{Using both wild-type and mutant constructs of human ZEB1 and ZEB2 3_UTRs carrying mutations in all miR-200b/c/429 targeting sites, we now demonstrate that ectopic miR-200c suppresses by directly affecting the predicted miR-200c targeting sites (Fig. 3C)}
{The endogenous wild-type IRS-1 is again down-regulated (about 50%) by miR145, but the truncated IRS-1 is not.}
{The endogenous wild-type IRS-1 is again down-regulated (about 50%) by miR145, but the truncated IRS-1 is not.}
{Next, the studies were repeated with random mutations in the recognition sequence, which resulted in abolition of the reporter activation by miR-370 precursor (Figure 5).}
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
{A reporter with mutations in the predicted miR-29 target site (Fig. 4b, white) was not suppressed by miR-29 miRNAs.}
{but a reporter with mutated target sites was not repressed by miR-29 (Fig. 4e, white). This suggests that these two sites mediate miR-29‚Äôs regulation of CDC42}
{but a reporter with mutated target sites was not repressed by miR-29 (Fig. 4e, white). This suggests that these two sites mediate miR-29‚Äôs regulation of CDC42}
{Transfection of a CDC34-m1&m2 vector resulted in luciferase activity at levels similar to the control plasmid in the presence or absence of exogenous let-7, demonstrating specific downregulation of reporter activity by let-7 through the Cdc34 3‚ÄôUTR}
{Cells transfected with miR-128 showed a reduction of 40% in luciferase activity compared to those transfected with empty vectors (Student'st test, p<0.05). When the hypothesized miR-128 binding site in the 3'UTR of E2F3a mRNA was mutated, luciferase activity was restored close to the control level (Fig. 3b).}
{mutation in the Bcl-2 3'UTR complimentary to the miR-34 root sequence abolished this effect, indicating that the observed reporter activity is miR-34 sequence-specific.}
{mutation in the Bcl-2 3'UTR complimentary to the miR-34 root sequence abolished this effect, indicating that the observed reporter activity is miR-34 sequence-specific}
{mutation in the Bcl-2 3'UTR complimentary to the miR-34 root sequence abolished this effect, indicating that the observed reporter activity is miR-34 sequence-specific.}
{In addition, to validate whether RECK is a direct target of miR-21, we mutated the miR-21 binding site in the 30UTR of RECK (in the reporter plasmid) and observed loss of repression (Figure 7d and e).}
{Results from triplicate experiments showed that reporter activity for each site was reduced by the ectopic expression of miR-155, with site 1 exhibiting a more significan tresponse than the two other sites. When these sites were mutated,the luciferase reporter was no longer inhibited by miR-155 (Fig. 5F and G).}
{Results from triplicate experiments showed that reporter activity for each site was reduced by the ectopic expression of miR-155, with site 1 exhibiting a more significan tresponse than the two other sites. When these sites were mutated,the luciferase reporter was no longer inhibited by miR-155 (Fig. 5F and G).}
{Results from triplicate experiments showed that reporter activity for each site was reduced by the ectopic expression of miR-155, with site 1 exhibiting a more significan tresponse than the two other sites. When these sites were mutated,the luciferase reporter was no longer inhibited by miR-155 (Fig. 5F and G).}
{Luciferase constructs were developed that contain each of the miR-181b putative binding sites identified in the mouse AID 3'UTR (BS1-3), or mutated versions of the same (mutBS1-3). Reporter constructs, which included a fi refly luciferase cassette to allow internal Renilla luciferase activity normalization, were transiently cotransfected together with miR-181b duplexes into 293T cells, and luciferase activity was analyzed 24 h later (Fig. S9 shows raw values of luciferase measurements). We found that transfection of the BS1 and BS2 constructs in the presence of miR-181b resulted in a signifi cant decrease of luciferase activity when compared with their mutant counterparts mutBS1 (85 ¬± 9%) and BS2 (45 ¬± 9%; Fig. 3 A ),}
{Luciferase constructs were developed that contain each of the miR-181b putative binding sites identified in the mouse AID 3'UTR (BS1-3), or mutated versions of the same (mutBS1-3). Reporter constructs, which included a fi refly luciferase cassette to allow internal Renilla luciferase activity normalization, were transiently cotransfected together with miR-181b duplexes into 293T cells, and luciferase activity was analyzed 24 h later (Fig. S9 shows raw values of luciferase measurements). We found that transfection of the BS1 and BS2 constructs in the presence of miR-181b resulted in a signifi cant decrease of luciferase activity when compared with their mutant counterparts mutBS1 (85 ¬± 9%) and BS2 (45 ¬± 9%; Fig. 3 A ),}
{let-7a precursor  inhibitor had no effect on the activity of pGL3-CASP3/mut-3'UTR reporter.}
{We then proceeded to identify the authenticmiRNAtarget sites bymutagenizing each putativemiRNA target site (except for the first miR-130 target site) conserved in vertebrates, as well as one miR-130 target site that is unique to humans (Supplementary Methods and Supplementary Figs. 6 and 7 online).Most of the conserved putativemiRNA target sites, except for the fourth miR-19 target site and the miR-130 target site unique to humans, were authentic (Fig. 1f and Supplementary Figs. 6 and 7).}
{We then proceeded to identify the authenticmiRNAtarget sites bymutagenizing each putativemiRNA target site (except for the first miR-130 target site) conserved in vertebrates, as well as one miR-130 target site that is unique to humans (Supplementary Methods and Supplementary Figs. 6 and 7 online).Most of the conserved putativemiRNA target sites, except for the fourth miR-19 target site and the miR-130 target site unique to humans, were authentic (Fig. 1f and Supplementary Figs. 6 and 7).}
{We then proceeded to identify the authenticmiRNAtarget sites bymutagenizing each putativemiRNA target site (except for the first miR-130 target site) conserved in vertebrates, as well as one miR-130 target site that is unique to humans (Supplementary Methods and Supplementary Figs. 6 and 7 online).Most of the conserved putativemiRNA target sites, except for the fourth miR-19 target site and the miR-130 target site unique to humans, were authentic (Fig. 1f and Supplementary Figs. 6 and 7).}
{This effect was abolished on a mutated construct in which the putative miR‚ÄìmRNA interaction sequence was deleted, confirming that this miRNA was effectively targeting Gli1 mRNA (Figure 3D).}
{As a control, no repression was observed with Smo-3'UTR mutant constructs deleted in the miRNA-binding sites (Figure 2D).}
{As a control, no repression was observed with Smo-3'UTR mutant constructs deleted in the miRNA-binding sites (Figure 2D).}
{As a control, no repression was observed with Smo-3'UTR mutant constructs deleted in the miRNA-binding sites (Figure 2D).}
{In contrast, miR-34a has a minimal effect on a reporter vector with a mutated miR-34a response element. Furthermore, miR-34a does not affect SIRT1 RNA (Fig. 3C).}
{Moreover, miR-19a and miR-19b mimics inhibited the expression of a reporter vector containing SOCS-1 3'UTR, while mutation of the predicted miRNA-binding site abrogated this effect (Fig. 3 B and C).}
{Moreover, miR-19a and miR-19b mimics inhibited the expression of a reporter vector containing SOCS-1 3'UTR, while mutation of the predicted miRNA-binding site abrogated this effect (Fig. 3 B and C).}
{Mutation of the predicted miRNA binding sites in the reporter vector abrogated this effect,indicating that these miRNAs directly interact with the PCAF 3'UTR. }
{Mutation of the predicted miRNA binding sites in the reporter vector abrogated this effect,indicating that these miRNAs directly interact with the PCAF 3'UTR.}
{Mutation of the predicted miRNA binding sites in the reporter vector abrogated this effect,indicating that these miRNAs directly interact with the PCAF 3'UTR.}
{Mutation of the predicted miRNA binding sites in the reporter vector abrogated this effect,indicating that these miRNAs directly interact with the PCAF 3'UTR.}
{Mutation of the predicted miRNA binding sites in the reporter vector abrogated this effect,indicating that these miRNAs directly interact with the PCAF 3'UTR.}
{Mutation of the predicted miRNA binding sites in the reporter vector abrogated this effect,indicating that these miRNAs directly interact with the PCAF 3'UTR.}
{Addition of 12 nM miR-659 to N2A cells transfected with vectorD18 failed to repress the luciferase activity (Fig. 4D), supporting the hypothesis that miR-659 binds to the predicted binding-site in the 30-UTR of GRN, overlapping with rs5848 at position 78.}
{Mutation of the seed sequence attenuates the repression effect (Fig. 4E).}
