothelial cells.}
{In contrast, the reduction of EDG-1 expression did not affect the chemotaxic response of the ECs to the EDG-1-ligand sphingosine-1-phosphate.}
{Since Jak1 was efficiently down-regulated on mRNA as well as on protein level by miR-17 and the closely related miR-20a (Fig. 5A/B/C)}
{Overexpression of individual miR-17-92 cluster members caused only a minor reduction of Bim expression (Fig. 4A).}
{Overexpression of individual miR-17-92 cluster members caused only a minor reduction of Bim expression (Fig. 4A).}
{Overexpression of individual miR-17-92 cluster members caused only a minor reduction of Bim expression (Fig. 4A).}
{Overexpression of individual miR-17-92 cluster members caused only a minor reduction of Bim expression (Fig. 4A).}
{Ectopic expression of miR-18a resulted in a substantial down-regulation (‚àº90%) of ESR1 protein levels within 24h of transfection.}
{Ectopic expression of miR-18a resulted in a substantial down-regulation (‚àº90%) of ESR1 protein levels within 24h of transfection.}
{Forced miR-19a expression also resulted in reduced ESR1 levels 24 h posttransfection.}
{Western blotting revealed that the protein level of Smad3 was reduced after transfection of miR-140 mimicking siRNA into C3H10T1/2 cells and was increased after suppressing endogenous miR-140 activity by antimiR-140 (Fig. 1B).}
{The single administration of miR-30a, miR-30b or miR-30d antagomir could slightly elevate p53 expression levels. However, the simultaneous administration of the three antagomirs led to a significant increase in p53 levels.}
{The single administration of miR-30a, miR-30b or miR-30d antagomir could slightly elevate p53 expression levels. However, the simultaneous administration of the three antagomirs led to a significant increase in p53 levels.}
{The single administration of miR-30a, miR-30b or miR-30d antagomir could slightly elevate p53 expression levels. However, the simultaneous administration of the three antagomirs led to a significant increase in p53 levels.}
{Immunoblot analysis revealed that p53 expression levels were elevated upon knockdown of miR-30c and miR-30e (Figure S2B).}
{Immunoblot analysis revealed that p53 expression levels were elevated upon knockdown of miR-30c and miR-30e (Figure S2B).}
{"To confirm the contributory role of miR-497 in the regulation of bcl-2 and bcl-w, both in vitro gain- and loss-of-miR-497 functionmethods were applied by transfecting N2A cells with a miR-497 mimic or inhibitor reagent. As shown in Fig. 3, miR-497 inhibitor effectively increases (Fig. 3B), whereas miR-497 mimic decreases (Fig. 3C) the expression of bcl-2 and bcl-w in mouse N2A cells subjected to 3 hours of OGD followed by 4 hours of recovery time."}
{"To confirm the contributory role of miR-497 in the regulation of bcl-2 and bcl-w, both in vitro gain- and loss-of-miR-497 functionmethods were applied by transfecting N2A cells with a miR-497 mimic or inhibitor reagent. As shown in Fig. 3, miR-497 inhibitor effectively increases (Fig. 3B), whereas miR-497 mimic decreases (Fig. 3C) the expression of bcl-2 and bcl-w in mouse N2A cells subjected to 3 hours of OGD followed by 4 hours of recovery time."}
{Western blot analysis showed that miR-34a concomitantly reduced the level of MEK1 protein (Fig. 6C).}
{As shown in Figure 2c, ectopic transfection of miR-138 reduced the protein level of RhoC in UM1 cells.}
{As shown in Figure 3c, ectopic transfection of miR-138 reduced the protein level of ROCK2 in UM1 cells.}
{The downregulation of 14-3-3Œ∂ and cIAP-2 and upregulation of Bim by pre375 were further validated by qRT-PCR (Fig. 3B). As shown in Fig. 3C, 14-3-3Œ∂ was also downregulated at the protein level.}
{Notably, secretion of MCP-2 was almost completely suppressed in cells overexpressing mir-146a.}
{A significant decrease of the HDAC6 protein level was observed in cells transfected with hsa-miR-433 in comparison to the mock transfected cells (Fig. 2A and B, compare lanes 1 and 2) (P =0.03; n =3).}
{We found that after transfection with miR-224, there was a significant decrease in KLK1 expression as expression decreased from 0.65 mg/l to 0.12 mg/l (p-0.001; Figure 3).}
{AGO proteins on Western blots following transfections with miR-107 and miR-128 were decreased compared to miR-124, miR-320, and a miRNA negative control transfection (Fig. 7B). }
{AGO proteins on Western blots following transfections with miR-107 and miR-128 were decreased compared to miR-124, miR-320, and a miRNA negative control transfection (Fig. 7B). }
{AGO proteins on Western blots following transfections with miR-107 and miR-128 were decreased compared to miR-124, miR-320, and a miRNA negative control transfection (Fig. 7B). }
{AGO proteins on Western blots following transfections with miR-107 and miR-128 were decreased compared to miR-124, miR-320, and a miRNA negative control transfection (Fig. 7B). }
{Overexpression of miR-24 clearly decreased the expression of DHFR protein.}
{In miR-181a mimic-treated U87MG cells, Bcl-2 was significantly decreased 72 h after transfection (Fig. 2B and C).}
{Western blot analysis revealed that, while miR-133a mimic treatment decreased NFATc4 protein expression.}
{Western blot analysis revealed that, while miR-133a mimic treatment decreased NFATc4 protein expression.}
{"The XTT cell-growth assay showed significant cellmiR-growth inhibitions in miR-145 and miR133a transfectant comparedwith the controls from BOY and T24 cell lines."}
{"The XTT cell-growth assay showed significant cellmiR-growth inhibitions in miR-145 and miR133a transfectant comparedwith the controls from BOY and T24 cell lines."}
{"The XTT cell-growth assay showed significant cellmiR-growth inhibitions in miR-145 and miR133a transfectant comparedwith the controls from BOY and T24 cell lines."}
{"The XTT cell-growth assay showed significant cellmiR-growth inhibitions in miR-145 and miR133a transfectant comparedwith the controls from BOY and T24 cell lines."}
{"The XTT cell-growth assay showed significant cellmiR-growth inhibitions in miR-145 and miR133a transfectant comparedwith the controls from BOY and T24 cell lines."}
{Western blot analysis in brain extracts from OL-specific Dicer null mice and wild-type littermates revealed that ELOVL7 levels are greatly increased in 6-month-old mutant mice compared with controls, whereas no difference was observed in 1-month-old mice (Fig 5).}
{Expression of ENPP6 was not changed at either age.}
{Overexpression of several miRs (miR-9,-27a, -96, -153, -183 and -186) effectively down-regulated FOXO1 expression (Fig. 5A).}
{Overexpression of several miRs (miR-9,-27a, -96, -153, -183 and -186) effectively down-regulated FOXO1 expression (Fig. 5A).}
{Overexpression of several miRs (miR-9,-27a, -96, -153, -183 and -186) effectively down-regulated FOXO1 expression (Fig. 5A).}
{Overexpression of several miRs (miR-9,-27a, -96, -153, -183 and -186) effectively down-regulated FOXO1 expression (Fig. 5A).}
{Overexpression of several miRs (miR-9,-27a, -96, -153, -183 and -186) effectively down-regulated FOXO1 expression (Fig. 5A).}
{Overexpression of several miRs (miR-9,-27a, -96, -153, -183 and -186) effectively down-regulated FOXO1 expression (Fig. 5A).}
{Western blot and qPCR experiments confirmed that miR-221 represses DDIT4 expression at both the protein and RNA levels in the different cell types (Fig. 5C and Fig. S6D).}
{Western blot and qPCR experiments confirmed that miR-221 represses DDIT4 expression at both the protein and RNA levels in the different cell types (Fig. 5C and Fig. S6D).}
{A similar decrease was observed for BNIP3L and TBK1 protein/RNA levels, although the effect of miR-221 was weaker for these two targets (Fig. 5C and Fig. S6D).}
{A similar decrease was observed for BNIP3L and TBK1 protein/RNA levels, although the effect of miR-221 was weaker for these two targets (Fig. 5C and Fig. S6D).}
{Transfectionwith 30 nMof premiR-494 resulted in a decrease in PTEN protein (Fig. 2A and B) while mRNA remained unchanged (Fig. 2C) in both 16HBE-T30 and 16HBEN30 cells.}
{Transfectionwith 30 nMof premiR-494 resulted in a decrease in PTEN protein (Fig. 2A and B) while mRNA remained unchanged (Fig. 2C) in both 16HBE-T30 and 16HBEN30 cells.}
{However, the miRs targeting R1 in the KRT967‚Äì1466 transcript produced significant reductions ( 50%) in KRT protein expression (Fig. 2D,E).}
{However, the miRs targeting R1 in the KRT967‚Äì1466 transcript produced significant reductions ( 50%) in KRT protein expression (Fig. 2D,E).}
{miR-138, which aligned with the KRT967‚Äì1466 transcript at a site lying between R2 and R3, had profound effects on KRT protein levels, with reductions of  85%.}
{miR-let-7b,which targeted R6 in the KRT967‚Äì1466 transcript, also produced substantial reductions ( 70%) in KRT protein expression (Fig. 2).}
{Consistent with previous research, we found that protein expression levels of both CDK6 and CDC25A were decreased with miR-125b overexpression in CD133-positive U251 cells compared to the control (Fig. 3B).}
{Consistent with previous research, we found that protein expression levels of both CDK6 and CDC25A were decreased with miR-125b overexpression in CD133-positive U251 cells compared to the control (Fig. 3B).}
{The results showed that enforced expression of miR-16-1 led to a significant decrease in endogenous CCNE1 proteins (Fig. 2a), suggesting that the endougenous expression of CCNE1 is downregulated by miR-16-1.}
{The results showed that enforced expression of miR-16-1 led to a significant decrease in endogenous CCNE1 proteins (Fig. 2a), suggesting that the endougenous expression of CCNE1 is downregulated by miR-16-1.}
{As the target of miR-675, the level of RB protein also appears to be negatively correlated with the levels of both H19 and miR-675 in the human colon cancer cells (Figure 4D).}
{Relative to the control, overexpression of miR-2861 downregulated endogenous HDAC5 protein (Figure 4C).}
{"In contrast, transfection of miR-218 precursors triggered a negligible change in Col1A1 and SP1 mRNA expression (Fig. 4A, left andright panels) but slightly reduced their protein level (Fig. 4B)."}
{Transient transfection of miR-29b precursors signifi-cantly inhibited type I collagen mRNA and protein expression (Fig. 4A, left panel, and B) in unstimulated human stellate cells.}
{"In contrast, transfection of miR-218 precursors triggered a negligible change in Col1A1 and SP1 mRNA expression (Fig. 4A, left andright panels) but slightly reduced their protein level (Fig. 4B)."}
{Moreover, the transfection of miR-29b precursors decreased SP1 mRNA and protein expression (Fig. 4A, right panel, and B).}
{These cells expressed PTEN, PDCD4, E2F1 and TGF-bRII mRNAs and proteins at varying levels (Figs 3 and 4) and gain- and loss-of-miR-21 function differentially altered their expression in a cell-specific manner when compared with scrambled-oligo-transfected controls.}
{These cells expressed PTEN, PDCD4, E2F1 and TGF-bRII mRNAs and proteins at varying levels (Figs 3 and 4) and gain- and loss-of-miR-21 function differentially altered their expression in a cell-specific manner when compared with scrambled-oligo-transfected controls.}
{These cells expressed PTEN, PDCD4, E2F1 and TGF-bRII mRNAs and proteins at varying levels (Figs 3 and 4) and gain- and loss-of-miR-21 function differentially altered their expression in a cell-specific manner when compared with scrambled-oligo-transfected controls.}
{These cells expressed PTEN, PDCD4, E2F1 and TGF-bRII mRNAs and proteins at varying levels (Figs 3 and 4) and gain- and loss-of-miR-21 function differentially altered their expression in a cell-specific manner when compared with scrambled-oligo-transfected controls.}
{Its protein levels were inhibited by miR-199b over-expression in JAR cell line (Fig. 3).}
{As anticipated, the modulation of SHIP1 mRNA levels by miR-155 or antimiR-155 is reflected by accompanying changes in SHIP1 protein expression (Fig 2B, left panel).}
{As expected, the exogenous expression of miR-129-2 results in reduction of SOX4 protein. Cells transfected with the negative control did not exhibit any change in SOX4 levels (Fig. 2B).}
{"In comparison with NC-transfectants, hsa-miR-155-transfected cells revealed a significant reduction (~80%) in the C/EBPÔÅ¢ protein level (Figure 4C, upper panel). Conversely, no fluctuation in C/EBPŒ≤mRNA levels was observed after miR-155 transfection (Figure 4C,lower panel)."}
{Ectopic expression of miR-139 results in a decrease in FoxO1 protein of approximately 30% (Fig. 1D) without affecting FoxO1 mRNA level (Fig. 1C).}
{However, in 433mir125a and DOVmir125a cells, there is approximately a 70% to 80% decrease in total ARID3B expression (Figure 3B).}
{"Introduction of the miRNAs resulted in downregulation of RECK protein (Figure 1d,Protein) with no appreciable changes in the level ofRECK mRNA."}
{"Introduction of the miRNAs resulted in downregulation of RECK protein (Figure 1d,Protein) with no appreciable changes in the level ofRECK mRNA."}
{"Introduction of the miRNAs resulted in downregulation of RECK protein (Figure 1d,Protein) with no appreciable changes in the level of RECK mRNA."}
{"Introduction of the miRNAs resulted in downregulation of RECK protein (Figure 1d,Protein) with no appreciable changes in the level of RECK mRNA."}
{Transfection of Mz-ChA-1 and KMCH cells with either miR-148a or miR-152 decreased DNMT-1 expression after 3 days (Fig. 4).}
{Transfection of Mz-ChA-1 and KMCH cells with either miR-148a or miR-152 decreased DNMT-1 expression after 3 days (Fig. 4).}
{Downregulation of FIH mRNA expression and protein expression was found in SAS-miR-31 cells compared with controls.}
{Downregulation of FIH mRNA expression and protein expression was found in SAS-miR-31 cells compared with controls.}
{"Western blot analysis revealed that the protein level of cyclin D1,was markedly reduced in the cells overexpressing miR-19acompared with the cell lines transfected with control vector."}
{OA chondrocytes with overexpression of miR-27b produced  38% less MMP-13 protein upon stimulation with IL-1  compared with nontransfected chondrocytes (P <  0.05) (Figure 3C).}
{Transfection of H69 cells with the anti-miR-221 caused a dose-dependent increase of ICAM-1 protein content (Fig. 4A).}
{Western blot analysis showed that the rescue of PPP2R2A protein expression could be consistently detected in both cell lines in response to anti‚ÄìmiR-222 transfection compared with the mock control (Fig. 4C).}