{Importantly, the mutant miR-26a responsive element was able to mitigate repression (Figure 5F), therefore suggesting a direct regulation of EZH2 via miR-26a}
{There was a decreased level of relative luciferase activity in cells expressing miR-302a compared to the level in the negative control after transfection with the WT cyclin D1 UTR plasmid (P <0.05), whereas the cells transfected with the mutant cyclin D1 UTR plasmid did not have a decrease in relative luciferase activity, suggesting that miR-302a directly regulates the expression of cyclin D1.}
{For CCNE1, mutation of the single conserved seed complementary site had only a partial effect (Figure 4D), and mutations of both seed complementary sites fully rescued repression for CCNE1, indicating a combination of both sites effects of miR-16}
{For CCND3, only mutation of thefirst putative binding site rescued repression, which suggestedthat miR-16 regulated CCND3 by targeting thefirst seed complementary site}
{{Luciferase assays indicated that overexpression of miR-16, miR-195 and miR-424 could reduce the luciferase activity from the reporter construct containing wild-type CCND1 30-UTR (containing both putative binding sites), whereas had no effectiveness on any deletant of CCND1 30-UTR (Figure 2D)}}
{{Luciferase assays indicated that overexpression of miR-16, miR-195 and miR-424 could reduce the luciferase activity from the reporter construct containing wild-type CCND1 30-UTR (containing both putative binding sites), whereas had no effectiveness on any deletant of CCND1 30-UTR (Figure 2D)}}
{Luciferase assays indicated that overexpression of miR-16, miR-195 and miR-424 could reduce the luciferase activity from the reporter construct containing wild-type CCND1 30-UTR (containing both putative binding sites), whereas had no effectiveness on any deletant of CCND1 3'-UTR (Figure 2D)}
{Luciferase assays indicated that overexpression of miR-16, miR-195 and miR-424 could reduce the luciferase activity from the reporter construct containing wild-type CCND1 30-UTR (containing both putative binding sites), whereas had no effectiveness on any deletant of CCND1 3'-UTR (Figure 2D)}
{Luciferase activity with the wild-type Dicer 3' UTR construct was significantly lower than that with the vector control or the mutant Dicer 3' UTR construct (Figure 3F).}
{Luciferase activity with the wild-type Dicer 3' UTR construct was significantly lower than that with the vector control or the mutant Dicer 3' UTR construct (Figure 3F).}
{Mutation of the miR-17/20 binding site abrogated repression of luciferase activity ( Fig. 4 E )}
{Mutation of the miR-17/20 binding site abrogated repression of luciferase activity ( Fig. 4 E )}
{Mutation of six nucleotides in the miR-126 "seed"region (miR-126 m) or its complementary sequence in the Spred-1 3' UTR (Spred-1 m UTR) relieved the repressive effect of miR-126 (Figure 6E)}
{Direct evidence that the mutant MREs in the transgene were indeed mir-338 resistant was obtained by transfection of synthetic miR-338 that silenced the MRE-wild-type transgene (lane 3) but not the MRE-mutant (lane 6).}
{Direct evidence that the mutant MREs in the transgene were indeed mir-338 resistant was obtained by transfection of synthetic miR-338 that silenced the MRE-wild-type transgene (lane 3) but not the MRE-mutant (lane 6).}
{Direct evidence that the mutant MREs in the transgene were indeed mir-338 resistant was obtained by transfection of synthetic miR-338 that silenced the MRE-wild-type transgene (lane 3) but not the MRE-mutant (lane 6).}
{Therefore we tested mir-520c, we observed that mir-520c over-expression significantly decreased luciferase activity when the putative mir-106a target site was included in the reporter compared to either a reporter lacking the putative target site or a reporter carrying a seed-region mutant ofthe putative mir-106a target site (Figure 1B)}
{(Figure 1B)We observed a signifi-cant ~50% decrease (p < 0.0001) in luciferase activity when the putative mir-106a target site was included in the reporter compared to either a reporter lacking the putative target site or reporter carrying a seed-region mutant of the putative mir-106a target site}
{The reporter experiments showed that pmiR150-GFP effectively repressed zebrafish MYB WT and mt1 reporters (to 48.2% and 45.5%,respectively) and did not significantly repress the zebrafish MYB mt2 and mt1mt2 plasmids, suggesting that target site 2, not target site 1, of 3# UTR of zebrafish c-Myb transcripts is the miR-150 target site (fig. 2B).}
{As shown in figure 1B, miR-150 repressed neither the empty reporter vector nor theMYBmt1mt2 reporter but effectively repressed MYB WT reporter (to 54.6%) and repressed the positive reporter control p150AS most effectively (to13.8%). Besides, miR-150 repressed both MYB mt1 (to75.8%) and mt2 (to 64.7%) reporters, albeit to a lesser degree than MYB mt1mt2, suggesting that both sites are the targets for miR-150.}
{As shown in figure 1B, miR-150 repressed neither the empty reporter vector nor theMYBmt1mt2 reporter but effectively repressed MYB WT reporter (to 54.6%) and repressed the positive reporter control p150AS most effectively (to13.8%). Besides, miR-150 repressed both MYB mt1 (to75.8%) and mt2 (to 64.7%) reporters, albeit to a lesser degree than MYB mt1mt2, suggesting that both sites are the targets for miR-150.}
{As shown in Figure 3C, miR-126 significantly reduced the luciferase activity of the wild-type p85b when compared to the mutant p85Œ≤ and this reduction in luciferase activity was dependent on the concentration of miR-126.}
{The transfection results in 293T cells showed that the luciferase activity from PTPRO 3'UTR WT reporter was approximately 50% of the empty vector, while the luciferase activity from PTPRO 3'UTR Mut reporter was rescued (Fig. 4C).}
{no changes in reporter activity were observed after transfection of a miR-30a-based non-silencing control (‚ÄòNSC30‚Äô, Fig. 3B), in which the seed sequence (nt 2‚Äì7, see Fig. 4D for details) was mutated in order to disrupt complementarity with BDNF 3'-UTR target sites.}
{In contrast, pGL3 carrying the mutant 3¬∂ UTR of HIF-1a with 4-bp deletions in the core of seed sequences of each target site for miR-17-5p and miR-20a did not show obvious decreases in luciferase activities when transfected into clones #31 and #57 (Fig. 2D).}
{the 3‚Ä≤ UTR of Itgb1 (Itgb1-WT) was inserted after a luciferase reporter gene. miR-124 repressed reporter activity approximately three-fold (Fig. 8B). The ability of miR-124 to inhibit the luciferase activity was attenuated by mutations in either or both predicted miR-124 binding sites on Itgb1 3‚Ä≤ UTR (Fig. 8B), indicating that these sites mediate miR-124 inhibition.}
{Mutation of site 1 alone (mutS1, position 1648‚Äì1654) resulted in luciferase activity that was 50% of control compared to 34% for the wild type sites [22] (Fig. 2c).Mutation of site 2 (mutS2, position 7788‚Äì7794) alone increased activity to 73% of control, and mutation of both sites (mutS1, S2) restored luciferase activity to 93% of control}
{Mutation of site 1 alone (mutS1, position 1648‚Äì1654) resulted in luciferase activity that was 50% of control compared to 34% for the wild type sites [22] (Fig. 2c).Mutation of site 2 (mutS2, position 7788‚Äì7794) alone increased activity to 73% of control, and mutation of both sites (mutS1, S2) restored luciferase activity to 93% of control}
{p-miR-20a/p-zbtb7a 3'UTR interaction reduced the fluorescence, while p-miR-20a/p-zbtb7a 3'UTRm interaction rescues the inhibition (Figure 1g) indicating that miR-20a binds directly zbtb7a 3'UTR.}
{MiR-9 and let-7a decreased normalized Renilla luciferase activities to 39.2%+-4.6%,and 41.7% +-3.8% of negative controls, respectively,whereas cotransfection with miR-155 showed no significant difference (Figure 3A)}
{Mutations in the predicted target sites for each of these miRNAs relieve repression of luciferase activities (Figure 3A).}
{Mutations in the predicted target sites for each of these miRNAs relieve repression of luciferase activities (Figure 3A).}
{Mutations in the predicted target sites for each of these miRNAs relieve repression of luciferase activities (Figure 3A).}
{miR132 transfection markedly suppressed luciferase activity of the p250GAP MRE luciferase construct. In contrast,coexpression of miR132 with the reverse complement control had no effect on luciferase activity (Fig. 3A).We next generated a p250GAP mutant (mt-p250GAP) that contained a two-nucleotide deletion in the miR132MRE seed sequence to test whether this site was required for miR132-mediated repression. Expression of miR132 attenuated expression of WT-p250GAP but had no effect on mt-p250GAP (Fig. 3B)}