{These results showed a slight reduction in PTEN expression when miR-205 was overexpressed as compared with the luciferase shRNA control vector (Fig. 4E).}
{These results showed a slight reduction in PTEN expression when miR-205 was overexpressed as compared with the luciferase shRNA control vector (Fig. 4E).}
{Western blot analysis of TOM1 in CFBE41o‚Äì cells showed that miR-126 overexpression caused a reduction in TOM1 protein production compared with nontransfected (NT) cells or cells transfected with a Scr (Fig. 6A).}
{Using qRT-PCR and immunoblotting of p16Ink4a and p19Arf we show that both are downregulated in Lmnb1Œî/Œî compared to WT cells and that transfecting Lmnb1Œî/Œî cells with anti-mir-31 results in the upregulation of both (Fig. 4A and B).}
{The downregulation of NOTCH4 and upregulation of p21 (WAF1/Cip1) were also observed at the protein level (Figure 5C).}
{A decrease of KLF4 protein level in KYSE140 and KYSE450 cells was observed.}
{As predicted by the increased expression of miR-205 in hypoxic trophoblasts, hypoxia reduced MED1 protein and MED1 RNA expression in primary human trophoblasts}
{As predicted by the increased expression of miR-205 in hypoxic trophoblasts, hypoxia reduced MED1 protein and MED1 RNA expression in primary human trophoblasts}
{As predicted by the increased expression of miR-205 in hypoxic trophoblasts, hypoxia reduced MED1 protein and MED1 RNA expression in primary human trophoblasts}
{Western blot analysis further demonstrated that the high expression of miR-30d dramatically suppressed the endogenous protein level of GNAI2.}
{Western blot analysis further demonstrated that the high expression of miR-30d dramatically suppressed the endogenous protein level of GNAI2.}
{When 100nM synthetic LNA‚ÄìmiR-124 was introduced into differentiated P19 cells, the intracellular level of mature miR-124 reduced significantly (Figure 2). In contrast, the level of Hes-1 protein increased in the presence of LNA‚ÄìmiR-124 (Figure 3).}
{As expected, a pronounced decrease in TIMP3 protein level (B56%) was observed in both Hep3B and SK-Hep1 cells expressing miR-181b.}
{miR-15b down-regulated both mRNA and protein of Arl2. }
{This overexpression of let-7a led to a discernible downregulation of K-Ras expression and increased radiosensitivity of A549 cells.}
{As shown in Fig. 2B (top), miR-145 suppressed both large and small isoforms of MUC1. }
{Notably, we found that FADD protein levels, expressed as mean Ô¨Çuorescence intensity (MFI), were 20% lower in pRRL-146a Jurkat cells compared with the control pRRL-ct Jurkat cells .}
{Concomitant knockdown of the 4 miRNAs elevated MKK4 protein abundance .}
{Concomitant knockdown of the 4 miRNAs elevated MKK4 protein abundance .}
{Concomitant knockdown of the 4 miRNAs elevated MKK4 protein abundance .}
{Concomitant knockdown of the 4 miRNAs elevated MKK4 protein abundance .}
{Ectopic expression of miR-143 in LNCaP, and C4-2 prostate cancer cell lines resulted in a significant decrease in ERK5 protein expression, compared to cells transfected with non-relevant miRNA.}
{Western blotting showed that endogenous RhoB expression was increased when miR-223 was inhibited (Figure 2d).}
{Western blotting showed that endogenous RhoB expression was increased when miR-223 was inhibited (Figure 2d).}
{Following transfection with anit-miRs, a 70-80% reduction in miR expression (data not shown) resulted in an average 3-fold (miR-99a) and 6-fold (miR-100) upregulation of FGFR3 protein (Figure 4).}
{Following transfection with anit-miRs, a 70-80% reduction in miR expression (data not shown) resulted in an average 3-fold (miR-99a) and 6-fold (miR-100) upregulation of FGFR3 protein (Figure 4).}
{At the protein level, TGFBR1 expression was significantly inhibited by let-7c.}
{At the protein level, TGFBR1 expression was significantly inhibited by let-7c.}
{We found that the mRNA level of SIP1 was not changed while the protein level of SIP1 was down-regulated after overexpression .}
{We found that the mRNA level of SIP1 was not changed while the protein level of SIP1 was down-regulated after overexpression .}
{p53 protein was reduced in miRNA-125a transfected cells but not in cells that had been transfected by an empty vector or one encoding an irrelevant miRNA.}
{ However, this fluorescence was dampened over 60% by co-injection of miR-145 mimic.}
{By using the miR-421 inhibitor, CBX7 and RBMXL1 in protein levels were increased in a dose-dependent fashion .}
{By using the miR-421 inhibitor, CBX7 and RBMXL1 in protein levels were increased in a dose-dependent fashion .}
{we conÔ¨Årmed by 3'-UTR dual-luciferase activity assay and by Western blot that the member of this family more clearly down-regulated during SIPS in HDF and HTMcells,miR-106a, also targets the P21cdkn1a mRNA.}
{Western blot analysis ofHDF andHTMcells transfectedwith miR-182 showed a marked decrease in RARG protein expression compared to cells transfected with a scrambled control.}
{We clearly showed that the Tv_MDH protein expression level in trophozoite stage transfected with miR-1 mimics was significantly decreased by 60%.}
{"Overexpression of miR-140 significantly decreased the expression of HDAC4 protein without considerableamount of mRNA degradation (Figure 5c, lane 3)."}
{The expression of 133a and 133b reduced the protein levels of KLF15, whereas they did not change the KLF15 mRNA level.}
{The expression of 133a and 133b reduced the protein levels of KLF15, whereas they did not change the KLF15 mRNA level.}
{Anti-miR-183 elevated SC35 protein levels by more than twofold.}
{Remarkably, anti-miR- 18a, anti-miR-20b, and anti-miR-19b molecules able to silence their miRNA function significantly increased reporter activity (Fig. 5B).}
{Remarkably, anti-miR- 18a, anti-miR-20b, and anti-miR-19b molecules able to silence their miRNA function significantly increased reporter activity (Fig. 5B).}
{Interestingly, we found that increased levels of miR-200a in meningioma cells significantly downregulated  -catenin at both mRNA and protein levels.}
{At the protein level, significant downregulation by LV-miR-124 of BDNF was also observed.}
{At protein levels, Western blots analysis showed a significant decrease of DA D3R.}
{It was shown that knockdown of miR-9 enhanced NF-jB1 protein expression, including its cytoplamsic precursor p105 and corresponding processing product p50.}
{Importantly, KGF release by lung fibroblasts in response to IL-1b was significantly decreased in cells transfected by miR-155 compared to control.}
{Importantly, KGF release by lung fibroblasts in response to IL-1b was significantly decreased in cells transfected by miR-155 compared to control.}
{We then confirmed by western blotting that CDK6 protein levels are also down-regulated by miR-107.}
{Transfection of miR-221 in SNU398 cells resulted in a 47% decrease of Bmf levels when compared with cells transfected with negative contron.}
{As shown in Figure 3A and 3B, over-expression of miR-93 or miR-98 significantly inhibited Fus1 protein expression.}
{As shown in Figure 3A and 3B, over-expression of miR-93 or miR-98 significantly inhibited Fus1 protein expression.}
{Furthermore, western blot analysis indicated that miR-210 overexpression effectively reduced MNT protein levels in all cell lines tested.}
{Furthermore, overexpression of miR-155 resulted in the down-regulation of the protein levels of IKK-Œµ , SMAD2, and FADD.}
{Furthermore, overexpression of miR-155 resulted in the down-regulation of the protein levels of IKK-Œµ , SMAD2, and FADD.}
{Furthermore, overexpression of miR-155 resulted in the down-regulation of the protein levels of IKK-Œµ , SMAD2, and FADD.}
{AntimiR-183 also elevated the expression of Œ≤TrCP protein in a dose-dependent manner.}
{More importantly, we also found an inverse correlation of DNMT3A protein and miR-143 in seven paired CRC tissues .}
{miR-7 reduced  -Syn expression dose-dependently: 40 Nm miR-7 resulted in 40% reduction of  -Syn expression, and 80 nM miR-7 reduced it by 50%.}
{For each genotype, we individually tested  25 carefully staged embryos (0‚Äì1 hr) and found a 2.5-fold average increase of TTK69 protein in  ‚ñ≥mir-184, supporting the idea that miR-184 downregulates TTK69 protein levels in vivo.}
{In Dmir-184 mutant ovaries, we find K10 protein expressed early (stage 2) and at much higher levels than in wild-type.}
{In the Dmir-184 mutant, we observe SAX protein present at much higher levels.}
{"In comparison with negative control 1 precursor miRNA, transfection of miR-15a and miR-16 into ovarian cell lines caused a significant decreasein protein levels of Bmi-1 as determined by Western blot (Fig. 3)."}
{"In comparison with negative control 1 precursor miRNA, transfection of miR-15a and miR-16 into ovarian cell lines caused a significant decreasein protein levels of Bmi-1 as determined by Western blot (Fig. 3)."}
{"In comparison with negative control 1 precursor miRNA, transfection of miR-15a and miR-16 into ovarian cell lines caused a significant decreasein protein levels of Bmi-1 as determined by Western blot (Fig. 3)."}
{With transfection of either the miR-155 or -802 mimic, endogenous miR-155 and -802 levels increased by 200‚Äì310% (Fig. 1G) and MeCP2 mRNA (Fig. 1G) and MeCP2 protein (Fig. 1H) levels were significantly decreased (30‚Äì50% at the mRNA level and 40‚Äì50% at the protein level).}
{With transfection of either the miR-155 or -802 mimic, endogenous miR-155 and -802 levels increased by 200‚Äì310% (Fig. 1G) and MeCP2 mRNA (Fig. 1G) and MeCP2 protein (Fig. 1H) levels were significantly decreased (30‚Äì50% at the mRNA level and 40‚Äì50% at the protein level).}
{With transfection of either the miR-155 or -802 mimic, endogenous miR-155 and -802 levels increased by 200‚Äì310% (Fig. 1G) and MeCP2 mRNA (Fig. 1G) and MeCP2 protein (Fig. 1H) levels were significantly decreased (30‚Äì50% at the mRNA level and 40‚Äì50% at the protein level).}
{With transfection of either the miR-155 or -802 mimic, endogenous miR-155 and -802 levels increased by 200‚Äì310% (Fig. 1G) and MeCP2 mRNA (Fig. 1G) and MeCP2 protein (Fig. 1H) levels were significantly decreased (30‚Äì50% at the mRNA level and 40‚Äì50% at the protein level).}
{"hCD98 expression at the protein level was also decreased upon transfection of cells with miR-7 with a maximal reduction level after 48 hof transfection (Fig. 3B). "}
{As shown in Fig. 3, D and E, miR-706 significantly down-regulated expression of mCD98 at both mRNA and protein levels after 1 and 2 days of transfection.}
{MiR-125 transfection reduced HuR protein level in MCF-7 and T47D cells, with miR-125a having a greater effect than miR-125b (Fig. 2B).}
{MiR-125 transfection reduced HuR protein level in MCF-7 and T47D cells, with miR-125a having a greater effect than miR-125b (Fig. 2B).}
{Furthermore, we observed down-regulation of Mcl-1 and Cdk6 proteins after transfection of synthetic miR-29a and -29b oligonucleotides, but not with the controls in K562 cells (Figure 4D).}
{Furthermore, we observed down-regulation of Mcl-1 and Cdk6 proteins after transfection of synthetic miR-29a and -29b oligonucleotides, but not with the controls in K562 cells (Figure 4D).}
{Furthermore, we observed down-regulation of Mcl-1 and Cdk6 proteins after transfection of synthetic miR-29a and -29b oligonucleotides, but not with the controls in K562 cells (Figure 4D).}
{Furthermore, we observed down-regulation of Mcl-1 and Cdk6 proteins after transfection of synthetic miR-29a and -29b oligonucleotides, but not with the controls in K562 cells (Figure 4D).}
{Furthermore, we observed down-regulation of Mcl-1 and Cdk6 proteins after transfection of synthetic miR-29a and -29b oligonucleotides, but not with the controls in K562 cells (Figure 4D).}
{Furthermore, we observed down-regulation of Mcl-1 and Cdk6 proteins after transfection of synthetic miR-29a and -29b oligonucleotides, but not with the controls in K562 cells (Figure 4D).}
{Furthermore, we observed down-regulation of Mcl-1 and Cdk6 proteins after transfection of synthetic miR-29a and -29b oligonucleotides, but not with the controls in K562 cells (Figure 4D).}
{Furthermore, we observed down-regulation of Mcl-1 and Cdk6 proteins after transfection of synthetic miR-29a and -29b oligonucleotides, but not with the controls in K562 cells (Figure 4D).}
{Furthermore, we observed down-regulation of Mcl-1 and Cdk6 proteins after transfection of synthetic miR-29a and -29b oligonucleotides, but not with the controls in K562 cells (Figure 4D).}
{Furthermore, we observed down-regulation of Mcl-1 and Cdk6 proteins after transfection of synthetic miR-29a and -29b oligonucleotides, but not with the controls in K562 cells (Figure 4D).}
{Furthermore, we observed down-regulation of Mcl-1 and Cdk6 proteins after transfection of synthetic miR-29a and -29b oligonucleotides, but not with the controls in K562 cells (Figure 4D).}
{"Transfection of miR132 increased expression of the microRNA in myofibroblasts by 300 fold (Figure 4C), which was associated with diminishedexpression of MeCP2 protein (Figure 4D)."}
{"Upon transfection of these cell lines with the miR-17-92 cluster microRNAs or the miR-15/16 family (a kind gift from Dr. Patrick Lewis,Merck Co., Inc.), we observed significant downregulation of endogenous cyclin D1 protein in the MCF-7 cell line but not in the MDA-MB-453 cell line with cyclin D1 3'UTR deletions (Fig. 3C, upper)."}
{"Upon transfection of these cell lines with the miR-17-92 cluster microRNAs or the miR-15/16 family (a kind gift from Dr. Patrick Lewis,Merck Co., Inc.), we observed significant downregulation of endogenous cyclin D1 protein in the MCF-7 cell line but not in the MDA-MB-453 cell line with cyclin D1 3'UTR deletions (Fig. 3C, upper)."}
{"Upon transfection of these cell lines with the miR-17-92 cluster microRNAs or the miR-15/16 family (a kind gift from Dr. Patrick Lewis,Merck Co., Inc.), we observed significant downregulation of endogenous cyclin D1 protein in the MCF-7 cell line but not in the MDA-MB-453 cell line with cyclin D1 3'UTR deletions (Fig. 3C, upper)."}
{"Upon transfection of these cell lines with the miR-17-92 cluster microRNAs or the miR-15/16 family (a kind gift from Dr. Patrick Lewis,Merck Co., Inc.), we observed significant downregulation of endogenous cyclin D1 protein in the MCF-7 cell line but not in the MDA-MB-453 cell line with cyclin D1 3'UTR deletions (Fig. 3C, upper)."}
{Transfection of miR-34a reduced the protein levels of c-Met protein in glioma and medulloblastoma cells and astrocytes.}
{Transfection of miR-34a reduced the protein levels of c-Met protein in glioma and medulloblastoma cells and astrocytes.}
{Transfection of miR-34a also reduced the protein levels of Notch-1, Notch-2, and CDK6 in glioma cells and stem cells (Fig. 1A).}
{Transfection of miR-34a also reduced the protein levels of Notch-1, Notch-2, and CDK6 in glioma cells and stem cells (Fig. 1A).}
{Transfection of miR-34a also reduced the protein levels of Notch-1, Notch-2, and CDK6 in glioma cells and stem cells (Fig. 1A).}
{I3C significantly reduced the level of miR-21 only; the expression of the other miRNAs was not affected (Figure 5A). In subsequent studies, we examined if I3C also modulates miR-21 target proteins in A549 cells. As in the immunoblot study with mouse lung tissues, the expressions of PTEN, PDCD4 and RECK were upregulated by I3C (Figure 5B).}
{MYC protein also decreased substantially (85%) (Figure 4C).}
{MYC protein also decreased substantially (85%) (Figure 4C).}
{MYC protein also decreased substantially (85%) (Figure 4C).}
{MYC protein also decreased substantially (85%) (Figure 4C).}
{MYC protein also decreased substantially (85%) (Figure 4C).}
{MYC protein also decreased substantially (85%) (Figure 4C).}
{As expected, E2F2 protein (Figure 5B) was also substantially reduced (9-fold).}
{detect a substantial decrease in the mean fluorescence intensity of cyclin D1 protein in the New Zealand Black B-cell line transfected with miR-16 compared with the negative control .}
{Transfection with miR-15a also resulted in a decrease in cyclin D1 protein levels in the New Zealand Black B-cell line (Table 1).}
{Furthermore, overexpression of miR- 206 markedly decreased the levels of endogenous mouse Notch3 protein (Fig. 1D) in mouse embryonic fibroblasts (MEFs).}
{As expected, ectopic expression of miR-206 resulted in a significant reduction of human Notch3 protein expression .}
{Western blot analysis showed that Reelin protein was clearly reduced in SH-SY5Y cells expressing high levels of miR-128, as compared with cells transfected with the empty vector (Fig. 3B).}
{Fig. 3C shows the great reduction of DCX  cells in the SH-SY5Y cell line stably transfected with DpA-miR-128.}
{In addition, PDCD4 expression in cardiac cells was up-regulated by LNAanti- miR-21 (30 nM), but down-regulated by pre-miR-21 (Fig. 7C).}
{Consistent with the results obtained in microarray analysis, Cdc25A levels were significantly up-regulated in miR-21 knockout cells (Fig. 2A and B).}
{Transfection of MDA-MB-231 cells with 30 nM miR-193b inhibitor caused elevated uPA protein expression (Figure 4a), without affecting the uPA mRNA amounts (Figure 4b).}
{we found that transfection of miR-133b down regulated not only b-III-tubulin but also Pitx3 and IGF-IR(Fig. 4A). Third,we found thatdownregulationof b-III-tubulin, Pitx3 and IGF-Ir by miR-133b occurred at the protein but not the mRNA level(Fig. 4B and C).}
{We found that miR-153 overexpression decreased both Bcl-2 and Mcl-1 protein levels compared to mock transfection or NC miRNA controls (Figs. 3a and 3c).}
{We found that miR-153 overexpression decreased both Bcl-2 and Mcl-1 protein levels compared to mock transfection or NC miRNA controls (Figs. 3a and 3c).}
{we used pre‚ÄìmiR-135 to increase the miR-135a levels in the cell. The analysis of JAK2 protein levels at 48 and 72 hours after transfection showed a dosedependent reduction of the protein expression by 28% (100 nM; 48 hours) and by 58% (100 nM; 72 hours; Figure 4A).}
{As shown in Figure 2, a significant increase in the expression level of RhoA protein was observed in the cells treated with antagomir-133a.}
{The red fluorescence intensity of individual cell bodies showed that transfection of miR-206 lowered the basal level of TPPP/ p25 by about 40% when compared with untransfected cells .}
{As expected, there was a significant reduction of E2F1 and cyclin D1 protein amounts in pre-miR-330-transfected but not control-miR-transfected cells.}
{Furthermore, transfection of miR-146a mimics decreased IRAK2 expression in macrophages at both the protein and mRNA levels.}
{Furthermore, transfection of miR-146a mimics decreased IRAK2 expression in macrophages at both the protein and mRNA levels.}
{Furthermore, transfection of miR-146a mimics decreased IRAK2 expression in macrophages at both the protein and mRNA levels.}
{Western blot confirmed a significant increase in CDKN1C/p57kip2 protein level in the antisense oligonucleotides oligonucleotides treated ESCs as compared with the control.}
{Western blotting analysis revealed that blockage of endogenous miR-181s in Hep3B cells, which highly express miR-181s, resulted in an induction of CDX2, GATA6 and NLK (Fig 5B).}
{Western blotting analysis revealed that blockage of endogenous miR-181s in Hep3B cells, which highly express miR-181s, resulted in an induction of CDX2, GATA6 and NLK (Fig 5B).}
{Western blotting analysis revealed that blockage of endogenous miR-181s in Hep3B cells, which highly express miR-181s, resulted in an induction of CDX2, GATA6 and NLK (Fig 5B).}
{Western blotting analysis revealed that blockage of endogenous miR-181s in Hep3B cells, which highly express miR-181s, resulted in an induction of CDX2, GATA6 and NLK (Fig 6B).}
{Western blotting analysis revealed that blockage of endogenous miR-181s in Hep3B cells, which highly express miR-181s, resulted in an induction of CDX2, GATA6 and NLK (Fig 7B).}
{Western blotting analysis revealed that blockage of endogenous miR-181s in Hep3B cells, which highly express miR-181s, resulted in an induction of CDX2, GATA6 and NLK (Fig 5B).}
{Western blotting analysis revealed that blockage of endogenous miR-181s in Hep3B cells, which highly express miR-181s, resulted in an induction of CDX2, GATA6 and NLK (Fig 6B).}
{Western blotting analysis revealed that blockage of endogenous miR-181s in Hep3B cells, which highly express miR-181s, resulted in an induction of CDX2, GATA6 and NLK (Fig 7B).}
{Western blotting analysis revealed that blockage of endogenous miR-181s in Hep3B cells, which highly express miR-181s, resulted in an induction of CDX2, GATA6 and NLK (Fig 8B).}
{Western blotting analysis revealed that blockage of endogenous miR-181s in Hep3B cells, which highly express miR-181s, resulted in an induction of CDX2, GATA6 and NLK (Fig 9B).}
{Western blotting analysis revealed that blockage of endogenous miR-181s in Hep3B cells, which highly express miR-181s, resulted in an induction of CDX2, GATA6 and NLK (Fig 10B).}
{Western blotting analysis revealed that blockage of endogenous miR-181s in Hep3B cells, which highly express miR-181s, resulted in an induction of CDX2, GATA6 and NLK (Fig 11B).}
{miR-34a overexpression in undifferentiated K562 cells reduced MYB protein by 74% and mRNA by 40% (Figure 4B).}
{miR-34a overexpression in undifferentiated K562 cells reduced MYB protein by 74% and mRNA by 40% (Figure 4B).}
{miR-34a overexpression in undifferentiated K562 cells reduced MYB protein by 74% and mRNA by 40% (Figure 4B).}
{"Overexpression of miR-29a in NB1691 substantially reduced B7-H3 protein expression with a reduction of ‚àº 60% when compared with negative controlNC (Fig 6C)."}
{we transfected human cancer cell lines with miR-559 or miR-661 or a negative control (designated con-miR) and MTA1 protein levels were determined by Western blot analysis. Both miRs inhibited the expression level of MTA1 but not MTA2 or vinculin. (Fig. 1B).}
{we transfected human cancer cell lines with miR-559 or miR-661 or a negative control (designated con-miR) and MTA1 protein levels were determined by Western blot analysis. Both miRs inhibited the expression level of MTA1 but not MTA2 or vinculin. (Fig. 1B).}
{miR-331-3p significantly downregulated expression of both ERBB-2 protein and RNA in each of the prostate cancer cell lines, independent of AR status (Figure 2A and 2B).}
{miR-331-3p significantly downregulated expression of both ERBB-2 protein and RNA in each of the prostate cancer cell lines, independent of AR status (Figure 2A and 2B).}
{Three of four hearts examined showed significantly reduced FOG-2 protein levels (Fig. 5B). Further, quantitation of protein levels found that FOG-2 was reduced by 75 to 80% in half of the embryos examined (Figure 5C).}
{Using NCodeTM quantitative PCR, it was found that the expression of miR-21 is approximately eight times higher in PC3 cells than in Lncap cells [Fig. 4(A)].On the other hand, the results of the western blot showed that the expression of BMPRII was lower in PC3 cells than in Lncap cells [Fig. 4(B,C)].}
{Using NCodeTM quantitative PCR, it was found that the expression of miR-21 is approximately eight times higher in PC3 cells than in Lncap cells [Fig. 4(A)].On the other hand, the results of the western blot showed that the expression of BMPRII was lower in PC3 cells than in Lncap cells [Fig. 4(B,C)].}
{Using NCodeTM quantitative PCR, it was found that the expression of miR-21 is approximately eight times higher in PC3 cells than in Lncap cells [Fig. 4(A)].On the other hand, the results of the western blot showed that the expression of BMPRII was lower in PC3 cells than in Lncap cells [Fig. 4(B,C)].}
{Using NCodeTM quantitative PCR, it was found that the expression of miR-21 is approximately eight times higher in PC3 cells than in Lncap cells [Fig. 4(A)].On the other hand, the results of the western blot showed that the expression of BMPRII was lower in PC3 cells than in Lncap cells [Fig. 4(B,C)].}
{We then overexpressed miR-23a in the cardiomyocytes to test if endogenous MuRF1 is regulated by miR-23a. MuRF1 protein but not mRNA levels were significantly decreased in the presence of miR-23a (Fig. 5E).}
{The results indicated that the suppression of miR-21 by miR-21 ASO significantly increased both the mRNA and protein level of LRRFIP1 (Figs. 3B and C).}
{The results showed that both the mRNA and the protein levels of BTG2 were elevated in HEp-2 cells transfected with miR-21 ASO compared with cells transfected with control ASO}
{A significant overexpression of miR-27b was observed in pre-miR-27b-transfected cells but not in pre-miR-negative control cells. Also, decreased levels of ST14 were observed (Fig. 3C), as shown by Western blotting as well as by QPCR analysis (Fig. 3D).}
{We transfected H1 human ES cells with anti-miR-92b molecules and then measured p57 protein by Western blot. We found that p57 protein was upregulated 1.7-fold in anti-miR-92b transfected human ES cells compared with negative control transfected cells.}
{Decreased levels of Pten were confirmed in glomeruli of diabetic db/db mice (Fig. 2b‚Äìd) and in MMC treated with TGF-Œ≤ or HG (Fig. 2e). miR-216a or miR-217 mimic decreased Pten levels in MMC (30~40% of negative control)(Fig. 2f,g).}
{Decreased levels of Pten were confirmed in glomeruli of diabetic db/db mice (Fig. 2b‚Äìd) and in MMC treated with TGF-Œ≤ or HG (Fig. 2e). miR-216a or miR-217 mimic decreased Pten levels in MMC (30~40% of negative control)(Fig. 2f,g).}
{As shown in Figure 6C and 6D, KLF5 expression was upregulated by 2'OMe-miR-145 and was downregulated by pre-miR-145 at the protein level, but not at the mRNA level.}
{we determined whether they are able to suppress the endogenous CCND1 protein levels. Western blot analysis indicated that only miR-503 was able to suppress CCND1 protein by over 40% (Fig. 4A and 4B) in UMSCC10B cells in addition to its ability to suppress the luciferase activity (Fig. 4D).}
{"As shown in Figure 2, transfection with the miR-30a mimic caused a 25‚Äì40% decrease in beclin 1 mRNA (Fig. 2A), and a 25‚Äì60% decrease in beclin1 protein (Fig. 2B)."}
{Being BLIMP-1 strongly downregulated by this miRNA.}
{Endogenous Fzd3, ITGA5, MMP16, RDX, and RhoA protein levels were assayed in miR-31- expressing 231 cells. miR-31 repressed the levels of these proteins by 40‚Äì60% (Figure 4C).}
{Endogenous Fzd3, ITGA5, MMP16, RDX, and RhoA protein levels were assayed in miR-31- expressing 231 cells. miR-31 repressed the levels of these proteins by 40‚Äì60% (Figure 4C).}
{Endogenous Fzd3, ITGA5, MMP16, RDX, and RhoA protein levels were assayed in miR-31- expressing 231 cells. miR-31 repressed the levels of these proteins by 40‚Äì60% (Figure 4C).}
{Endogenous Fzd3, ITGA5, MMP16, RDX, and RhoA protein levels were assayed in miR-31- expressing 231 cells. miR-31 repressed the levels of these proteins by 40‚Äì60% (Figure 4C).}
{Endogenous Fzd3, ITGA5, MMP16, RDX, and RhoA protein levels were assayed in miR-31- expressing 231 cells. miR-31 repressed the levels of these proteins by 40‚Äì60% (Figure 4C).}
{"As shown in Figure 4A, the levels of Cdk9 protein were decreased in ES cells overexpressing miR-1 compared with those expressing controlmiRNA."}
{Similarly, HEK293T cells transfected with p(23a,27a,24-2) showed reduced FADD protein by western blot (Fig. 4B).}
{The diminished cytokine response cells transfected with miR-105 antagomir up-regulated the TLR-2 protein levels in either challenge (Fig. 4A) compared with mock transfected cells.}
{WT miR-1 significantly reduced the levels of Bcl-2 Mrna in cardiomyocytes in the absence and presence of H2O2.}
{"The results again using relative qPCR analysis but with E-cadherin primers are shown in Figure 2C. This demonstrates that the upregulation ofE-cadherin mRNA is seen in cells in which the ectopic expression of miR-200c correlates with loss of TCF8 (ZEB1)."}
{In Figure 1A and B, we confirmed that miR145 targets both IGF-IR and IRS-1.}
{miR 223, 96 and 217 also downregulate IRS-1.}
{miR 223, 96 and 217 also downregulate IRS-1.}
{miR 223, 96 and 217 also downregulate IRS-1.}
{whereas the IGF-IR was downregulated by miR145 and 182 (Fig. 1B).}
{Results showed that the protein level was  3-fold higher in GK muscle tissue than that in Wistar rats, which indicated that miR-24 might downregulate rat p38 MAPK in vivo (Fig. 4).}
{Results showed that the protein level was  3-fold higher in GK muscle tissue than that in Wistar rats, which indicated that miR-24 might downregulate rat p38 MAPK in vivo (Fig. 4).}
{Western blot and zymographic analysis of the conditioned media of transfected cells revealed a significant reduction of uPA expression and its enzymatic activity.}