{Co-transfection of U251 cells with a WT CDK6-3'UTR (CDK6-WT) reporter and the miR-137 mimic resulted in a significant decrease in luminescence (P < 0.0001) relative to cells co-transfected with CDK6-WT and a negative control miRNA mimic (Figure 6D). Mutation of the CDK6 miR-137 seed region rendered the reporter construct insensitive to inhibition by miR-137 (Figure 6D)}
{Importantly, the FL ABCG2 3'UTR construct with a deletion of the putative hsa-miR-519c binding site (3820- 3841) restored the luciferase activity to about 40% of the pGL3-control level.}
{Luciferase activity assays indicated that mutation of the essential target site abolished the effect of miR-328, while mutation of the non-essential site produced little effect of miR-328‚Äôs repression activity (Fig 4D). These}
{Interestingly, the relative luciferase activity of the reporter that contained wild-type 3'-UTR was significantly suppressed when pc3-miR-101 was cotransfected(Fig. 3B). In contrast, the luciferase activity of mutant reporter was unaffected by simultaneous transfection of pc3-miR-101 (Fig. 3B)}
{Consequently, pmiR-221 and pmiR-222, but not pDNR-CMV or pmiR-145, inhibited luciferase activity (Figure 5B). In the mutated control groups,the inhibitory effect of pmiR-221 and miR-222 disappeared(Figure 5B). The results suggested that miR-221 and miR-222 were able to bind to p27(Kip1) and p57(Kip2) directly and inhibit their expression}
{Consequently, pmiR-221 and pmiR-222, but not pDNR-CMV or pmiR-145, inhibited luciferase activity (Figure 5B). In the mutated control groups,the inhibitory effect of pmiR-221 and miR-222 disappeared(Figure 5B). The results suggested that miR-221 and miR-222 were able to bind to p27(Kip1) and p57(Kip2) directly and inhibit their expression}
{Cotransfection of the intact SOCS1 3' UTR with miR155 resulted in an appreciable repression of the reporter activity, whereas mutation of the miR155 seed-binding sequence abolished this repression (Figure 6E)}
{Mutant luciferase reporter was unaffected by either overexpression or knockdown of miR-143 (Figure 3f).}
{Mutant luciferase reporter was unaffected by either overexpression or knockdown of miR-143 (Figure 3f).}
{precursor molecules for miR106b were cotransfected with reporter constructs containing the ITCH 3‚Äô UTR with the miR106b predicted binding site and either the 3‚ÄôUTR in the antisense orientation or a mutated construct lacking the first seven bases of the binding site, into HeLa cells. Our results indicate that overexpression of miR106b specifically inhibited the expression of the reporter construct containing the ITCH 3‚ÄôUTR (Figure 4B) suggesting that miR106b directly interacts with the 3‚ÄôUTR of the Itch mRNA.}
{In these experiments, miR-7 reduced the expression of a reporter construct that carried both of the predicted miR-7 target sites from the Raf1 3‚Äô-UTR but not of a reporter containing an analogous insert with three point mutations in each seed match region (Fig. 5C).}
{In these experiments, miR-7 reduced the expression of a reporter construct that carried both of the predicted miR-7 target sites from the Raf1 3‚Äô-UTR but not of a reporter containing an analogous insert with three point mutations in each seed match region (Fig. 5C).}
{Using A549 cells, miR-7 reduced the expression of reporters bearing putative target sites B and C, but not of the corresponding mutant reporters (Fig. 1F). In contrast, putative target site A did not mediate a change in reporter expression by miR-7 (Fig. 1F)}
{Using A549 cells, miR-7 reduced the expression of reporters bearing putative target sites B and C, but not of the corresponding mutant reporters (Fig. 1F). In contrast, putative target site A did not mediate a change in reporter expression by miR-7 (Fig. 1F)}
{After introducing mutation into the targeting sites of M1 or M2 (Fig. 3C, right panel), luciferase activity of M1(mut)-luc or M2(mut)-luc were not suppressed by mir-320 (Fig. 3D).}
{After introducing mutation into the targeting sites of M1 or M2 (Fig. 3C, right panel), luciferase activity of M1(mut)-luc or M2(mut)-luc were not suppressed by mir-320 (Fig. 3D).}
{Transfection of LV-miR196a-infected hASC with the parental luciferase construct (without the HOXC8 3'UTR) or mutant did not significantly alter the expression of the reporter.}
{We found that luciferase activity in cells transfected with luciferase containing wild-type E2f5 3'UTR and siRNA449 was significantly lower than in cellstransfected with the luciferase plasmid without siRNA449.Confirming that this reduction was due to the presence of the miR449 target site at the 3‚Ä≤UTR, we did not see a difference for the mutant construct when it was transfected with or without siRNA449. Results of these experiments are presented in Fig. 5}
{Moreover, we did not observe variations of the luciferase activity in cells cotransfected with pGL3-mut and miR-101, in comparison to cells transfected only with pGL3-mut (Fig. 3B).}
{Co-transfection of pRL-S3'UTR and miR-27a into HEK293 cells resulted in a pronounced (~50%) reduction of luciferase activity compared with miR-negativecontrol (miR-NC)-transfected cells (Fig. 2A). Mutations introduced in the 3'UTR miR-27a binding sites, either the first (Mut1; also see Fig. S2) or the second (Mut2), significantly reduced the inhibitory effect of miR-27a, and mutation in both sites [Mut (1+2)] completely abolished it (Fig. 2A)}
{(A) miR-142-3p targets 3'-UTR of AC9 messenger RNA (mRNA). miR-142-3p inhibited the expression of the luciferase gene containing AC9 3'-UTR but not mutated AC9 3'-UTR; *<0.05, compared with AC9 3'-UTR group}
{To confirm this result, we mutated the miR-205 binding site on SHIP2 3'UTR (Fig. 1B, SHIP2.mut1). The mutation prevented miR-205from interfering With luciferase activity, indicating that the 3'UTR of SHIP2 is indeed a target of miR-205 (Fig. 1C)}
{if the 8-mer seed sequence was removed from the 3'UTR construct, luciferase levels did not vary from those of the control reporter (Fig. 1D).}
{As expected, miR-223 had no effects on LMO2 mut-pRL-TK that carried the mutant miR-223 binding sites (Fig. 7B, C).}
{We cloned these three 3'UTRs and their relevant mutants into luciferase report vector pcDNA3.1-Luc. Luciferase activity assay showed that ectopically expressed miR-21 decreased the luciferase activity of 30UTRs with wild-type binding sites, but ectopically expressed miR-21 had no effect on 3'UTRs with mutant type binding sites (Fig. 2B).}
{We cloned these three 3'UTRs and their relevant mutants into luciferase report vector pcDNA3.1-Luc. Luciferase activity assay showed that ectopically expressed miR-21 decreased the luciferase activity of 3'UTRs with wild-type binding sites, but ectopically expressed miR-21 had no effect on 3'UTRs with mutant type binding sites (Fig. 2B).}
{We cloned these three 3'UTRs and their relevant mutants into luciferase report vector pcDNA3.1-Luc. Luciferase activity assay showed that ectopically expressed miR-21 decreased the luciferase activity of 3'UTRs with wild-type binding sites, but ectopically expressed miR-21 had no effect on 3'UTRs with mutant type binding sites (Fig. 2B).}
{A 338-bp fragment encompassing this region inserted 3'of a luciferase expression cassette significantly reduced luciferase expression after pre-miR-128-1 transfection in a dose-dependent manner (Fig.2B). mutagenesis of the predicted miR-128 binding site blocked this effect (Fig. 2B)}
{To examine whether miR-101 regulates the 3'UTR of EZH2, we generated luciferase reporters encoding the normal, antisense, and mutated versions of the EZH2 3'UTR. Overexpression of miR-101, but not miR-217 or control miRNA, decreased the activity of the luciferase reporter encoding the 3'UTR of EZH2 (fig. S1). Similarly, the antisense and mutant EZH2 3'UTR activities were not inhibited by miR-101}
{To examine whether miR-101 regulates the 3'UTR of EZH2, we generated luciferase reporters encoding the normal, antisense, and mutated versions of the EZH2 3'UTR. Overexpression of miR-101, but not miR-217 or control miRNA, decreased the activity of the luciferase reporter encoding the 3'UTR of EZH2 (fig. S1). Similarly, the antisense and mutant EZH2 3'UTR activities were not inhibited by miR-101}
{(A) Sequence of miR-138 and its binding site in mouse cspg2 3'UTR with complementary nucleotides indicated in red. Luciferase activity in Cos cells on introduction of wild type or mutated (mut) cspg2 3'UTR sequences downstream of a CMV-driven luciferase (LUC) reporter with or without miR-138 is shown.}
{Point mutations in the miR-138 binding site, located where nucleotides 2‚Äì7 of the miRNA are predicted to bind, abolished repression,indicating that miR-138 can directly target this binding site in the 3'UTR of aldh1a2.}
{we observed a 3 to 4.5-fold increase in Rluc activity from the reporter genes carrying mutated BS for both miRNAs, as compared to the wild-type (WT) sequence (Fig. 6B, left and right panels).}
{we observed a 3 to 4.5-fold increase in Rluc activity from the reporter genes carrying mutated BS for both miRNAs, as compared to the wild-type (WT) sequence (Fig. 6B, left and right panels).}
{In the case of the miR-33 and miR-7 mutants, that do not bind to the original target, no significant difference of GUS stainingwas observed when comparedwith the control (Fig. 5A-4/5)}
{In the case of the miR-33 and miR-7 mutants, that do not bind to the original target, no significant difference of GUS stainingwas observed when comparedwith the control (Fig. 5A-4/5)}
{This was specific to miR-29 binding since luciferase was less affected when transfections were repeated with an irrelevant miR or with a mutant lacking the miR-29 binding site in the YY1 3'UT}
{This was specific to miR-29 binding since luciferase was less affected when transfections were repeated with an irrelevant miR or with a mutant lacking the miR-29 binding site in the YY1 3'UT}
{This was specific to miR-29 binding since luciferase was less affected when transfections were repeated with an irrelevant miR or with a mutant lacking the miR-29 binding site in the YY1 3'UT}
{no reduction was observed with the Mut cyclin E 3'UTR,confirming that miR-15a directly inhibits cyclin E1 translation via binding to its 3'-UTR at the predicted sites.}
{In addition, a luciferase reporter containing the 3' UTR of the CDC42 mRNA was suppressed by ~40% when the cells were transfected with miR-29 miRNAs (Fig. 4e, black), but a reporter with mutated target sites was not repressed by miR-29 (Fig. 4e, white)}
{In addition, a luciferase reporter containing the 3' UTR of the CDC42 mRNA was suppressed by ~40% when the cells were transfected with miR-29 miRNAs (Fig. 4e, black), but a reporter with mutated target sites was not repressed by miR-29 (Fig. 4e, white)}
{A reporter with mutations in the predicted miR-29 target site (Fig. 4b, white) was not suppressed by miR-29 miRNAs.}
{but a reporter with mutated target sites was not repressed by miR-29 (Fig. 4e, white). This suggests that these two sites mediate miR-29‚Äôs regulation of CDC42}
{but a reporter with mutated target sites was not repressed by miR-29 (Fig. 4e, white). This suggests that these two sites mediate miR-29‚Äôs regulation of CDC42}
{A reporter with mutations in the predicted miR-29 target site (Fig. 4b, white) was not suppressed by miR-29 miRNAs.}
{Moreover, miR-1-dependent repression of luciferase activity was abrogated upon the deletion of both miR-1 cognate sites, whereas deletion of each site individually inhibited luciferase activity by 50%.}
{Moreover, miR-1-dependent repression of luciferase activity was abrogated upon the deletion of both miR-1 cognate sites, whereas deletion of each site individually inhibited luciferase activity by 50%.}
{Luciferase activity in respective cells was then measured. Compared with the decrease in luciferase activity observed when the authentic c-myb 3'UTR was cotransfected with miR-15a, deletion of the miR-15a binding sites in the c-myb 3'UTR resulted in a 2- to 3-fold increase in luciferase activity, indicating that miR15a was no longer able to bind the UTR with the same avidity (Figure 4C).}
{Luciferase activity in respective cells was then measured. Compared with the decrease in luciferase activity observed when the authentic c-myb 3'UTR was cotransfected with miR-15a, deletion of the miR-15a binding sites in the c-myb 3'UTR resulted in a 2- to 3-fold increase in luciferase activity, indicating that miR15a was no longer able to bind the UTR with the same avidity (Figure 4C).}
{As shown in Fig. 3C, miR-130a directly interacts with the two binding sites, over achieving the most significant repression of luciferase expression when both the site interactions are allowed, while site-specific mutants, in which the seed sequence has been disrupted, prevented the interaction}
{To test whether miR-8 directly targets the 3'UTR of wls, we generated a sensor for wls by cloning the wls 3'UTRdownstream of the coding region for lacZ. The wls 3'UTRsensor was suppressed by miR-8 in Kc167 cells, and mutation of the seed region (base pairs2‚Äì8) of the putativemiR-8‚Äìbinding site partially blocked the ability of miR-8 to inhibit the wls sensor (Fig. 3A)}
{Expression of miR-8 inhibited the CG32767 sensor 3-fold in Kc167 cells (Fig. 5B). This inhibition was blocked by mutation of the base pairs of both sites predicted to bind the seed region of miR-8 (Fig. 5B). Mutation of either site alone gave only a partial rescue, suggesting that both sites are functionally important(data not shown).}
{Expression of miR-8 inhibited the CG32767 sensor 3-fold in Kc167 cells (Fig. 5B). This inhibition was blocked by mutation of the base pairs of both sites predicted to bind the seed region of miR-8 (Fig. 5B). Mutation of either site alone gave only a partial rescue, suggesting that both sites are functionally important(data not shown).}
{while luciferase activity with mutated IRS-1 3'-UTR reporter was not inhibited (Fig. 3B and C)}
{Furthermore, the luciferase reporter conjugated with mutant Foxg1 3'UTR (luc-Mm-Foxg1MT), in which the target sequence was transversely mutated, was not effectively suppressed by miR-9-1WT, miR-9-2WT, or miR-9-3WT(Fig. 2A).}
{Luciferase expression from luciferase reporters conjugated with chick or Xenopus tropicalis Foxg1 3'UTR (luc-Gg-Foxg1WT and luc-Xt-Foxg1WT,respectively) were effectively suppressed by mouse miR-9-2WT,and not by miR-9-2MT (Fig. 2D).}
{miR-451‚Äìmediated repression on the gata2-3'UTR was abolished when both seed sequences were mutated (GFP-3'UTRgata2-mut), shown by comparable GFP-fluorescence of this reporter construct alone and in the presence of miR-451 (Figure 4C,D).}
{miR-451‚Äìmediated repression on the gata2-3'UTR was abolished when both seed sequences were mutated (GFP-3'UTRgata2-mut), shown by comparable GFP-fluorescence of this reporter construct alone and in the presence of miR-451 (Figure 4C,D).}
{The WT Cyclin D2 reporter showed an approximately three fold inhibition of Renilla luciferase activity relative to a mutant reporter (mutant; Mut) with mismatched sequences inserted into the ‚Äúseed‚Äù sequences of the predicted miR-302b-binding site (Fig.4B).}
{We found that when the cells were transfected with a reporter construct harboring wild-type CDKN1A, luciferase activity was significantly repressed, compared with cells transfected with a construct harboring a CDKN1A mutant (Figure 3D).}
{Luciferase activity was significantly lower in cells cotransfected with luc-Cxcl12-wt and siR-140 than in cells cotransfected with luc-Cxcl12-wt and siR-96, luc-Cxcl12-mut and siR-140 or luc-Cxcl12 without siRNA (Fig. 4B).}
{As shown in Figure 7B, in PCK-CCL cotransfected with miR15a precursor and Cdc25A WT construct, luciferase reporter translation wasdecreased by 75.1% compared with that in PCK-CCL transfected with Cdc25A WT construct only. However, in PCK-cCL cotransfected with miR15a precursor and Cdc25A mutant construct, expression of luciferase reporter was not inhibited.}
{As expected, the mutations of the complementary bases abolished the interaction between MIRN155 and Ets-1 3'-UTR.}
{As expected, the mutations of the complementary bases abolished the interaction between MIRN155 and Ets-1 3'-UTR.}
{Reporter construct with MIRN155 binding site deleted (‚ñ≥/155) was produced as additional control. As expected, ‚ñ≥/155 construct was resistant to MIRN155-mediated repression(Fig 5B).}
{no difference in luciferase activity was found in cells transfected with the 3'UTR mutant compared with the empty vector control (Fig. 3C).}
{Accordingly, pmiR-21, but not pDNR-CMV or pmiR-145 inhibited luciferase activity (Fig. 6B). In the mutated control group, the inhibitory effect of pmiR-21 and pmiR-222 disappeared (Fig. 6B).}
{Mef2a has two conserved miR-1 recognition sequences in its 3' UTR (Fig. 7a). These sequences conferred miR-1 responsiveness to a luciferase reporter (Fig. 7b).}
{Mef2a has two conserved miR-1 recognition sequences in its 3' UTR (Fig. 7a). These sequences conferred miR-1 responsiveness to a luciferase reporter (Fig. 7b).}
{Both contain highly conserved miR-1 seed match sequences in their 3'UTRs (Fig. 5a). When cloned downstream of luciferase, these sequences mediated downregulation of luciferase activity by miR-1 (Fig. 5b).}
{Both contain highly conserved miR-1 seed match sequences in their 3'UTRs (Fig. 5a). When cloned downstream of luciferase, these sequences mediated downregulation of luciferase activity by miR-1 (Fig. 5b).}
{We further showed that the miR-145 binding site in c-Myc 3'UTR was required for this regulation; deletion of the binding site (Luc-c-Myc-UTR-d) abolished its suppression (Fig. 4F).}
{As shown in Figure 2B, the deletion of miR-29b interaction sites abrogated the miR-29b/DNMT3A and miR-29b/DNMT3B interaction evidenced by no change in the normalized luciferase activity.}