{Western blot and zymographic analysis of the conditioned media of transfected cells revealed a significant reduction of uPA expression and its enzymatic activity.}
{Forty-eight hours after transfection, c-met protein expression was inhibited by 48% at 100 nm miR-23b, as detected by a semiquantitative western blotting analysis.}
{Forty-eight hours after transfection, c-met protein expression was inhibited by 48% at 100 nm miR-23b, as detected by a semiquantitative western blotting analysis.}
{Forty-eight hours after transfection, c-met protein expression was inhibited by 48% at 100 nm miR-23b, as detected by a semiquantitative western blotting analysis.}
{Forty-eight hours after transfection, c-met protein expression was inhibited by 48% at 100 nm miR-23b, as detected by a semiquantitative western blotting analysis.}
{Furthermore, ectopic transfection of hsa-miR-222 reduced the protein level of MMP1 in UM1 cells as shown by western blots (Figure 3C).}
{Furthermore, ectopic transfection of hsa-miR-222 reduced the expression of SOD2 in UM1 cells as shown by western blots (Figure 4C).}
{Inhibition of miR-106b or miR-93 resulted in enhanced E2F1 protein expression while overexpression of the miRNAs led to reduced expression (Fig. 6b,c).}
{Inhibition of miR-106b or miR-93 resulted in enhanced E2F1 protein expression while overexpression of the miRNAs led to reduced expression (Fig. 6b,c).}
{"As shown in Fig. 4B, fndc3b mRNA levels were not significantly affected, whereas protein levels decreased substantially after treatmentwith miR-143."}
{Compared with control, BMF protein level was significantly down-regulated by the miR-125b-1 or combination of both (P<0.05; Fig. 6A) and measurably decreased by miR-125b-2.}
{Compared with control, BMF protein level was significantly down-regulated by the miR-125b-1 or combination of both (P<0.05; Fig. 6A) and measurably decreased by miR-125b-2.}
{We confirmed that ITGA5 and eNOS protein levels were significantly reduced in HUVECs overexpressing miR-92a (Fig. 4B and fig. S21, A and B).}
{However, transfection with either miR-141 or -200a reduced the expression levels of Dlx5 and Osx to almost the same levels, as those in the control cells without BMP-2 stimulation at the indicated times (Fig. 4C).}
{However, transfection with either miR-141 or -200a reduced the expression levels of Dlx5 and Osx to almost the same levels, as those in the control cells without BMP-2 stimulation at the indicated times (Fig. 4C).}
{Transfection of miR-195 induced an obvious decrease in all three proteins in both MHCC-97L and U-2OS cells, and in CDK6 and E2F3 in HCT-116.}
{Transfection of miR-195 induced an obvious decrease in all three proteins in both MHCC-97L and U-2OS cells, and in CDK6 and E2F3 in HCT-116.}
{Transfection of miR-195 induced an obvious decrease in all three proteins in both MHCC-97L and U-2OS cells, and in CDK6 and E2F3 in HCT-116.}
{Transfection of miR-195 induced an obvious decrease in all three proteins in both MHCC-97L and U-2OS cells, and in CDK6 and E2F3 in HCT-116.}
{Transfection of miR-195 induced an obvious decrease in all three proteins in both MHCC-97L and U-2OS cells, and in CDK6 and E2F3 in HCT-116.}
{"ALL-derived TOM-1 and NALM-20 cells nucleofected with pre-miR-124a showed an increase in expression of hsa-miR-124a (Fig. 2A) and a decrease in CDK6 mRNA and CDK6 and phosphorylated retinoblastoma (Rb-P) protein (Fig. 3B) levels in comparison with cells transfected with pre-miRscr ambledcontrol or empty vector."}
{Western blotting revealed that p21 protein expression was sharply reduced by miR-93 and miR-106b mimics.}
{Western blotting revealed that p21 protein expression was sharply reduced by miR-93 and miR-106b mimics.}
{Western blotting revealed that the expression of both Bim isoforms was reduced by a miR-25 mimic and increased by a miR-25 inhibitor (Figures 6A and 6B).}
{Figure 5B shows that FOXP3 protein level was significantly reduced in lenti-miR-31 transduced cells compared with lenti-miR-ctrl and non transduced cells.}
{we found that overexpression of miR-124a substantially suppressed the expression of CDK-2 in both cell types (Figure 4C).}
{In contrast, MCP-1 and angiogenin levels were significantly decreased in the presence of miR-124a overexpression.}
{The overexpression of the miR-17/92 cluster resulted in a significant decrease of BMPR2 on the protein level as analyzed by Western blot.}
{The overexpression of the miR-17/92 cluster resulted in a significant decrease of BMPR2 on the protein level as analyzed by Western blot.}
{The overexpression of the miR-17/92 cluster resulted in a significant decrease of BMPR2 on the protein level as analyzed by Western blot.}
{The overexpression of the miR-17/92 cluster resulted in a significant decrease of BMPR2 on the protein level as analyzed by Western blot.}
{The overexpression of the miR-17/92 cluster resulted in a significant decrease of BMPR2 on the protein level as analyzed by Western blot.}
{The overexpression of the miR-17/92 cluster resulted in a significant decrease of BMPR2 on the protein level as analyzed by Western blot.}
{The overexpression of the miR-17/92 cluster resulted in a significant decrease of BMPR2 on the protein level as analyzed by Western blot.}
{During DC maturation, after LPS stimulation, there is a correlation between the increase in miR-155 levels and the decrease in PU.1 (Fig. 1).}
{whereas knockdown of endogenous miR-320 by AdasmiR-320 infection increased the levels of Hsp20 protein by ‚àº60% (Fig. 6A).}
{Immunoblot also showed that the protein expression of various KRT7 isotypes was markedly decreased in miR-199a* transfection and slightly decreased in miR-145- and miR-30a-3p transfection (Fig. 4c).}
{Immunoblot also showed that the protein expression of various KRT7 isotypes was markedly decreased in miR-199a* transfection and slightly decreased in miR-145- and miR-30a-3p transfection (Fig. 4c).}
{Immunoblot also showed that the protein expression of various KRT7 isotypes was markedly decreased in miR-199a* transfection and slightly decreased in miR-145- and miR-30a-3p transfection (Fig. 4c).}
{Immunoblot also showed that the protein expression of various KRT7 isotypes was markedly decreased in miR-199a* transfection and slightly decreased in miR-145- and miR-30a-3p transfection (Fig. 4c).}
{Transfection with anti-miR-18a  inhibitor but not the control inhibitor increased the K-Ras protein level in A431 cells, HT-29 cells and WRL-68 cells as detected by western blot analysis (Figure 2C).}
{"Western blot analysis, RTKN protein was down-regulated 48 h after transfection while there was no effect for the control transfection(Fig. 3D)."}
{To test this, we transfected 5637 cells (carrying endogenous p63) with synthetic microRNA 302b. Again, immunoblot analysis revealed strong reduction of p63 levels (Fig. 2B).}
{To test this, we transfected 5637 cells (carrying endogenous p63) with synthetic microRNA 302b. Again, immunoblot analysis revealed strong reduction of p63 levels (Fig. 2B).}
{The 3 '-UTR reporter assays and HDAC4 andTGFŒ≤3 endogenous protein levels were both repressed by miR-29b (Fig. 3B).}
{The 3 '-UTR reporter assays and HDAC4 andTGFŒ≤3 endogenous protein levels were both repressed by miR-29b (Fig. 3B).}
{As expected, we observed a dose-dependent downregulation of pVHL level in 293T cells with the exogenous introduction of the microRNA (Figure 3D).}
{RUNX3 protein expression was also upregulated in anti‚ÄìmiR-532 miRNA‚Äìtransfected melanoma cells compared with nontransfected cells as shown by flow cytometry (Fig. 5).}
{Overexpression of miR-146a consistently reduced the expression of IRF-5 and STAT-1 at the protein level (Figure 3C).}
{Overexpression of miR-146a consistently reduced the expression of IRF-5 and STAT-1 at the protein level (Figure 3C).}
{To test this hypothesis, we analyzed whether endogenous MIF mRNA and protein levels fluctuated when miR-451 was overexpressed. We found that miR-451 expression in gastric and colorectal cancer cells caused a significant reduction of endogenous MIF mRNA (Fig. 5B) and MIF protein levels (Fig. 5C).}
{Blocking the activity of miR-21 by anti-miR-21 led to a significant increase in MARCKS protein in a dose-dependent manner, both in DU-145 cells and in PC-3 cells (Fig. 4C).}
{The knockdown of ADAM17 in SK-HEP-1 and Mahlavu cells (Fig. 4C) showed significant reduction in cell migration and invasion (Fig. 4D) as well as lower anchorage-independent growth in soft agar (Fig. 4E); this is similar to the phenotype detected with miR-122 restoration in these two lines of HCC cells (Fig. 2B,C).}
{Induction of miR-605 reduced SEC24D mRNA and protein levels, while anti-miR-605 increased SEC2D mRNA and protein levels, confirming miR-605‚Äôs effect on endogenous SEC24D (Fig. 5).}
{Indeed, knockdown of endogenous miR-124 with AS-124 resulted in up-regulation of Sox9 protein in dividing neuroblasts both in vitro (Fig. 7a, b) and in vivo in migratory chains (Fig. 7c‚Äìe).}
{Aggregate data (Figure 5A left) reveal an 86% increase in NFAT1 protein expression when the cells were treated with antisense to miR-184.}
{The results indicated that the level of the protein was significantly reduced by the transfection with miR-143m ( fig. 2 c).}
{We found that transfection of miR-491 decreased Bcl-XL protein expression to the same extent to Bcl-XL siRNA transfection (Fig. 3b).}
{As shown in Fig. 3, miR-497 inhibitor effectively increases (Fig. 3B), whereas miR-497 mimic decreases (Fig.3C) the expression of bcl-2 and bcl-w in mouse N2A cells subjected to 3 hours of OGD followed by 4 hours of recovery time. Of}
{Elevation of miR-696 expression was seen after transfection of its precursor into C2C12 cells, and PGC-1  protein was suppressed compared with the level in control cells at 24 h after transfection (Fig. 4A)}
{"By western blot, we found a three-fold reduction in Cdk6 protein level in both E10 and H1299 cells in which miR-129 is overexpressed (Fig. 4A). However, our array analysis showed no difference in the mRNA levels of Cdk6 between miR-129 overexpressingand control E10 cells"}
{As shown in Fig. 4D, miR-148a precursor significantly decreased the MSK1 protein level in PC3 cells on days 2, 3, 4, and 5 after transfection,}
{Western blot analysis revealed that, while miR-133a mimic treatment decreased NFATc4 protein expression, the opposite result was observed byfollowing miR-133a inhibitor treatment (Fig. 3B),}
{Western blot analysis revealed that, while miR-133a mimic treatment decreased NFATc4 protein expression, the opposite result was observed byfollowing miR-133a inhibitor treatment (Fig. 3B),}
{Introduction of the miRNAs resulted in downregulation of RECK protein (Figure 1d,Protein) with no appreciable changes in the level of RECK mRNA (Figure 1d, mRNA).}
{Introduction of the miRNAs resulted in downregulation of RECK protein (Figure 1d,Protein) with no appreciable changes in the level of RECK mRNA (Figure 1d, mRNA).}
{Introduction of the miRNAs resulted in downregulation of RECK protein (Figure 1d,Protein) with no appreciable changes in the level of RECK mRNA (Figure 1d, mRNA).}
{Introduction of the miRNAs resulted in downregulation of RECK protein (Figure 1d,Protein) with no appreciable changes in the level of RECK mRNA (Figure 1d, mRNA).}
{Introduction of the miRNAs resulted in downregulation of RECK protein (Figure 1d,Protein) with no appreciable changes in the level of RECK mRNA (Figure 1d, mRNA).}
{Transfection with (AS)miR-221 reduced miR-221 expression, and increased both the mRNA and protein levels of p85a (Fig. 5A,B).}
{overexpression of miR-33 resulted in reduced expression of ABCA1, but not ABCG1, mRNA (Fig. 2E,lanes 1‚Äì4), as well as reduced ABCA1 protein expression in these human hepatoma cells (Fig. 2F);}
{overexpression of miR-33 resulted in reduced expression of ABCA1, but not ABCG1, mRNA (Fig. 2E,lanes 1‚Äì4), as well as reduced ABCA1 protein expression in these human hepatoma cells (Fig. 2F);}
{overexpression of miR-33 resulted in reduced expression of ABCA1, but not ABCG1, mRNA (Fig. 2E,lanes 1‚Äì4), as well as reduced ABCA1 protein expression in these human hepatoma cells (Fig. 2F);}
{PANC-1 and MIAPaCa-2 cells were transfected with pre-miR-217, prescramble control, 217-vector or pcDNA 3.1 and the results showed that upregulation of miR-217 led to a dramatic reduction in KRAS expression at the protein level (Figure 4E and F, Supplementary Figure 1C is available at Carcinogenesis Online) but not at mRNA level}
{Both Suv39h1 mRNA levels (Fig. 3A) and protein levels (Fig. 3B) were significantly decreased in db/  cells toward that seen in db/db cells after transfection with the miR-125b relative to NC-M.}
{Cells transfected with miR-155 precursor displayed a slower kinetics of CTLA-4 expression and, on day3 after transfection and stimulation, expressed significantly less intracellular CTLA-4 compared with cells transfected with a control precursor (Fig 5, C). }
{"In murine 70Z/3 cells, we observed a similar repression of Foxp1 at the protein level within 4 days of transduction (Figure 3E). Analysis of Foxp1 RNA in B cell subsets showed an inverse correlation with miR-34a amounts at the pro- and pre-B cell stages (Figure 3F; compare with Figure2C). Having observed repression of Foxp1 in cell lines, we also examined Foxp1 mRNA expression in whole bone marrow from miR-34a mice. Foxp1 mRNA is significantly downregulatedin the bone marrow of mice transduced with miR-34a ("}
{However, we observed that CUGBP2 was downregulated by ~20% in AdmiR-144-infected cardiomyocytes and by ~30% in Ad.miR-451-cardiomyocytes (Fig. 4B). More strikingly, protein levels of CUGBP2 were decreased by ~80% in AdmiR-144/451-cells (Fig. 4B}
{However, we observed that CUGBP2 was downregulated by ~20% in AdmiR-144-infected cardiomyocytes and by ~30% in Ad.miR-451-cardiomyocytes (Fig. 4B). More strikingly, protein levels of CUGBP2 were decreased by ~80% in AdmiR-144/451-cells (Fig. 4B}
{Compared with the control, overexpression of miR-21 knocked down FASLG protein by 32 ¬± 11% (n=4; P<0.05; Fig. 3B),}
{"Additionally, inosteosarcoma MG63 and U2OS cells, endogenous expressionof Bcl-2 protein was suppressed by miR-143 transfection"}
{"Additionally, inosteosarcoma MG63 and U2OS cells, endogenous expressionof Bcl-2 protein was suppressed by miR-143 transfection"}