{Concurrent mutations of sites 1 and 2 (i.e., 3370, 3763) or sites 1, 2 and 3 (i.e., 3370, 3763, 3818) predicted miR-29b binding sites abrogated the miR-29b/Sp1 interaction evidenced by no change in the normalized luciferase activity, Mutations of only site 1 (i.e., 3370) did not suffice in preventing this interaction (Figure 3B).}
{As shown in Figure 2B, the deletion of miR-29b interaction sites abrogated the miR-29b/DNMT3A and miR-29b/DNMT3B interaction evidenced by no change in the normalized luciferase activity.}
{The results of these reporter assays showed that miR-302d and miR-372 were indeed able to suppress specifically the luciferase activities of the reporter vectors harboring the predicted binding sites of wild-type 3UTR of KLF13 and MBNL2, but not mutant-types (Fig. 1D). }
{The results of these reporter assays showed that miR-302d and miR-372 were indeed able to suppress specifically the luciferase activities of the reporter vectors harboring the predicted binding sites of wild-type 3UTR of KLF13 and MBNL2, but not mutant-types (Fig. 1D). }
{The results of these reporter assays showed that miR-302d and miR-372 were indeed able to suppress specifically the luciferase activities of the reporter vectors harboring the predicted binding sites of wild-type 3UTR of KLF13 and MBNL2, but not mutant-types (Fig. 1D). }
{The results of these reporter assays showed that miR-302d and miR-372 were indeed able to suppress specifically the luciferase activities of the reporter vectors harboring the predicted binding sites of wild-type 3UTR of KLF13 and MBNL2, but not mutant-types (Fig. 1D). }
{The results of these reporter assays showed that miR-302d and miR-372 were indeed able to suppress specifically the luciferase activities of the reporter vectors harboring the predicted binding sites of wild-type 3'UTR of KLF13 and MBNL2, but not mutant-types (Fig. 1D). }
{The results of these reporter assays showed that miR-302d and miR-372 were indeed able to suppress specifically the luciferase activities of the reporter vectors harboring the predicted binding sites of wild-type 3UTR of KLF13 and MBNL2, but not mutant-types (Fig. 1D). }
{The results of these reporter assays showed that miR-302d and miR-372 were indeed able to suppress specifically the luciferase activities of the reporter vectors harboring the predicted binding sites of wild-type 3UTR of KLF13 and MBNL2, but not mutant-types (Fig. 1D). }
{Mutating the binding site had abolished the ability of miR-126 to inhibit the expression of the luciferase reporter}
{As shown in Fig. 5C, deletion of the first binding site impaired the suppression of luciferase activity, but we still detected about 30% suppression; deletion of the second binding site had a similar effect. When both sites were deleted, however, miR-30e‚Äìmediated suppression of luciferase activity was abolished.These results suggest that both binding sites are critical for microRNA regulation}
{As shown in Fig. 5C, deletion of the first binding site impaired the suppression of luciferase activity, but we still detected about 30% suppression; deletion of the second binding site had a similar effect. When both sites were deleted, however, miR-30e‚Äìmediated suppression of luciferase activity was abolished.These results suggest that both binding sites are critical for microRNA regulation}
{As shown in Fig. 5C, deletion of the first binding site impaired the suppression of luciferase activity, but we still detected about 30% suppression; deletion of the second binding site had a similar effect. When both sites were deleted, however, miR-30e‚Äìmediated suppression of luciferase activity was abolished.These results suggest that both binding sites are critical for microRNA regulation}
{As shown in Fig. 5C, deletion of the first binding site impaired the suppression of luciferase activity, but we still detected about 30% suppression; deletion of the second binding site had a similar effect. When both sites were deleted, however, miR-30e‚Äìmediated suppression of luciferase activity was abolished.These results suggest that both binding sites are critical for microRNA regulation}
{ As expected, this suppressive effect could be abolished by partially deleting the perfectly complementary sequences in the 3'-UTR of Bcl-2 mRNA (pGL3-mut Bcl-2-3'-UTR), which disrupted the interaction between miR-15b/miR-16 and Bcl-2(Fig. 7).}
{ As expected, this suppressive effect could be abolished by partially deleting the perfectly complementary sequences in the 3'-UTR of Bcl-2 mRNA (pGL3-mut Bcl-2-3'-UTR), which disrupted the interaction between miR-15b/miR-16 and Bcl-2(Fig. 7).}
{As shown in Figure 5F, we similarly demonstrated that miR-205 directly targets VEGF-A by interacting with the putative miR-205 binding site in its 3‚Ä≤-UTR}
{miR-205-mediated suppression of the luciferase activity was completely abolished in Luc-ErbB3-3‚Ä≤-UTR-d (Figure5C), suggesting that the miR-205 binding site is critical for miR-205-mediated suppression.}
{The dual luciferase assay demonstrated that miR-1, miR-206 could significantly reduce the luciferase activities mediated by pGL3-Mmbs2 (p < 0.05), and miR-1, miR-206 could reduce luciferase activities mediated by pGL3-mbs12 a little more efficiently than those mediated by pGL3-Mmbs2 without significant differences (p > 0.05) (Fig. 3).}
{The dual luciferase assay demonstrated that miR-1, miR-206 could significantly reduce the luciferase activities mediated by pGL3-Mmbs2 (p < 0.05), and miR-1, miR-206 could reduce luciferase activities mediated by pGL3-mbs12 a little more efficiently than those mediated by pGL3-Mmbs2 without significant differences (p > 0.05) (Fig. 3).}
{co-transfection of miR-29a led to a strong reduction of the luciferase signal (Fig 2C, WT).}
{For HMGA1, two mutants were constructed for HMGA1 RE1 and RE2, denoted as m1 and m2 respectively. Only the mutation of the RE1 (m1) completely corrected the inhibition of the luciferase activity (Fig. 2 A).}
{For caprin-1, the mutation of the sole RE completely abolished the inhibition of luciferase activity (Fig. 2 B)}
{We could restore the luciferase activity only by concomitantly mutating the two miR-16 binding sites (m3 mutant), while independent mutation of each of them was quasi Inefficient (Fig. 2 C).}
{We could restore the luciferase activity only by concomitantly mutating the two miR-16 binding sites (m3 mutant), while independent mutation of each of them was quasi Inefficient (Fig. 2 C).}
{miR-199a* significantly inhibited activity of the wildtype reporter gene pGL3Smad1, whereas mutation of either seed site largely abolished miR-199a*-mediated repression of reporter gene activity.}
{miR-199a* significantly inhibited activity of the wildtype reporter gene pGL3Smad1, whereas mutation of either seed site largely abolished miR-199a*-mediated repression of reporter gene activity.}
{In contrast, exogenous miR-34b expression did not reduce LUC activity with the mutated CREB 3'-UTR, further suggesting that CREB expression is typically regulated by miR-34b (Fig. 2A).}
{ Further characterization of the Lmnb1 3‚Ä≤UTR revealed that there are three separate miR-23 binding sites within this region (see supplementary material Fig. S1 and Table S1). }
{ Further characterization of the Lmnb1 3‚Ä≤UTR revealed that there are three separate miR-23 binding sites within this region (see supplementary material Fig. S1 and Table S1). }
{ Further characterization of the Lmnb1 3‚Ä≤UTR revealed that there are three separate miR-23 binding sites within this region (see supplementary material Fig. S1 and Table S1). }
{ Further characterization of the Lmnb1 3‚Ä≤UTR revealed that there are three separate miR-23 binding sites within this region (see supplementary material Fig. S1 and Table S1). }
{ Further characterization of the Lmnb1 3‚Ä≤UTR revealed that there are three separate miR-23 binding sites within this region (see supplementary material Fig. S1 and Table S1). }
{ Further characterization of the Lmnb1 3‚Ä≤UTR revealed that there are three separate miR-23 binding sites within this region (see supplementary material Fig. S1 and Table S1). }
{Importantly,mutation of the four candidate miR-150 binding sites abrogated miR-150 repression,}
{Inclusion of this sequence within the 3'UTR of a luciferase gene rendered it a target of miR-199a, as demonstrated by the inhibition of its activity on overexpression of miR-199a (Figure 2b)}
{Inclusion of this target sequence within the 3'UTR of a luciferase gene rendered it a target of miR-199a, as demonstrated demonstrated by the inhibition of its activity on overexpression of miR-199a, relative to a mutant sequence (Figure 5b).}
{Luciferase  activity in U373 cells co-transfected with pre-miR-146b and  pGL3-MMP16 vector was decreased when compared with controls (Fig. 4C).}
{Luciferase  activity in U373 cells co-transfected with pre-miR-146b and  pGL3-MMP16 vector was decreased when compared with controls (Fig. 4C).}
{disruption of miR-328 MRE within ABCG2 3‚Äô-UTR,particularly the segment complementary to 8-bp seed sequence (Fig. 1A), resulted in a 300% increase in luciferase activity in MCF-7 cells (Fig. 2C).}
{However, the activity of the reporter construct deleted at the seed sequences of microRNA-21 target site was unaffected by a simultaneous transfection with anti‚ÄìmicroRNA-21 (Fig. 5F)}
{Moreover, the mutation of the miR-205 binding site in HER3-3'UTR prevented the down-regulation of luciferase expression,}
{Importantly, mutation of six bases (Figure3A, top panel) in the candidate miR-223 binding site totally abrogated miR-223-dependent repression}
{An inhibitory effect was not suggested with mutant 1, whereas minimal luciferase inhibition by approximately 18% was indicated with mutant 2 (P =.19) (Figure 4C).}
{However, luciferase activity was significantly up-regulated after 3T3 cells were transfected with the Reck-pGL3-Mutant plasmid containing the mutated binding site and the miR-21 precursor (Fig. 7E).}
{Activity of the FL‚ÄìMut2/3, MutS2 or MutS3 reporter vector was not affected, (Fig. 3d), indicating that miR-146a interferes with CXCR4 mRNA translation through a direct interaction with S2 and S3 sites in the 3¬¥UTR‚ÄìFL.}
{Activity of the FL‚ÄìMut2/3, MutS2 or MutS3 reporter vector was not affected, (Fig. 3d), indicating that miR-146a interferes with CXCR4 mRNA translation through a direct interaction with S2 and S3 sites in the 3¬¥UTR‚ÄìFL.}
{Cotransfection experiments revealed that the DM is completely refractory to miR-135a&b‚Äìmediated repression, indicating the direct suppression of APC by miR-135a&b (Fig. 2D).}
{Cotransfection experiments revealed that the DM is completely refractory to miR-135a&b‚Äìmediated repression, indicating the direct suppression of APC by miR-135a&b (Fig. 2D).}
{Introducing a mutation (exchange of two nucleotides) into the hsa-let-7a target site led to an increase of luciferase expression of 70% (¬±5%) in HMB2-MIA5 cells compared with the wild-type 3'-UTR construct}
{Importantly, the mutant miR-26a responsive element was able to mitigate repression (Figure 5F),}
{A reporter construct with mutated miR-26a target site, m(236-257), also lost the inhibition to the reporter construct (Fig. 3D).}
{Introduction of excess wild-type human miR-33a/b precursor oligonucleotides resulted in further repression of the wild-type ABCA1 3‚Ä≤UTR reporter (Fig. 2E). By contrast, miR-33a/b precursors mutated in the seed base-pairing sequence had no effect, as compared with precursor control (PC) (Fig. 2E and fig. S5C).}
{Introduction of excess wild-type human miR-33a/b precursor oligonucleotides resulted in further repression of the wild-type ABCA1 3‚Ä≤UTR reporter (Fig. 2E). By contrast, miR-33a/b precursors mutated in the seed base-pairing sequence had no effect, as compared with precursor control (PC) (Fig. 2E and fig. S5C).}
{"Our results clearly demonstrated a significant decrease of luciferase activity compared to either mutant or empty vector controlsupon transient over-expression of miR-215 (Figure 1C)."}
{"Our results clearly demonstrated a significant decrease of luciferase activity compared to either mutant or empty vector controls upon transient over-expression of miR-215 (Figure 1C)."}
{"Our results clearly demonstrated a significant decrease of luciferase activity compared to either mutant or empty vector controls upon transient over-expression of miR-215 (Figure 1C)."}
{Luciferase activity with p53 3' UTR mutant constructs decreased less than with the wild-type construct, especially in mutants of the first target site (Fig. 2C). Furthermore, the inhibitory effect of miR-1285 on luciferase activity was abrogated when a p53 3' UTR containing the two mutant binding sites was used (Fig. 2C).}
{Luciferase activity with p53 3' UTR mutant constructs decreased less than with the wild-type construct, especially in mutants of the first target site (Fig. 2C). Furthermore, the inhibitory effect of miR-1285 on luciferase activity was abrogated when a p53 3' UTR containing the two mutant binding sites was used (Fig. 2C).}
{Cotransfection of a pMir-Reporter containing the AKT2 binding site for miR-184 and mature miR-184 mimics significantly (p < 0.003) diminished luciferase activity while co-transfection of the reporter with a negative control oligonucleotide had no effect (Figure 4d). A three base pair mutation introduced into the seed region of the miR-184 binding site in the AKT2 3' UTR completely abolished the ability of mature miR-184 mimics to affect luciferase activity.}
{Luciferase activity was markedly diminished in cells transfected with miR-650 precursor and wildtype 3'UTR reporter plasmid transfected cells, compared to luciferase activity in cells transfected with miR-650 precursor and mutant 3'UTR reporter plasmids (Fig. 3C, p < 0.05).}
{Suppression of luciferase activity by miR-148a was seen with the MSK1 3 -UTR MT2 reporter gene, which was almost similar to that with the WT reporter gene (Fig. 5C). However, miR-148 showed little or no effects on luciferase activity with the MT1 and MT3 reporter genes.}
{Suppression of luciferase activity by miR-148a was seen with the MSK1 3 -UTR MT2 reporter gene, which was almost similar to that with the WT reporter gene (Fig. 5C). However, miR-148 showed little or no effects on luciferase activity with the MT1 and MT3 reporter genes.}
{Suppression of luciferase activity by miR-148a was seen with the MSK1 3 -UTR MT2 reporter gene, which was almost similar to that with the WT reporter gene (Fig. 5C). However, miR-148 showed little or no effects on luciferase activity with the MT1 and MT3 reporter genes.}
{miR-17/20 repressed IL-8 luciferase reporter gene activity, and mutation of themiR-17/20 binding site of the IL-8 3‚Ä≤ UTR reduced the repression (Fig. 5 B and C).}
{miR-17/20 repressed IL-8 luciferase reporter gene activity, and mutation of themiR-17/20 binding site of the IL-8 3‚Ä≤ UTR reduced the repression (Fig. 5 B and C).}
{Our data show that in resting RAW264.7 cells, miR-466l downregulated the wildtype IL-10 39UTR luciferase activity, but had no effect on that of mutant IL-10 3'UTR reporter gene. Down regulation of luciferase activity is unchanged if the first, second, or fourth potential miR-466l binding sites are mutated, whereas downregulation of luciferase activity is abolished if the third binding site is mutated (Fig. 3B). In LPS-stimulated RAW264.7 cells, miR-466l upregulated the wildtypeIL-10 3'UTR luciferase activity, but had no effect on that of mutant IL-10 3'UTR reporter gene. The first and fourth mutation of potential miR-466l binding sites did not block the upregulation of luciferase activity, whereas the second and third mutation abolished the upregulation of luciferase activity caused by miR-466l (Fig. 3B).}
{"We also cotransfected these cells with an miR-15a mimic or mimic control and measured luciferase activity. As shown in Figure 6C,the miR-15a mimic at a concentration of 50 nM decreases luciferase activity of the reporter vector containing the miR-15a binding sequence (Fig. 6C). In contrast, the miR-15a mimic has no significant effect on a reporter vector with a mutated miR-15a binding sequence."}
{"The relative luciferase activity of the reporter that contained wild-type 30UTR was significantly suppressed whenmiR-375was cotransfected (Fig. 2A). In contrast, the luciferase activity of the mutant reporter was unaffected by cotransfection ofmiR-375 (Fig. 2A)."}
{Subsequently, we generated two Luciferase report vectors that contain the putative miR-155 binding sites within 30UTR and mutant 30UTR (30MUT), as shown in Fig. 1B, the relative luciferase activity was reduced by 50% following cotransfection with miR-155 mimics compared with transfection with miR-155 control. In contrast, no change of luciferase was observed in cells transfected with 30MUT constructs or miR-155 inhibitors.}
{Inhibition of miR-34c with a 20 O-methyl oligonucleotide (anti-miR) relieves repression of a luciferase reporter harboring the wild-type c-myc 30 'UTR (pLSVM30) relative to a version with a point mutation in the miR-34c target-site (pLSVM30mut) (Fig. 1A).}
{Both pcDNA3.0‚ÄìmiR-22 and miR-22 duplex reduced luciferase activities expressed in pGL3m‚ÄìESR1‚Äì3¬¢-UTR‚ÄìWT by   50%, but such a reduction was completely abolished in pGL3m‚ÄìESR1-3¬¢‚ÄìUTR‚ÄìMUT (Fig. 2B, C).}
{To determine whether miR-9 targets CDH1 directly, we performed reporter assays and found that miR-9 decreased the activity of a luciferase reporter fused to the wild-type CDH1 3¬¥ UTR but not to a mutant 3¬¥ UTR (Fig. 1d);}