{In total, we identified ten proteins with differential expression with miR-122a overexpression. PRDXII was downregulated in HepG2 cells infected with Lenti-miR-122a as revealed by 2-DE (Fig. 3A).}
{Expression of miR-380-5p resulted in a significant ~40% decrease in basal p53 protein levels}
{we utilized a reporter construct, which encodes the FBXW7Œ± cDNA with an N-terminal epitope tag and the full-length 3‚Ä≤-UTR of FBXW7. We found that miR-223 overexpression led to significant reduction in ectopic Fbw7Œ± abundance. Despite repressing the activity of the FBXW7 3‚Ä≤-UTR reporter and mRNA levels, miR-25 did not significantly reduce Fbw7 protein abundance (Fig. 2D).}
{we utilized a reporter construct, which encodes the FBXW7Œ± cDNA with an N-terminal epitope tag and the full-length 3‚Ä≤-UTR of FBXW7. We found that miR-223 overexpression led to significant reduction in ectopic Fbw7Œ± abundance. Despite repressing the activity of the FBXW7 3‚Ä≤-UTR reporter and mRNA levels, miR-25 did not significantly reduce Fbw7 protein abundance (Fig. 2D).}
{Transfection of AS-192, but not AS-215, led to a significant 1.4-fold increase in L-WNK1 protein expression compared with the scramble }
{Transfection of AS-192, but not AS-215, led to a significant 1.4-fold increase in L-WNK1 protein expression compared with the scramble }
{we knocked-down miR-221/222 in U251 cells, which exhibit elevated level of miR-221/222, and ectopically expressed miR-221/222 in H4 cells with low endogenous miR-221/222 expression (Fig. ‚Äã(Fig.1A).1A). Western blot analysis showed that PUMA expression was up-regulated in U251 cells with knockdown of miR-221/222 (Fig. ‚Äã(Fig.2B),2B),}
{we knocked-down miR-221/222 in U251 cells, which exhibit elevated level of miR-221/222, and ectopically expressed miR-221/222 in H4 cells with low endogenous miR-221/222 expression (Fig. ‚Äã(Fig.1A).1A). Western blot analysis showed that PUMA expression was up-regulated in U251 cells with knockdown of miR-221/222 (Fig. ‚Äã(Fig.2B),2B),}
{miR-155 has no effect on the mRNA expression level of MyD88. In contrast, overexpression of miR-155 mimics decreased MyD88 protein content in a dose and time-dependent manner (Fig. 2B and C). }
{Western blot analysis showed that transfection of miR-1 significantly decreased the expression level of endogenous Pim-1 protein (Fig. 5A & B). Additionally, there was no significant decrease in the mRNA level of Pim-1 upon miR-1 transfection}
{Quantitative RT-PCR of SATB2 and western blot analysis confirmed the higher expression in CAFs (Fig. 4B and C). }
{overexpression of mir-29a significantly (p < 0.05) decreased p85Œ± subunit protein levels (Fig. 6A and B) that was prevented in the presence of the specific miR-29a inhibitor. }
{Staining signals of antibodies to endogenous Sp1 and Sirt1 are reduced in miR-34a or miR-93-transfected HEK 293 cells, compared to those transfected with control plasmid. }
{Staining signals of antibodies to endogenous Sp1 and Sirt1 are reduced in miR-34a or miR-93-transfected HEK 293 cells, compared to those transfected with control plasmid. }
{Staining signals of antibodies to endogenous Sp1 and Sirt1 are reduced in miR-34a or miR-93-transfected HEK 293 cells, compared to those transfected with control plasmid. }
{we transfected MGC-803 cells with miR-148b mimics and the NC. Forty-eight hours after transfection, we examined the CCKBR protein levels in cells by western blot. Obviously, CCKBR protein levels were suppressed by miR-148b mimics in MGC-803 cells (Figure ‚Äã(Figure4C).4C). Despite the effect of miR-148b on CCKBR protein levels, no effect on the CCKBR mRNA level was detected by qRT-PCR (Figure ‚Äã(Figure4D4D).}
{More importantly, both mRNA and protein levels of IGF-II were reduced by 60‚Äì70% on days 2 and 3 of differentiation when a chemically modified miR-125b mimic (miRIDIAN) was transfected into the cells (Fig. 4 A). }
{Upregulation of miR-98 significantly downregulated cyclin D2 in cultured cardiomyocytes at baseline and in response to Ang II stimulation (Fig. 7FG). Ang II-induced upregulation of cyclin D2 was inhibited by miR-98 and enhanced by anti-miR-98 introduced by adenovirus-mediated gene transfer in the mouse heart (Fig. 7H, supplemental Figure S6AB). }
{Elevated miR-7 levels in a mouse schwannoma cell line, NF2S-1, also produced a marked reduction in the protein levels of Ack1, Pak1, and EGFR }
{Elevated miR-7 levels in a mouse schwannoma cell line, NF2S-1, also produced a marked reduction in the protein levels of Ack1, Pak1, and EGFR }
{We found that miR-7 transfection decreased both mRNA (data not shown) and protein levels (Fig. 4F) of Ack1 compared to GAPDH mRNA and actin in both cell types.}
{As shown in Fig. 4, induced overexpression of miR-155 resulted in reduced CD62L expression as compared to the mock or miR-23a transfected cells. }
{As a result, miR-27a decreased TRŒ≤1 protein levels without affecting TRŒ≤1 mRNA levels in NRVMs, and the miR-27a decoy increased TRŒ≤1 protein levels (Fig. 5A and B). }
{As a result, miR-27a decreased TRŒ≤1 protein levels without affecting TRŒ≤1 mRNA levels in NRVMs, and the miR-27a decoy increased TRŒ≤1 protein levels (Fig. 5A and B). }
{Moreover, we observed that ectopic expression of miR-15a significantly inhibited the UCP-2 protein level in MIN6 cells, and miR-15a inhibitor increased UCP-2 expression (Fig. 4D). }
{Our analysis showed an enhancement of Khc-73 levels in KT40 mutants by an average of 50% (n=3, normalized to actin, p=0.034) (Figure 3D). }
{Our analysis showed an enhancement of Khc-73 levels in KT40 mutants by an average of 50% (n=3, normalized to actin, p=0.034) (Figure 3D). }
{Our analysis showed an enhancement of Khc-73 levels in KT40 mutants by an average of 50% (n=3, normalized to actin, p=0.034) (Figure 3D). }
{Our analysis showed an enhancement of Khc-73 levels in KT40 mutants by an average of 50% (n=3, normalized to actin, p=0.034) (Figure 3D). }
{miR-132 inhibitor-transfected cells expressed greater levels of all five candidate target genes (Fig. 1E). }
{miR-132 inhibitor-transfected cells expressed greater levels of all five candidate target genes (Fig. 1E). }
{miR-132 inhibitor-transfected cells expressed greater levels of all five candidate target genes (Fig. 1E). }
{miR-132 inhibitor-transfected cells expressed greater levels of all five candidate target genes (Fig. 1E). }
{miR-132 inhibitor-transfected cells expressed greater levels of all five candidate target genes (Fig. 1E). }
{miR-132 inhibitor-transfected cells expressed greater levels of all five candidate target genes (Fig. 1E). }
{These reduced miR-10a and -10b levels led to an increase of the endogenous SFRS1 protein expression, as compared with the negative control anti-miR-transfected cells (Fig. 7C). }
{These reduced miR-10a and -10b levels led to an increase of the endogenous SFRS1 protein expression, as compared with the negative control anti-miR-transfected cells (Fig. 7C). }
{Consistent with this result, suppression of the real target genes in mirPSCs was also confirmed by Western blot analysis (Fig. 4C). }
{Consistent with this result, suppression of the real target genes in mirPSCs was also confirmed by Western blot analysis (Fig. 4C). }
{Consistent with this result, suppression of the real target genes in mirPSCs was also confirmed by Western blot analysis (Fig. 4C). }
{Consistent with this result, suppression of the real target genes in mirPSCs was also confirmed by Western blot analysis (Fig. 4C). }
{Consistent with this result, suppression of the real target genes in mirPSCs was also confirmed by Western blot analysis (Fig. 4C). }
{Consistent with this result, suppression of the real target genes in mirPSCs was also confirmed by Western blot analysis (Fig. 4C). }
{Consistent with this result, suppression of the real target genes in mirPSCs was also confirmed by Western blot analysis (Fig. 4C). }
{Consistent with this result, suppression of the real target genes in mirPSCs was also confirmed by Western blot analysis (Fig. 4C). }
{To determine whether miR-155 targeted AT1R mRNA for degradation, real-time RT-PCR demonstrated that AT1R mRNA levels remained unchanged following miR-155 transfection (Fig. 2B). However, western blot assay revealed significantly reduced AT1R protein expression in AFs transfected with pSUPER/miR-155 compared with controls (Fig. 2C). }
{we first transfected miRNA mimics into human HEK 293T cells. As detected by western blot analysis, miR-18 downregulated the protein levels of HSF2 (Fig. 2A). HSF2 was similarly downregulated by overexpressing miR-18 in rat ST15A cells (data not }
{As shown in Fig. 2A‚áì , the protein levels of Smarca5 and Jarid1b are significantly decreased on overexpression of miRs 100 and 137, respectively. On the contrary, overexpression of miR-204 had no effects on Sirt1 protein level (Supplemental Fig. 2). }
{As shown in Fig. 2A‚áì , the protein levels of Smarca5 and Jarid1b are significantly decreased on overexpression of miRs 100 and 137, respectively. On the contrary, overexpression of miR-204 had no effects on Sirt1 protein level (Supplemental Fig. 2). }
{Expression of endogenous TBP in mRNA as well as protein level was decreased in STHdhQ111/HdhQ111 cells expressing exogenous miR-146a (Fig. 4B and C). }
{As shown in Figure 2B, despite the fact that STAT5A was also a predicted target of miR-221, miR-222, but not miR-221, overexpression was associated with down-regulation of STAT5A protein levels}
{Consistent with several previous reports (36, 37), we found that elevated glucose levels increased VEGF secretion (Fig. 6A). In contrast, transfection of podocytes with miR-93 mimics significantly reduced high glucose-induced VEGF release in the medium. Conversely, inhibition of miR-93 in podocytes with anti-miR-93 increased the secretion of VEGF in the medium, whereas inhibition of miR-690, as a control, did not have any effect (Fig. 6B). Thus, these findings suggest that overexpression or knockdown of miR-93 modulates VEGF production in podocytes.}
{Neurons infected by sh-miR-137 expressing virus had a 13% decrease in endogenous Mib1 expression compared with neurons infected by control virus (sh-Con, n = 3, p < 0.05) (Figure 5G). }
{Consistent with the changes in mRNA levels, the level of N-Ras protein was also altered by 214in and 214mi (Fig. 5B). }
{Expression of miR-214 also led to a decrease in PTEN protein expression in HIO80 cells when compared with cells transfected with control plasmid alone (Fig. 5C), }
{hAT1R levels in cells transfected with miR-802 showed a small but significant decrease (18.1 ¬± 3.2%) compared with control (Fig. 5A). }
{Conversely, overexpression of the miR-221 precursor was found to suppress precartilage condensation, chondrogenic differentiation (Fig. 4E), and Mdm2 expression (Fig. 4F) in the tested cells.}
{To see whether miR-133a regulated the CASP9 protein during IR, miR-133a mimic or AMO-133a was transferred into the myocardium before IR. It was found that the expression of CASP9 protein was uperegulated by AMO-133a and down-regulated by miR-133a mimic (P < 0.05, Figure ‚ÄãFigure88).}
{Indeed, reducing the levels of endogenous miR-708 with the antagomir resulted in a 6.9-fold increase of steady-state levels of rhodopsin protein (Fig. 4 B, left). Furthermore, expression of a miR-708 mimic resulted in a 2.5-fold decrease in rhodopsin expression (Fig. 4 B, right)}
{To demonstrate the effect of the miRNA on the PODXL level, we transfected NT2 cells with different concentrations of miR-199a-5p or miR-199a-3p mimics. Seventy-two hours after transfection, the PODXL protein was significantly decreased following miR-199a-5p, but not miR-199a-3p treatment.}
{However, results from our real-time PCR and western blot analysis revealed that with treatment of miR-23b mimics (150 pmol in 24-well plate) in BRL-3A cells, Smad3 expression notably decreased in both mRNA and protein level; }
{First, we demonstrated that overexpression of miR-100 represses mTOR expression on both the mRNA and protein levels in endothelial cells, whereas miR-100 inhibition resulted in a modest but significant upregulation of mTOR }
{Transfection of the pre-miR-3960, rather than the control, resulted in a decrease in the Hoxa2 protein levels after 48 h (Fig. 4C). However, the Hoxa2 mRNA levels were not affected (Fig. 4D). }
{ Indeed, miR-93 and miR-106b expression efficiently decreased both Tgfbr2 and p21 protein levels (Figure 5A and D) and p21 mRNA levels were decreased by ~25‚Äì30% while Tgfbr2 was decreased by ~60‚Äì70% }
{ Indeed, miR-93 and miR-106b expression efficiently decreased both Tgfbr2 and p21 protein levels (Figure 5A and D) and p21 mRNA levels were decreased by ~25‚Äì30% while Tgfbr2 was decreased by ~60‚Äì70% }
{ Indeed, miR-93 and miR-106b expression efficiently decreased both Tgfbr2 and p21 protein levels (Figure 5A and D) and p21 mRNA levels were decreased by ~25‚Äì30% while Tgfbr2 was decreased by ~60‚Äì70% }
{ Indeed, miR-93 and miR-106b expression efficiently decreased both Tgfbr2 and p21 protein levels (Figure 5A and D) and p21 mRNA levels were decreased by ~25‚Äì30% while Tgfbr2 was decreased by ~60‚Äì70% }
{we transiently transfected miR-1258 mimic, miR-1258 inhibitor, or negative control oligo into 231BR3 and SUM-149 cells, and studied heparanase expression and activity which were reduced by miR-1258 mimic, but augmented by miR-1258 inhibitor (Figs. 2A and 2B).}
{To further confirm that miR-9 affects FoxP1 expression at a protein level, miR-9-1 and miR-9-2 were co-transfected with FoxP1 cDNA sequences containing both the coding region and the 3‚Ä≤-UTR in Neuro2A cells. MiR-9-1 and miR-9-2, but not miR-9-Mut, significantly reduced FoxP1 protein but not FoxP1 mRNA }
{In contrast, CD44 mRNA (Supplementary Fig. 4e) and protein (Supplementary Fig. 11c) were increased in anti-34a transfected tumors. }
{In contrast, CD44 mRNA (Supplementary Fig. 4e) and protein (Supplementary Fig. 11c) were increased in anti-34a transfected tumors. }
{Thus, overexpressing mir-200c and mir-141 in the PC-3 cells, or mir-141 alone, produced a 60% and 30% decrease in the JAG1 protein, respectively (Figure 4A, upper blot), without significantly altering JAG1 mRNA levels }
{Thus, overexpressing mir-200c and mir-141 in the PC-3 cells, or mir-141 alone, produced a 60% and 30% decrease in the JAG1 protein, respectively (Figure 4A, upper blot), without significantly altering JAG1 mRNA levels }