{An enhanced level of miR-22 expression decreased the WT-vector luciferase activity (P < 0.05); however, no difference was observed between MT-vectors with precursor (Fig. 4D).}
{Injection of the WT sensor mRNA alone resulted in bright GFP fluorescence, which was significantly dampened by miR-143 (P =0.002),Additionally, a single point mutation in the predicted miR-143 binding site blocked the observed GFP silencing by miR-143 (Fig. 6D).}
{Cotransfection of cells with pGL3-F plus the miR-512-3p decreased the luciferase activity in a dose-dependent manner.To further determine the suppression of miR-512-3p on c-FLIP by binding to c-FLIP 3'UTR, the seed region of c-FLIP 3'UTR was deleted and subcloned downstream of luciferase gene (designated as pGL3-FM). As we expected, the luciferase activity in pGL3-FM was unchanged by overexpression of miR-512-3p (Fig. 3A).}
{As shown in Fig. 2E, the reporter activities of wildtype (WT) but not mutated (MUT) pMIR-FOXO3a are reversely correlated with miR-155 expression levels, i.e., the activity is high in miR-155 low cells (e.g., MDA-MB-468 and BT-474) and low in miR-155 high cells (e.g., (HS578T and MDA-MB-157). Further, expression of miR-155 represses WT-pMIR-FOXO3a but not MUT-pMIR-FOXO3a reporter activity in BT-474 (Fig. 2F).}
{Using a reporter assay, we found that the reporter fused to the A24G-mutated SOCS1 3‚Ä≤UTR was not repressed by miR-155 (Fig. 3B).}
{"Compared with the control, transfection with pre-miR-34a reduced the luciferase activities significantly (Figure 2A). However, the pre-miR scramble or pre-miR-34a did not affect the luciferase activity in the mutant construct and their luciferase activitiesdid not have any significant differences when compared with the control."}
{"Compared with the control, transfection with pre-miR-34a reduced the luciferase activities significantly (Figure 2A). However, the pre-miR scramble or pre-miR-34a did not affect the luciferase activity in the mutant construct and their luciferase activitiesdid not have any significant differences when compared with the control."}
{We performed luciferase assays in which the miR-17 target sequence in the Jak1 3¬¥UTR was cloned in the luciferase 3¬¥UTR. Overexpression of miR-17 reduced luciferase activity, but exhibited no effect on a mutated construct (Fig. 5F) showing that miR-17 directly targets the Jak1 3¬¥UTR.}
{We transfected a ESR1 3‚Ä≤-UTR (3‚Ä≤-ESR1-wt) luciferase reporter construct (Fig. S4B) together with miR-18a precursors into HEK-293 cells and noticed an ‚àº60% reduction in luciferase activity compared to cells transfected with the scramble control (Fig. 3B). Conversely, no reduction in luciferase activity was detected with the miR-18a seed-modified ESR1 3‚Ä≤- UTR (3‚Ä≤-ESR1-mut) reporter construct.}
{We transfected a ESR1 3‚Ä≤-UTR (3‚Ä≤-ESR1-wt) luciferase reporter construct (Fig. S4B) together with miR-18a precursors into HEK-293 cells and noticed an ‚àº60% reduction in luciferase activity compared to cells transfected with the scramble control (Fig. 3B). Conversely, no reduction in luciferase activity was detected with the miR-18a seed-modified ESR1 3‚Ä≤- UTR (3‚Ä≤-ESR1-mut) reporter construct.}
{Luciferase activity was significantly lower  following the siRNA transfection suggesting that Smad3 is a direct target of miR-140 (Fig. 2). Mutations were introduced to all three potential target sites, which rendered the construct insensitive to the siRNA (Fig. 2).}
{miR-150 suppressed the luciferase activity of the Luc-EGR2 3'UTR by approximately 50% compared to the vector control, while mutation or removal of the miR-150 binding site blocked this suppression.}
{Concomitantly, the expression of p53 with wild type but not the mutated 39UTR could be significantly suppressed by miR-30a (Figure 2E) or miR-30b (data not shown).}
{Concomitantly, the expression of p53 with wild type but not the mutated 39UTR could be significantly suppressed by miR-30a (Figure 2E) or miR-30b (data not shown).}
{We then transfected this luciferase reporter vector with either wild-type or mutant miR-497 binding sequences into N2A cells. We also co-transfected these cells with a miR-497 mimic or mimic control and measured luciferase activity. As shown in Fig. 4, the miR-497 mimic administered at a concentration of 50 nM decreases luciferase activity of the reporter vector containing miR-497 binding sequences (Fig. 4). As expected, the miR-497 mimic has no significant effect on the reporter vector with mutated miR-497 binding sequences. }
{We then transfected this luciferase reporter vector with either wild-type or mutant miR-497 binding sequences into N2A cells. We also co-transfected these cells with a miR-497 mimic or mimic control and measured luciferase activity. As shown in Fig. 4, the miR-497 mimic administered at a concentration of 50 nM decreases luciferase activity of the reporter vector containing miR-497 binding sequences (Fig. 4). As expected, the miR-497 mimic has no significant effect on the reporter vector with mutated miR-497 binding sequences. }
{"We examined whether MEK1 was directly regulated by miR-34a. Transfection of the miR-34a precursor oligonucleotide significantly reduced luciferase expression from a reporter plasmid that contained the 3 ' UTR of MEK1 compared with the transfection of ascrambled RNA control (Fig. 5B)."}
{The miR-221 or miR-222 precursor oligonucleotide reduced the activity of a reporter plasmid that contained the 3 ' UTR of c-fos (Fig. 5D).}
{The miR-221 or miR-222 precursor oligonucleotide reduced the activity of a reporter plasmid that contained the 3 ' UTR of c-fos (Fig. 5D).}
{As illustrated in Figure 2b, when cells were transfected with miR-138, the luciferase activity was significantly diminished when compared to the cells transfected with negative control. When the seed region of the targeting site was mutated (pGL3-RhoCm), the miR-138 effect on luciferase was abolished.}
{As illustrated in Figure 3b, when cells were transfected with miR-138 mimic, the luciferase activity was significantly reduced when compared to the cells transfected with negative control. However, when the seed region of the targeting site was mutated (pGL3-ROCK2m), the miR-138 effect on luciferase was abolished.}
{"When pre375 was cotransfected, the relative luciferase activity of a reporter containing WT-3‚Ä≤ UTR was significantly suppressedby 30% (P < 0.001) compared with that of a reporter containing MUT-3‚Ä≤ UTR (Fig. 3D)."}
{Instead, coexpression of 3 UTR MCP-2 with mir-146a (MCP-2 146a) resulted in a reproducible 25% reduction of R/L values (P 0.05). The control reporter plasmid carrying the MCP-2 3 UTR mutated in the mir-146a seeding site was not affected by the addition of mir-146a (mutMCP-2 146a).}
{eGFP expression was analysed by flow cytometry 72 h later. As shown in Figure 4A, expression of eGFP-Var or eGFP-GLO remained unchanged in the three conditions tested. However, a significant decrease in expression of eGFP-WT was observed in MG63 cells transfected with hsa-miR-433 compared with the mock transfected cells (39% decrease, Fig. 4A).}
{As indicated in Fig. 2B, the introduction of miR-133a mimic decreased luciferase activity 40% compared with the cells treated with a control mimic (P  < 0.05). In a parallel experiment, the inhibitory effect of miR-133a in cells transfected with the mutant reporter vector (the two putative targeting sites were mutated) was completely abolished.}
{As indicated in Fig. 2B, the introduction of miR-133a mimic decreased luciferase activity 40% compared with the cells treated with a control mimic (P  < 0.05). In a parallel experiment, the inhibitory effect of miR-133a in cells transfected with the mutant reporter vector (the two putative targeting sites were mutated) was completely abolished.}
{We observed a significant dose-dependent miR-21-mediated decrease (P <0.05) in luciferase activity (Fig. 3B).mutant Peli1 reporter levels in cultured cells did not decrease upon pre-miR-21 cotransfections (Fig. 4).}
{Deletion as well as mutation of the miR-27 site completely abolished the LPS-mediated reduction of luciferase activity (Fig. 3C).}
{Furthermore, to determine the specific sites targeted by the two microRNAs, we constructed vectors covering four conserved sites for miR-145 and one site for miR-133a (Figure 2B). The luminescence intensity was significantly decreased at the two sites targeted by miR-145 (positions 377‚Äì383 and 1140‚Äì1146) and one site targeted by miR-133a (position 745‚Äì751) (Figure 2B).}
{Furthermore, to determine the specific sites targeted by the two microRNAs, we constructed vectors covering four conserved sites for miR-145 and one site for miR-133a (Figure 2B). The luminescence intensity was significantly decreased at the two sites targeted by miR-145 (positions 377‚Äì383 and 1140‚Äì1146) and one site targeted by miR-133a (position 745‚Äì751) (Figure 2B).}