{Our results show that indeed Bcl-2 expression is repressed in all three miRNA-transfected cultures, by individual cell immnocytochemistry of transfected cultures with anti-body to this protein (Figure 3A-B). }
{Our results show that indeed Bcl-2 expression is repressed in all three miRNA-transfected cultures, by individual cell immnocytochemistry of transfected cultures with anti-body to this protein (Figure 3A-B). }
{Our results show that indeed Bcl-2 expression is repressed in all three miRNA-transfected cultures, by individual cell immnocytochemistry of transfected cultures with anti-body to this protein (Figure 3A-B). }
{Western blot analyses confirmed the ability of miR-449 to down-regulate MET, GMNN, MYC, SIRT1, cyclin E2 (CCNE2) and HDAC1 at the protein level to an extent similar to that achieved by re-introduction of miR-34a (figure ‚Äã(figure3c).3c). }
{Western blot analyses confirmed the ability of miR-449 to down-regulate MET, GMNN, MYC, SIRT1, cyclin E2 (CCNE2) and HDAC1 at the protein level to an extent similar to that achieved by re-introduction of miR-34a (figure ‚Äã(figure3c).3c). }
{Western blot analyses confirmed the ability of miR-449 to down-regulate MET, GMNN, MYC, SIRT1, cyclin E2 (CCNE2) and HDAC1 at the protein level to an extent similar to that achieved by re-introduction of miR-34a (figure ‚Äã(figure3c).3c). }
{Western blot analyses confirmed the ability of miR-449 to down-regulate MET, GMNN, MYC, SIRT1, cyclin E2 (CCNE2) and HDAC1 at the protein level to an extent similar to that achieved by re-introduction of miR-34a (figure ‚Äã(figure3c).3c). }
{Neurons infected by sh-miR-137 expressing virus had a 13% decrease in endogenous Mib1 expression compared with neurons infected by control virus (sh-Con, n = 3, p < 0.05) (Figure 5G). }
{Consistent with the changes in mRNA levels, the level of N-Ras protein was also altered by 214in and 214mi (Fig. 5B). }
{Consistent with the changes in mRNA levels, the level of N-Ras protein was also altered by 214in and 214mi (Fig. 5B). }
{"As shown by qRT-PCR and immunocytochemistry, overexpression of mature hsa-miR-381 in U251/LRRC4+ cells resulted in the down-regulation of LRRC4 at the mRNA transcriptional (Fig. 4C) and posttranscriptional levels (Fig. 4D)."}
{"Overexpressionof miR-375 in AGS and MGC-803 cells greatlyreduced the protein level of JAK2, whereas the level ofJAK2 mRNA was not significantly affected (Figure 4Band Supplementary information, Figure S4)"}
{"Overexpressionof miR-375 in AGS and MGC-803 cells greatlyreduced the protein level of JAK2, whereas the level ofJAK2 mRNA was not significantly affected (Figure 4Band Supplementary information, Figure S4)"}
{"E2F3 protein expression was significantly decreased inT24 and 5637 cells after transfection with miR-125b (Fig.8b), whereas the E2F3 mRNA was almost unchanged (Fig.8a), which suggested that miR-125b may regulate the level ofE2F3 protein at the post-transcriptional level but not themRNA level."}
{"Overexpressionof miR-448 reduced the protein level of KLF5, whereas itdid not change the Klf5 mRNA level (Fig. 4C and SupplementalFig. 3B)."}
{"As shown in Fig. 4C, when HepG2, 7721, and Sk-hep-1cells were transfected with miR-122 mimics, the CUTL1protein was significantly reduced."}
{Furthermore, Western blot analysis showed that PTEN was significantly upregulated in AS-miR-221/222 transfected cells. In contrast, PTEN expression was downregulated in pMSCV-miR-221/222 infected cells (Figure ‚Äã(Figure3B).3B). Together, these data demonstrated that PTEN is a target gene of the miR-221/222 cluster.}
{Furthermore, Western blot analysis showed that PTEN was significantly upregulated in AS-miR-221/222 transfected cells. In contrast, PTEN expression was downregulated in pMSCV-miR-221/222 infected cells (Figure ‚Äã(Figure3B).3B). Together, these data demonstrated that PTEN is a target gene of the miR-221/222 cluster.}
{MOR expression was determined by western blotting. When SH-SY5Y cells were transfected with increasing amounts of the LNA-let-7 inhibitor, MOR expression was up-regulated in an antisense dose-dependent fashion (Fig. 2).}
{MiR-21 overexpression resulted in significantly reduced basal protein expression levels of PPARŒ± as shown in Fig. 7A (P < 0.05). }
{To test whether exogenous miR-216a addition inhibits endogenous levels of Ybx1, miR-216a mimic oligonucleotides were transfected into MMC using nucleofection (10), and protein levels of Ybx1 were examined. Significant decrease of Ybx1 was observed in MMC transfected with miR-216a mimic in a dose-dependent manner (Fig. 2, A and B). }
{"Wefound that inhibition of miR-107 expression in SGC-7901 andMKN-45 cells increased the protein level of DICER1 (Fig. 3D).Those results really demonstrated that the endogenous DICER1 isdirect target of miR-107."}
{"The levels of RB1 mRNAand protein in the anti-miR-106a group were both above thelevels seen in the NC inhibitor groups. In contrast, the levels ofRB1 mRNA and protein in the miR-106a mimic group wereboth below the levels seen in the NC mimic group (Figs. 7Cand D)."}
{"In wild-type glands, we detected low levels of MMP-9 throughoutthe whole fat pad, with slightly higher levels in distal regions than inproximal regions. Around the TEBs, there were several fibroblastsinwhich MMP-9 was highly expressed (arrows in Fig. 7a). We foundlow, diffuse staining in the periductal stroma of distal ducts; however,proximal ducts had almost no MMP-9 in their periductal stroma(Fig. 7b,c). Some scattered cells in the fat pad had high MMP-9 levels(blue arrows in Fig. 7c), consistent with the results of the in situhybridizationexperiments25. By contrast, MMP-9 was much more highly expressed throughout the miR-212/132‚àí/‚àí glands."}
{"In wild-type glands, we detected low levels of MMP-9 throughoutthe whole fat pad, with slightly higher levels in distal regions than inproximal regions. Around the TEBs, there were several fibroblastsinwhich MMP-9 was highly expressed (arrows in Fig. 7a). We foundlow, diffuse staining in the periductal stroma of distal ducts; however,proximal ducts had almost no MMP-9 in their periductal stroma(Fig. 7b,c). Some scattered cells in the fat pad had high MMP-9 levels(blue arrows in Fig. 7c), consistent with the results of the in situhybridizationexperiments25. By contrast, MMP-9 was much more highly expressed throughout the miR-212/132‚àí/‚àí glands."}
{"Western blot analysis (Fig. 3D) revealedthat SULF1 expression was simply the inverse of that ofmiR-516a-3p by Northern blot analysis (Fig. 1)."}
{"The ability of miR-328 to repress the expression of thesegenes was further verified by Western blot analysis of thelevels of the proteins encoded by their corresponding mRNAs in neonatal rat atrial myocytes transfected with miR-328(Figure 5B). This downregulation was efficiently preventedby AMO-328. The efficacy of AMO-328 to knock down themature miR-328 in all our experimental settings was confirmed(Figure II in the online-only Data Supplement)."}
{"The ability of miR-328 to repress the expression of thesegenes was further verified by Western blot analysis of thelevels of the proteins encoded by their corresponding mRNAs in neonatal rat atrial myocytes transfected with miR-328(Figure 5B). This downregulation was efficiently preventedby AMO-328. The efficacy of AMO-328 to knock down themature miR-328 in all our experimental settings was confirmed(Figure II in the online-only Data Supplement)."}
{"As shown in Fig. 2A and 2B, transfection with miR-101 mimicdid not significantly change the mRNA level of MKP-1 as com-pared with control dsRNA. We also have analyzed the effect ofmiR-101 on the protein level of MKP-1 using Western blot analyses.The results reveal that transfection of miR-101 mimic but notcontrol dsRNA markedly inhibits the production of MKP-1 protein(Fig. 2C). These results suggest that miR-101 may function at thetranslational level."}
{"To evaluate the expression level of miR-126 in humanbreast tumors, Northern blot of miR-126 was performedand the results showed that expression of miR-126 wasdown-regulated in human breast tumors compared with theadjacent normal tissues (Fig. 5a). On the other hand,Western blot showed that PIK3R2, VEGFA and AKTphosphorylation level were dramatically increased in breasttumor samples (Fig. 5b)."}
{"To evaluate the expression level of miR-126 in humanbreast tumors, Northern blot of miR-126 was performedand the results showed that expression of miR-126 wasdown-regulated in human breast tumors compared with theadjacent normal tissues (Fig. 5a). On the other hand,Western blot showed that PIK3R2, VEGFA and AKTphosphorylation level were dramatically increased in breasttumor samples (Fig. 5b)."}
{Semi-quantitative western blot analysis exhibited a 10-fold increase in PSIP1-P75 protein abundance in the cells expressing shRNA targeting miR-135b (Figure 4E; as compared to transfection with an anti-miR negative control. n=3, P<<0.005; Student's t-test). }
{ Furthermore, endogenous DDX6 protein levels were down-regulated by pre-miR-130a and -130b (Figure 3C). }
{In addition, real time PCR and western blot analysis revealed that the expression of RB1 mRNA and protein was impaired by the treatment with miR-192 mimics in A549 cells (Figure 3C and D). }
{To investigate the influence of miR-622 on ING1 expression, we searched for changes in ING1 protein and mRNA levels in HEK 293T cells transfected with the miR-622 precursor or the miR-622 inhibitor or in control-transfected cells. As shown in Figure ‚ÄãFigure4B,4B, miR-622 precursor transfection led to a significant decrease in ING1 protein levels (P < 0.05, Figure ‚ÄãFigure4B).4B).}
{Semi-quantitative analysis of western blot data showed a significant inhibition of NOBOX expression in the miR-196a-transfected cells }
{Next, we pursued the ability of miR-148a to regulate the expression of endongenous p27. miR-148a overexpression with mimic in AGS cells dramatically repressed p27 expression (Fig. ‚Äã(Fig.3C).3C). }
{To directly examine the regulation of Abcc3 by mmu-miR-665, RAW 264.7 cells were transfected with vehicle, or a mmu-miR-665 precursor, or a control miRNA precursor. As shown in Figure 3A and B, mmu-miR-665 significantly inhibited Abcc3 expression at both mRNA and protein levels as assessed by qRT-PCR and Western blotting, }
{Furthermore, the overexpression of miR-29a was able to markedly downregulate the expression of endogenous PTEN at the levels of mRNA and protein in HepG2 or MHCC-97L cells. }
{Furthermore, the overexpression of miR-29a was able to markedly downregulate the expression of endogenous PTEN at the levels of mRNA and protein in HepG2 or MHCC-97L cells. }
{To determine whether these miRNAs affect endogenous Runx2 expression in distinct cell types, we transfected these miRNAs into MC3T3-E1 osteoblasts and ATDC5 chondrocytes and examined Runx2 mRNA and protein levels. Except for miR-30c in MC3T3 and miR-133a in ATDC5 cells, these miRNAs repress Runx2 expression at the protein level but not the mRNA level (Fig. S3).}
{To determine whether these miRNAs affect endogenous Runx2 expression in distinct cell types, we transfected these miRNAs into MC3T3-E1 osteoblasts and ATDC5 chondrocytes and examined Runx2 mRNA and protein levels. Except for miR-30c in MC3T3 and miR-133a in ATDC5 cells, these miRNAs repress Runx2 expression at the protein level but not the mRNA level (Fig. S3).}
{To determine whether these miRNAs affect endogenous Runx2 expression in distinct cell types, we transfected these miRNAs into MC3T3-E1 osteoblasts and ATDC5 chondrocytes and examined Runx2 mRNA and protein levels. Except for miR-30c in MC3T3 and miR-133a in ATDC5 cells, these miRNAs repress Runx2 expression at the protein level but not the mRNA level (Fig. S3).}
{To determine whether these miRNAs affect endogenous Runx2 expression in distinct cell types, we transfected these miRNAs into MC3T3-E1 osteoblasts and ATDC5 chondrocytes and examined Runx2 mRNA and protein levels. Except for miR-30c in MC3T3 and miR-133a in ATDC5 cells, these miRNAs repress Runx2 expression at the protein level but not the mRNA level (Fig. S3).}
{To determine whether these miRNAs affect endogenous Runx2 expression in distinct cell types, we transfected these miRNAs into MC3T3-E1 osteoblasts and ATDC5 chondrocytes and examined Runx2 mRNA and protein levels. Except for miR-30c in MC3T3 and miR-133a in ATDC5 cells, these miRNAs repress Runx2 expression at the protein level but not the mRNA level (Fig. S3).}
{To determine whether these miRNAs affect endogenous Runx2 expression in distinct cell types, we transfected these miRNAs into MC3T3-E1 osteoblasts and ATDC5 chondrocytes and examined Runx2 mRNA and protein levels. Except for miR-30c in MC3T3 and miR-133a in ATDC5 cells, these miRNAs repress Runx2 expression at the protein level but not the mRNA level (Fig. S3).}
{To determine whether these miRNAs affect endogenous Runx2 expression in distinct cell types, we transfected these miRNAs into MC3T3-E1 osteoblasts and ATDC5 chondrocytes and examined Runx2 mRNA and protein levels. Except for miR-30c in MC3T3 and miR-133a in ATDC5 cells, these miRNAs repress Runx2 expression at the protein level but not the mRNA level (Fig. S3).}
{To determine whether these miRNAs affect endogenous Runx2 expression in distinct cell types, we transfected these miRNAs into MC3T3-E1 osteoblasts and ATDC5 chondrocytes and examined Runx2 mRNA and protein levels. Except for miR-30c in MC3T3 and miR-133a in ATDC5 cells, these miRNAs repress Runx2 expression at the protein level but not the mRNA level (Fig. S3).}
{To determine whether these miRNAs affect endogenous Runx2 expression in distinct cell types, we transfected these miRNAs into MC3T3-E1 osteoblasts and ATDC5 chondrocytes and examined Runx2 mRNA and protein levels. Except for miR-30c in MC3T3 and miR-133a in ATDC5 cells, these miRNAs repress Runx2 expression at the protein level but not the mRNA level (Fig. S3).}
{To determine whether these miRNAs affect endogenous Runx2 expression in distinct cell types, we transfected these miRNAs into MC3T3-E1 osteoblasts and ATDC5 chondrocytes and examined Runx2 mRNA and protein levels. Except for miR-30c in MC3T3 and miR-133a in ATDC5 cells, these miRNAs repress Runx2 expression at the protein level but not the mRNA level (Fig. S3).}
{To determine whether these miRNAs affect endogenous Runx2 expression in distinct cell types, we transfected these miRNAs into MC3T3-E1 osteoblasts and ATDC5 chondrocytes and examined Runx2 mRNA and protein levels. Except for miR-30c in MC3T3 and miR-133a in ATDC5 cells, these miRNAs repress Runx2 expression at the protein level but not the mRNA level (Fig. S3).}
{qRT-PCR and westernblot analysis revealed that miR-34a drastically inhibited the expression of INHBB and Met on both mRNA and protein levels (Figure 3A,B). }
{" In addition, col2a1 expression was found repressed significantly if miR-29a/b mimics were transiently transfected to the chondrocytes, demonstrated by RT-PCR (Fig. 6A). The repression on protein level was further confirmed by immunostaining (Fig. 6B) and western blotting (Fig. 6C). Therefore, it was concluded that miR-29a/b directly targeted 3‚Ä≤ UTR of col2a1 to repress its expression."}
{"In addition, col2a1 expression was found repressed significantly if miR-29a/b mimics were transiently transfected to the chondrocytes, demonstrated by RT-PCR (Fig. 6A). The repression on protein level was further confirmed by immunostaining (Fig. 6B) and western blotting (Fig. 6C). Therefore, it was concluded that miR-29a/b directly targeted 3‚Ä≤ UTR of col2a1 to repress its expression."}
{Consistently, miR-1 transgenic hearts showed significantly reduced Cdc42 mRNA and protein levels (Fig. 5, E and F), suggesting a repressive relationship between miR-1 and Cdc42 in vivo.}
{We tested if miR-485 controlled SV2A expression in hippocampal neurons by miR-485 overexpression and inhibition. miR-485 overexpression decreased both transcript abundance (n = 3; P < 0.05) (Fig. 2B) and protein expression (Fig. 2C) of SV2A relative to both an untreated control and negative controls for the miR-M and miR-I containing random sequences validated to not affect miR function. }
{Western blot analysis further showed that ectopic miR-146a in malignant astrocytes induced a dramatic reduction of Notch1 protein, especially the processed intracellular NICD, which is the most predominant form in these glia cells (Fig. 5C). }
{Overexpression of miR-206 in normoxic conditions significantly reduced TIMP-3 mRNA (Figure 9A) and protein levels (Figure 9B). }
{"Furthermore, transfection of a miR-15a mimic or inhibitor (50 nM each) into GC-1 cells resulted in a marked reduction or increase of Ccnt2 protein levels, respectively (Fig. 2G, Supplementary Fig. 2)."}
{"In order to validate whether miR-19b is capable of regulating NeuroD1 expression, we cloned the DNA sequence corresponding to the entire NeuroD1 mRNA into pEGFP-C1 to construct EGFP-NeuroD1 fusion protein expression vector (pEGFP-C1-NeuroD1), then the vector was co-transfected with either synthesized miR-19b or miR-19b inhibitor into 3T3 cells. The overexpression of miR-19b was confirmed by qRT-PCR (Fig. 3A). Also, qRT-PCR results showed that overexpression of miR-19b decreased NeuroD1 mRNA level (Fig. 3B). Western blotting results showed that the NeuroD1 protein was significantly inhibited by miR-19b (Fig. 3D). Meanwhile, the expression of EGFP protein was repressed because of the reducing NeuroD1 (Fig. 3C). Similar results were observed in 293GP cells which were co-transfected with pEGFP-C1-NeuroD1, miR-19b and/or miR-19b inhibitor (Fig. S3)."}
{"Lenti.miR-503reduced cdc25A and CCNE1 at mRNA and protein level inHUVECs in comparison with GFP-HUVECs."}
{"Lenti.miR-503reduced cdc25A and CCNE1 at mRNA and protein level inHUVECs in comparison with GFP-HUVECs."}
{"Lenti.miR-503reduced cdc25A and CCNE1 at mRNA and protein level inHUVECs in comparison with GFP-HUVECs."}
{"Western blot analysis indicated that there was remarkabledownregulation of p27 and p57 in OECM-1-miR-221 ‚ÅÑ miR-222 and SAS-miR-222 cells. However, only aslight downregulation of p27 and p57 in SAS-miR-221cells was noted."}
{"Western blot analysis indicated that there was remarkabledownregulation of p27 and p57 in OECM-1-miR-221 ‚ÅÑ miR-222 and SAS-miR-222 cells. However, only aslight downregulation of p27 and p57 in SAS-miR-221cells was noted."}
{"Western blot analysis indicated that there was remarkabledownregulation of p27 and p57 in OECM-1-miR-221 ‚ÅÑ miR-222 and SAS-miR-222 cells. However, only aslight downregulation of p27 and p57 in SAS-miR-221cells was noted."}
{Compared to the scramble ON transfection, miR-19a antisense ON transfection up-regulated 19 genes >2-fold. TNF-Œ± was up-regulated 12-fold (data not shown). We also observed a strong increase in TNF-Œ± protein level (Fig. 5A) after transfection with miR-19a AS.}
{"To obtain direct evidence for a functionalrole of miR-33, we used Raw264.7 cells to assess theeffects of gain and loss of function of miR-33 on RIP140expression. Figure 3D, E shows that RIP140 mRNA andprotein levels were both reduced in Raw264.7 cellstransfected with miR-33 mimic even without depletingcholesterol (by simvastatin treatment),"}
{"H1299, MDA-MB-231, HCT116 and Rkocells were transfected with control-miR, miR-34a ormiR-199a. As miR-199a and miR-199b have similarseed sequences and binds with more or less equal freeenergy to the Axl 30-UTR, miR-199a was used forfurther transfection experiments. qRT‚ÄìPCR resultsshowed that both the miRs (miR-34a and miR-199a)significantly reduced Axl-transcript levels (Figure 2b).Furthermore, western blot analysis confirmed the downregulationof Axl protein amounts (Figure 2c)."}
{"H1299, MDA-MB-231, HCT116 and Rkocells were transfected with control-miR, miR-34a ormiR-199a. As miR-199a and miR-199b have similarseed sequences and binds with more or less equal freeenergy to the Axl 30-UTR, miR-199a was used forfurther transfection experiments. qRT‚ÄìPCR resultsshowed that both the miRs (miR-34a and miR-199a)significantly reduced Axl-transcript levels (Figure 2b).Furthermore, western blot analysis confirmed the downregulationof Axl protein amounts (Figure 2c)."}
{"Western blotting revealedan approximately 30%-40% reduction of STAT3 protein expressionin 32D/miR-125b compared with 32D/miR-ctrl cells(Figure 4D)."}
{"Western blotting revealedan approximately 30%-40% reduction of STAT3 protein expressionin 32D/miR-125b compared with 32D/miR-ctrl cells(Figure 4D)."}
{"we determinedthe mRNA and protein levels of STAT3 in hASCs overexpressingmiR-21 or cells transfected with a miR-21-specificinhibitor. Real-time PCR and Western blot analyses showedthat miR-21-over-expressing hASCs exhibited less total andphospho-STAT3 expression and that anti-miR-21-transfectedhASCs exhibited increased STAT3 expression (Fig. 4C,D)."}
{"The fact that c-Fos protein was increased toa greater extent than c-Fos mRNA (25-fold versus 2- to 3-fold)suggested that miR155 represses c-Fos expression mainly at thelevel of translation."}
{"The fact that c-Fos protein was increased toa greater extent than c-Fos mRNA (25-fold versus 2- to 3-fold)suggested that miR155 represses c-Fos expression mainly at thelevel of translation."}
{"To determine whether miR-133b regulates RhoA during spinal cordregeneration in adult zebrafish, we examined the RhoA expressionlevels in the brain and lesioned spinal cord after treatment withantisense miR-133b or control MO at 9 days after SCI by Western blotanalysis and immunofluorescence. In slices of brain including theNMLF, RhoA relative expression levels increased in miR-133b MOtreatedfish (1.3 ¬± 0.06, P = 0.008) compared to control MO-treatedfish (1.0 ¬± 0.04; Fig. 4C and E)."}
{"To determine whether miR-133b regulates RhoA during spinal cordregeneration in adult zebrafish, we examined the RhoA expressionlevels in the brain and lesioned spinal cord after treatment withantisense miR-133b or control MO at 9 days after SCI by Western blotanalysis and immunofluorescence. In slices of brain including theNMLF, RhoA relative expression levels increased in miR-133b MOtreatedfish (1.3 ¬± 0.06, P = 0.008) compared to control MO-treatedfish (1.0 ¬± 0.04; Fig. 4C and E)."}
{"we examined the levels of Fbxw7 mRNAand protein expression upon treatment with an miR-27amimic. The results showed that the level of Fbxw7mRNA in HBELH cells transfected with 200 nMmiR-27a mimic was decreased by 60% compared withmimic NC measured by quantitative RT‚ÄìPCR, indicatingthat miR-27a regulates Fbxw7 expression by mRNAdegradation (Figure 6c). Consistent with the reductionat the mRNA level, treatment of HBELH cells with200 nM miR-27a mimic led to a 40% decrease in Fbxw7 protein expression (Figure 6c)."}
{"we examined the levels of Fbxw7 mRNAand protein expression upon treatment with an miR-27amimic. The results showed that the level of Fbxw7mRNA in HBELH cells transfected with 200 nMmiR-27a mimic was decreased by 60% compared withmimic NC measured by quantitative RT‚ÄìPCR, indicatingthat miR-27a regulates Fbxw7 expression by mRNAdegradation (Figure 6c). Consistent with the reductionat the mRNA level, treatment of HBELH cells with200 nM miR-27a mimic led to a 40% decrease in Fbxw7 protein expression (Figure 6c)."}
{"First, we examined the effect ofthe repression of miR-142-3p. The miR-142-3p LNA-knockdownprobe or miR-223-LNA knockdown probe was transfected intoBMDCs and the level of IL-6 was measured by ELISA. Asignificantly higher level of IL-6 was detected in DCs aftermiR-142-3p knockdown for 24 or 48 hours (P   .05 and P   .01)compared with the scrambled or miR-223-LNA knockdown transfectedDCs (Figure 3A)."}
{"we transiently transfected EA.hy926cells with miR-9 or miR-181b. Western blot analysis revealedthat the Nrp1 protein level was reduced in cells transfectedwith miR-9 or miR-181b compared with the control cells."}
{"we transiently transfected EA.hy926cells with miR-9 or miR-181b. Western blot analysis revealedthat the Nrp1 protein level was reduced in cells transfectedwith miR-9 or miR-181b compared with the control cells."}
{"The results showed that miR-126-3p inhibitorcould promote effectively endogenic miR-126-3pincreased by qRT-PCR (Fig. 4a P\0.01). However, PgrmRNA levels were not altered compared to control(Fig. 4b P[0.05). Meanwhile, we examined protein levelsof PGR in MMECs after miR-126-3p mimics, inhibitor,or control into cells. As showed in Fig. 5, transfection ofmiR-126-3p mimics led to a significant down-regulation ofPGR protein (P\0.05). Alternatively, transfection ofmiR-126-3p inhibitor increased PGR protein levels comparedto control-transfected cells (P\0.05)."}
{"The results showed that miR-126-3p inhibitorcould promote effectively endogenic miR-126-3pincreased by qRT-PCR (Fig. 4a P\0.01). However, PgrmRNA levels were not altered compared to control(Fig. 4b P[0.05). Meanwhile, we examined protein levelsof PGR in MMECs after miR-126-3p mimics, inhibitor,or control into cells. As showed in Fig. 5, transfection ofmiR-126-3p mimics led to a significant down-regulation ofPGR protein (P\0.05). Alternatively, transfection ofmiR-126-3p inhibitor increased PGR protein levels comparedto control-transfected cells (P\0.05)."}
{"Western blot and reverse transcription(RT)‚ÄìPCR demonstrated that overexpression of miR-342dramatically suppressed endogenous DNMT1 both at protein andmRNA levels compared with negative control miRNA (Figure 2B)."}
{"Transfection of miR-743a into HT22cells reduced endogenous MDH mRNA levels by 23%(Fig. 6a), and MDH activity by 31% as compared to cellstransfected with a negative control miRNA (Fig. 6b), whileno effect on expression of oxoglutarate dehydrogenase(ogdh), gene encoding one of the subunits of KGDHC(Fig. 6c) was observed."}
{"To further confirm the EPB41L3 is afunctional target of miR-223, we used the Western Blot to examine the EPB41L3 protein level afteroverexpression or knock-down of miR-223. Compared with the control, inhibition miR-223 level inXGC-9811L cells (with a high endogenous miR-223 expression) could increase EPB41L3 proteinlevel (Fig.4D1)."}
{"Enhanced miR-296-5p expression resulted in amarked decrease of PUMA cellular mRNA and protein levels( Fig. 2B, C )."}
{"Enhanced miR-296-5p expression resulted in amarked decrease of PUMA cellular mRNA and protein levels( Fig. 2B, C )."}
{"Overexpression of miR-103 also led to an approximately twofolddecrease in endogenous Cav1 levels in HEK 293 cells compared tocontrols (Fig. 4d)."}
{"Overexpression of miR-103 also led to an approximately twofolddecrease in endogenous Cav1 levels in HEK 293 cells compared tocontrols (Fig. 4d)."}
{"To determine the protein levels of IFNGR1 at the fourstages of CL, western blot analysis was performed on thetotal protein extracts from these different stages. As shownin Fig. 4, the level of IFNGR1 protein gradually decreasedfrom early to late stage and notably increased in the regressed stage, which is inversely linked to the expressionof miR-378 and the IFNGR1 gene."}
{Meanwhile, the protein levels of ROCK2 and EZH2 were also substantially decreased after ectopic overexpression of miR-124 in LM9 and HepG2 cell lines as evidenced by western blot assays}
{Meanwhile, the protein levels of ROCK2 and EZH2 were also substantially decreased after ectopic overexpression of miR-124 in LM9 and HepG2 cell lines as evidenced by western blot assays}
{"Transfection with mimics of miR-200b and miR-15b synergistically reduced BMI1 expression inCAL27-res (Figure 4c) and SCC25-res cells (Figure 4d),"}
{"Transfection with mimics of miR-200b and miR-15b synergistically reduced BMI1 expression inCAL27-res (Figure 4c) and SCC25-res cells (Figure 4d),"}
{"Transfection of miR-208 mimics decreased p21 proteinexpression in a concentration-dependent manner,determined by immunoblotting (Fig. 5a)."}
{"As shown in Fig. 6A, overexpression oflet-7i inhibited SOCS1 protein expression."}
{ As expected for the mechanisms of miRNAs regulation, measured by western blot assay, Sox9 protein level was notably decreased in the cells of miR-145 over-expression and increased in the cells of miR-145 suppression at 1 d and 7 d but not at 14 d by TGF-Œ≤3 treatment (Figure 3A). }
{However, cells pretreated with anti-miR-155 demonstrated sustained increase in HIF-1Œ± protein levels compared to the transient HIF-1Œ± activation in control anti-miR treatment (Fig. 6). }
{ Likewise, in anti-miR-144 transfected HeLa and 3T3L1 cells, the relative expression of miR-144 was reduced to 0.019¬±0.09 and 0.033¬±0.11 respectively while a significant increase in IRS1 mRNA level was observed (HeLa: 5.607¬±0.217; 3T3L1 Regulation of IRS1 by miR-144 was further verified by immunocytochemistry and complementarily, the IRS1 protein expression correlated to that of the gene expression studies.}
{Next, we evaluated whether overexpression of miR-34a resulted in decreased SIRT1 at the protein level. SIRT1 protein was significantly decreased after miR-34a overexpression (p<0.05) (Fig. 2D), suggesting that the effect of miR-34a on the number of postmitotic neurons and neurite elongation might be mediated by SIRT1.}
{The western blot revealed that Sp1 protein levels were obviously increased in the miR-140 knockdown (Fig. 5B), while Sp1 mRNA levels did not significantly change (Fig. 5C). }
{The western blot revealed that Sp1 protein levels were obviously increased in the miR-140 knockdown (Fig. 5B), while Sp1 mRNA levels did not significantly change (Fig. 5C). }
{Ischemic progenitor cells were delivered with miR-124a mimics and incubated in the growth medium for 3 days. JAG1 and Notch intracellular domain (NICD) were assayed by real-time RT-PCR and Western blot. Compared with the miRNA mimic control, nanoparticle-delivered mature miR-124a resulted in a substantial decrease of JAG1 transcript and protein (Fig. 5F and G). }
{"To determinewhether miR-365 inhibits HDAC4 protein expression,we quantified HDAC4 protein levels by Western blotanalysis. Expression of miR-365 mimic significantly inhibitedHDAC4 protein levels in both chicken and mouse"}
{"Furthermore, endogenousCAMTA1 mRNA as well as protein levels wereelevated, when miR-9 or miR-9* was inhibited (Figure 4Cand D)."}
{"Furthermore, endogenousCAMTA1 mRNA as well as protein levels wereelevated, when miR-9 or miR-9* was inhibited (Figure 4Cand D)."}
{"Finally, weanalyzed the effect of miR-16/-424 on VEGFR2, FGFR1, andVEGF expression. HUVECs were transfected with miR-16 ormiR-424 mimics, and the effects on protein and mRNA levelswere analyzed 36 hours posttransfection. As shown in Figure2A to 2C, both miR-16 and miR-424 significantly decreasedVEGFR2, FGFR1, and VEGF protein and mRNA levels."}
{"Finally, weanalyzed the effect of miR-16/-424 on VEGFR2, FGFR1, andVEGF expression. HUVECs were transfected with miR-16 ormiR-424 mimics, and the effects on protein and mRNA levelswere analyzed 36 hours posttransfection. As shown in Figure2A to 2C, both miR-16 and miR-424 significantly decreasedVEGFR2, FGFR1, and VEGF protein and mRNA levels."}
{"Finally, weanalyzed the effect of miR-16/-424 on VEGFR2, FGFR1, andVEGF expression. HUVECs were transfected with miR-16 ormiR-424 mimics, and the effects on protein and mRNA levelswere analyzed 36 hours posttransfection. As shown in Figure2A to 2C, both miR-16 and miR-424 significantly decreasedVEGFR2, FGFR1, and VEGF protein and mRNA levels."}
{"Finally, weanalyzed the effect of miR-16/-424 on VEGFR2, FGFR1, andVEGF expression. HUVECs were transfected with miR-16 ormiR-424 mimics, and the effects on protein and mRNA levelswere analyzed 36 hours posttransfection. As shown in Figure2A to 2C, both miR-16 and miR-424 significantly decreasedVEGFR2, FGFR1, and VEGF protein and mRNA levels."}
{"Finally, weanalyzed the effect of miR-16/-424 on VEGFR2, FGFR1, andVEGF expression. HUVECs were transfected with miR-16 ormiR-424 mimics, and the effects on protein and mRNA levelswere analyzed 36 hours posttransfection. As shown in Figure2A to 2C, both miR-16 and miR-424 significantly decreasedVEGFR2, FGFR1, and VEGF protein and mRNA levels."}
{"Finally, weanalyzed the effect of miR-16/-424 on VEGFR2, FGFR1, andVEGF expression. HUVECs were transfected with miR-16 ormiR-424 mimics, and the effects on protein and mRNA levelswere analyzed 36 hours posttransfection. As shown in Figure2A to 2C, both miR-16 and miR-424 significantly decreasedVEGFR2, FGFR1, and VEGF protein and mRNA levels."}
{"Finally, weanalyzed the effect of miR-16/-424 on VEGFR2, FGFR1, andVEGF expression. HUVECs were transfected with miR-16 ormiR-424 mimics, and the effects on protein and mRNA levelswere analyzed 36 hours posttransfection. As shown in Figure2A to 2C, both miR-16 and miR-424 significantly decreasedVEGFR2, FGFR1, and VEGF protein and mRNA levels."}
{"CPCs were transduced with these 3miRNAs to evaluate whether miRNAs could regulate Bimexpression by Western blotting (Figure 6C). Compared withcontrol, the levels of Bim isoforms proteins were significantlydownregulated by miRNA cocktails."}
{"CPCs were transduced with these 3miRNAs to evaluate whether miRNAs could regulate Bimexpression by Western blotting (Figure 6C). Compared withcontrol, the levels of Bim isoforms proteins were significantlydownregulated by miRNA cocktails."}
{"CPCs were transduced with these 3miRNAs to evaluate whether miRNAs could regulate Bimexpression by Western blotting (Figure 6C). Compared withcontrol, the levels of Bim isoforms proteins were significantlydownregulated by miRNA cocktails."}
{"We verified that miR-17 can binddirectly to the 3 UTR of tgfbr2 (position 2388) and creb1 (position6984) mRNAs (Figure 6B-C) using luciferase reporter assays. InCD4  T cells lacking mir-17-92, the expression levels of TGF RIIand CREB1 were significantly elevated at both the mRNA andprotein levels, and these elevations were completely abolishedwhen the mir-17 gene was reintroduced (Figure 6D-E)."}
{"We verified that miR-17 can binddirectly to the 3 UTR of tgfbr2 (position 2388) and creb1 (position6984) mRNAs (Figure 6B-C) using luciferase reporter assays. InCD4  T cells lacking mir-17-92, the expression levels of TGF RIIand CREB1 were significantly elevated at both the mRNA andprotein levels, and these elevations were completely abolishedwhen the mir-17 gene was reintroduced (Figure 6D-E)."}
{"We verified that miR-17 can binddirectly to the 3 UTR of tgfbr2 (position 2388) and creb1 (position6984) mRNAs (Figure 6B-C) using luciferase reporter assays. InCD4  T cells lacking mir-17-92, the expression levels of TGF RIIand CREB1 were significantly elevated at both the mRNA andprotein levels, and these elevations were completely abolishedwhen the mir-17 gene was reintroduced (Figure 6D-E)."}
{"The cells were transiently transfectedwith miR-150 mimic and the expression of MUC4 was examined. Ourdata revealed no apparent change in MUC4 expression at the transcriptlevel (Figure 1C), whereas it was significantly downregulated atthe protein level (Figure 1D) in miR-150 mimic transfected cells ascompared with non-targeting control (miR-NC) mimic transfectedcells. Our data suggest that miR-150 downregulates MUC4 expressionthrough a posttranscriptional mechanism."}
{"Further analysis demonstrated 50% reduction of endogenous TIMP3 mRNA levelin OHTR cells as well as miR-221/222-overexpressing cellswhen compared with MCF-7 cells and when miR-181b wastransiently overexpressed (Fig. 1E). In addition, a markedreduction (75%) in TIMP3 protein was observed in OHTRcells when compared with an  50% reduction when individualmiRs were overexpressed (Fig. 1F)."}
{"Further analysis demonstrated 50% reduction of endogenous TIMP3 mRNA levelin OHTR cells as well as miR-221/222-overexpressing cellswhen compared with MCF-7 cells and when miR-181b wastransiently overexpressed (Fig. 1E). In addition, a markedreduction (75%) in TIMP3 protein was observed in OHTRcells when compared with an  50% reduction when individualmiRs were overexpressed (Fig. 1F)."}
{"Further analysis demonstrated 50% reduction of endogenous TIMP3 mRNA levelin OHTR cells as well as miR-221/222-overexpressing cellswhen compared with MCF-7 cells and when miR-181b wastransiently overexpressed (Fig. 1E). In addition, a markedreduction (75%) in TIMP3 protein was observed in OHTRcells when compared with an  50% reduction when individualmiRs were overexpressed (Fig. 1F)."}
{"Further analysis demonstrated 50% reduction of endogenous TIMP3 mRNA levelin OHTR cells as well as miR-221/222-overexpressing cellswhen compared with MCF-7 cells and when miR-181b wastransiently overexpressed (Fig. 1E). In addition, a markedreduction (75%) in TIMP3 protein was observed in OHTRcells when compared with an  50% reduction when individualmiRs were overexpressed (Fig. 1F)."}
{"To confirm that Lats2 is a target gene of rno-miR-31 in culturedVSMCs, both gain-of-function and loss-of-functionapproaches were applied. As expected, LATS2 was up-regulatedby miR-31 inhibitor (Fig. 6D) and down-regulated by AdmiR-31 (Fig. 6E) at the protein level. It is well known that miRNAscould regulate their target gene expression via inhibitingtranslation and/or promoting degradation of target genes (1, 2).To determine whether rno-miR-31 could cause the degradationof its target Lats2, we detected Lats2 mRNA levels incultured VSMCs transfected with vehicle, Ad-GFP, orAd-miR-31 respectively. Interestingly, overexpression of rnomiR-31 by Ad-miR-31 decreased the Lats2 mRNA level,"}
{"To confirm this possibility wedetermined whether over-expression or inhibition ofmiR-155 would lead to significant changes in SOCS-1mRNA and protein levels."}
{"To test whethermiR-26brepresses Ctdsp2 protein expression in vivo, a syntheticmiR-26b mimic was injected into one-cell stage embryos.Thirty-one hours post-fertilization (hpf), Ctdsp2 expressionwas analyzed by Western blotting of whole-embryoextracts using affinity-purified Ctdsp2 antibodies. ExogenousmiR-26b led to reduced Ctdsp2 expression whencompared with animals injected with a control miRNA(Fig. 1A)."}
{The PTN mRNA and protein expression were examined by RT-PCR (Fig. 3C) or western blotting (Fig. 3D). MiR-143 significantly suppressed PTN protein level without affecting mRNA expression, }
{"Furthermore, the introduction of let-7a-mimics or let-7a-inhibitor into DLD1 cells caused downregulation and upregulation of NIRF expression, respectively, as demonstrated by qRT-PCR and Western blot analysis (Fig. 2B and C). Taken together, these data indicate that let-7a may regulate NIRF expression through let-7a-binding sites at the 3‚Ä≤-UTR of NIRF."}
{To identify whether Srebf1 could be a true target mRNA of miR-21 we introduced miR-21inhibitor (LNA-21) and control oligonucleotides (LNA-scr) into INS-1E cells, and extracted RNA and protein from these. MessengerRNA levels of Srebf1 increased by about 30% after miR-21 knock-down (Fig. 5E). Also SREBP1 protein levels increased approximately 50% after miR-21 knock-down compared with a LNA-scr (Fig. 5G, H). }
{ In addition, knockdown of miR-150 in MNCs by lentiviral vector significantly increased CXCR4 expression (Figure 4C).}
{" Western blots for sirt1 protein showed that its levels decreased significantly in hippocampal neurons by overexpression of miR-199a, while increased by knockdown of miR-199a (Figs. 5A, B). Taken together, these results demonstrated that sirt1 is negatively regulated by miR-199a in neurons."}
{Interestingly, we found that EphA5 protein was reduced by JNK inhibitor and this reduction was overcome by co-treatment of miR-34a inhibitor (Fig. 3A). }
{The transfection of miR-124 inhibitor remarkably increased BACE1 expression in both intact and AŒ≤ injured groups. Moreover, a higher level of BACE1 was still revealed in the miR-124 inhibitor plus AŒ≤ injury group than that of inhibitor transfection alone (P < 0.01, Fig. 2C and D). }
{"As shown inFigure 4A and B, the miR-622 mimic did not affect the levels ofE2f1, c-myc or Cdk6 proteins but exhibited a significant 0.35 ¬±0.08-fold reduction in the level of K-Ras protein in 16HBE-T cellstransfected with the miR-622 mimic,However, the miR-622 mimic did not affectthe K-Ras mRNA expression levels, suggesting that miR-622 regulatesK-Ras mRNA translation without affecting accumulation levels(Figure 4C)."}
{"As shown inFigure 4A and B, the miR-622 mimic did not affect the levels ofE2f1, c-myc or Cdk6 proteins but exhibited a significant 0.35 ¬±0.08-fold reduction in the level of K-Ras protein in 16HBE-T cellstransfected with the miR-622 mimic,However, the miR-622 mimic did not affectthe K-Ras mRNA expression levels, suggesting that miR-622 regulatesK-Ras mRNA translation without affecting accumulation levels(Figure 4C)."}
{"As shown inFigure 4A and B, the miR-622 mimic did not affect the levels ofE2f1, c-myc or Cdk6 proteins but exhibited a significant 0.35 ¬±0.08-fold reduction in the level of K-Ras protein in 16HBE-T cellstransfected with the miR-622 mimic,However, the miR-622 mimic did not affectthe K-Ras mRNA expression levels, suggesting that miR-622 regulatesK-Ras mRNA translation without affecting accumulation levels(Figure 4C)."}
{"Compared with control subjectswithout any treatment, transfection of miR-125b into BEAS-2Bcells suppressed 4E-BP1 protein expression (Figure 3A);"}
{"Such miR-145 overexpressiongreatly decreased the levels of SOX9 both at the mRNA andparticularly at the protein level (Fig. 2, B andC)."}
{"In addition, overexpression of selected miR was alsoperformed in the Y1 mouse adrenocortical cell line. Upontransfection of miR-101a, a significant down-regulationof the Nr3c1 expression ratio could be demonstrated bothat the mRNA (control: 1 0.107, vs. 0.688 0.122; P 0.05) (Fig. 5B) and the protein level (control: 1 0.07, vs.0.782   0.01; P   0.05) (Fig. 5C)."}
{"Similarly, transfectionof miR-142-3p resulted in a reduction of the Nr3c1 expressionratio in both the mRNA (control: 1   0.107, vs.0.891   0.017; P   0.05) and the protein level (control:1 0.107, vs. 0.933 0.055; P 0.05),"}
{"Furthermore, we showed that miR-210 can suppressthe messenger RNA (mRNA) and protein levels ofVMP1 in HCC cells (Fig. 4D,E) and that there isdose-dependence between the expression of VMP1 andmiR-210 (Supporting Fig. 3)."}
{In NIH3T3 cells transfected with either PCMV-Prm2 or -TP2 plasmid, mRNA of Prm2 or TP2 was not altered by the overexpression of miR-469 (p>0.05) (Fig. 7A). However, the level of protein expression in either case was significantly decreased (Fig. 7B) by overexpression of miR-469.}
{In NIH3T3 cells transfected with either PCMV-Prm2 or -TP2 plasmid, mRNA of Prm2 or TP2 was not altered by the overexpression of miR-469 (p>0.05) (Fig. 7A). However, the level of protein expression in either case was significantly decreased (Fig. 7B) by overexpression of miR-469.}
{In NIH3T3 cells transfected with either PCMV-Prm2 or -TP2 plasmid, mRNA of Prm2 or TP2 was not altered by the overexpression of miR-469 (p>0.05) (Fig. 7A). However, the level of protein expression in either case was significantly decreased (Fig. 7B) by overexpression of miR-469.}