{Furthermore, to determine the specific sites targeted by the two microRNAs, we constructed vectors covering four conserved sites for miR-145 and one site for miR-133a (Figure 2B). The luminescence intensity was significantly decreased at the two sites targeted by miR-145 (positions 377‚Äì383 and 1140‚Äì1146) and one site targeted by miR-133a (position 745‚Äì751) (Figure 2B).}
{Furthermore, to determine the specific sites targeted by the two microRNAs, we constructed vectors covering four conserved sites for miR-145 and one site for miR-133a (Figure 2B). The luminescence intensity was significantly decreased at the two sites targeted by miR-145 (positions 377‚Äì383 and 1140‚Äì1146) and one site targeted by miR-133a (position 745‚Äì751) (Figure 2B).}
{Furthermore, to determine the specific sites targeted by the two microRNAs, we constructed vectors covering four conserved sites for miR-145 and one site for miR-133a (Figure 2B). The luminescence intensity was significantly decreased at the two sites targeted by miR-145 (positions 377‚Äì383 and 1140‚Äì1146) and one site targeted by miR-133a (position 745‚Äì751) (Figure 2B).}
{To ascertain the direct effect of miR-221 on DDIT4, mutant constructs targeting S1, S2, or S1+S2 were generated from human and murine DDIT4 3‚Ä≤UTR. Mutations that disrupted the two binding sites for miR-221 entirely restored luciferase activity (Fig.5B and Fig. S6C).}
{To ascertain the direct effect of miR-221 on DDIT4, mutant constructs targeting S1, S2, or S1+S2 were generated from human and murine DDIT4 3‚Ä≤UTR. Mutations that disrupted the two binding sites for miR-221 entirely restored luciferase activity (Fig.5B and Fig. S6C).}
{Compared to antimiR-nc, wild-type luciferase vector co-transfection with anti-miR-494 led to a significant increase in luciferase/renilla activity. However, no significant changes were observed in mutated vector co-transfection groups (Fig. 1E).}
{Compared to antimiR-nc, wild-type luciferase vector co-transfection with anti-miR-494 led to a significant increase in luciferase/renilla activity. However, no significant changes were observed in mutated vector co-transfection groups (Fig. 1E).}
{"As shown in Fig. 3b, suppression of the reporter activity by miR-16-1 was partially relieved by mutation of the single conserved seedcomplementary site, and mutations of both seed complementary sites almost fully rescued the repression for CCNE1,"}
{"As shown in Fig. 3b, suppression of the reporter activity by miR-16-1 was partially relieved by mutation of the single conserved seedcomplementary site, and mutations of both seed complementary sites almost fully rescued the repression for CCNE1,"}
{Our results showed that the relative luciferase activity of the pMIR-RB-3'-UTR construct with anti-miR-675 was significantly increased in Clone A, HT-29 and MIP101 cells.}
{Overexpression of miR-2861 suppressed the luciferase activity of the HDAC5 CDS reporter gene (Figure 4B). Mutation of 2 nucleotides within the putative target site in the HDAC5 CDS (MUT-pGL3-HDAC5) abolished this repression, confirming the specificity of the action (Figure 4B).}
{When cotransfected with synthetic miR-495, a 50% decrease in luciferase activity, relative to control, was observed (Fig. 3A). The luciferase activity was rescued when the miR-495 target site was rendered ineffective by mutating two nucleotides in the seed sequence (Fig. 3A).}
{When a construct with wild-type 30UTR was cotransfected with synthetic miR-218, a decrease in relative luciferase activity was observed, indicating that this 30UTR region was targeted by miR-218 (Fig. 3B). We then generated constructs bearing mutations in the different sites and verified their activity in the presence of miR-218.}
{When a construct with wild-type 30UTR was cotransfected with synthetic miR-218, a decrease in relative luciferase activity was observed, indicating that this 30UTR region was targeted by miR-218 (Fig. 3B). We then generated constructs bearing mutations in the different sites and verified their activity in the presence of miR-218.}
{When a construct with wild-type 30UTR was cotransfected with synthetic miR-218, a decrease in relative luciferase activity was observed, indicating that this 30UTR region was targeted by miR-218 (Fig. 3B). We then generated constructs bearing mutations in the different sites and verified their activity in the presence of miR-218.}
{We performed a luciferase reporter assay and observed a significant decrease in luciferase activity for the reporter vector of the 3'UTR of ECOP in AGS cells treated with pre-miR-218 compared with mutant controls (Fig. 5A),}
{As anticipated, cotransfection of miR-155 attenuated expression of luciferase from the WT 3‚Ä≤-UTR-luc reporter (Fig 2C, bars 1 and 3), whereas no inhibition was observed when a control miR (miR-CTL) was used (Fig 2C, bar 2). Similarly, a mutated miR-155 (miR-155mut) whose seed sequence had been altered, failed to suppress the luciferase activity originating from WT 3‚Ä≤-UTR-luc plasmid (Fig 2C, bar 4).}
{When the wild-type or mutation-type vector was transfected with miR-129-2, the luciferase activity of wild-type vector was significantly decreased (P < 0.001) compared with mutation-type vector (Fig. 2C).}
{"As shown in Figure 4B, co-transfection with miR-155 expression vector (pc3-miR-155) significantly decreased the luciferase activityof the reporter vector containing the wild-type 3‚ÄôUTR of C/EBPÔÅ¢, but had no effect on the mutant 3‚ÄôUTR of C/EBPÔÅ¢ (Figure 4B),"}
{"Luciferase activity of cells co-transfected with pre-miR-139 and pLUC-3'UTR-FoxO1 was decreased compared to cells co-transfected with control pNeg and pLUC-3'UTR-FoxO1. With the negative control constructs, p-LUC-Empty or pLUC-3'UTR mut-FoxO1 expressing a mutated sequence of the FoxO1 3'UTR target site, no reduced luciferase activity was observed when cells were co-transfected with premiR-139 compared to pNeg (Fig. 2A)."}
{Luciferase activity was strongly suppressed when the reporter gene was linked to the wild-type RECK 3'UTR (3'RECK-wt) (Figure 1e, bar 2). This effect was relieved when the predicted miRNA-target sites were mutated.}
{Luciferase activity was strongly suppressed when the reporter gene was linked to the wild-type RECK 3'UTR (3'RECK-wt) (Figure 1e, bar 2). This effect was relieved when the predicted miRNA-target sites were mutated.}
{Luciferase activity was strongly suppressed when the reporter gene was linked to the wild-type RECK 3'UTR (3'RECK-wt) (Figure 1e, bar 2). This effect was relieved when the predicted miRNA-target sites were mutated.}
{Luciferase activity was strongly suppressed when the reporter gene was linked to the wild-type RECK 3'UTR (3'RECK-wt) (Figure 1e, bar 2). This effect was relieved when the predicted miRNA-target sites were mutated.}
{Transfection with precursors of miR-148a and miR-152 significantly modulated reporter activity in Mz-ChA-1 cells.Next, the studies were repeated with random mutations in the shared recognition sequence, which resulted in abolition of the reporter activation by miR-148a and miR-152 precursors.}
{Transfection with precursors of miR-148a and miR-152 significantly modulated reporter activity in Mz-ChA-1 cells.Next, the studies were repeated with random mutations in the shared recognition sequence, which resulted in abolition of the reporter activation by miR-148a and miR-152 precursors.}
{In SAS-miR-31 cells, the A site mutation resulted in a considerable reversion of the Lac Z repression compared with the B site mutation (Fig. 3C). Mutations in both A and B sites resulted in a complete reversion of LacZ repression.}
{In SAS-miR-31 cells, the A site mutation resulted in a considerable reversion of the Lac Z repression compared with the B site mutation (Fig. 3C). Mutations in both A and B sites resulted in a complete reversion of LacZ repression.}
{"A marked reduction in luciferase activity levels in cells overexpressing miR-27b was observed (P < 0.05 versus nontransfectedcontrol) (Figure 3A). In contrast, cotransfection of the mutant reporter plasmid with miR-27b mimic or the reporter plasmid with the negative control microRNA had no effect on luciferase activity in the transfected cells (Figure 3A)."}
{As shown in Fig. 3B, a significant decrease of luciferase activity was detected in cells transfected with the ICAM-1 3‚ÄôUTR construct containing the potential binding site compared with mutant control vector, suggesting endogenous translational repression of the construct with the ICAM-1 3‚ÄôUTR.}
{Transfection of anti‚ÄìmiR-222 into Hep3B was able to restore the luciferase activity of WT construct by 30% (P = 0.0066; Fig. 4Biii). By contrast, no effect on the luciferase signal was detected with the deleted construct.}
{As expected, decreased reporter activity was observed in the cells expressing the luciferase shRNA (positive control).Accordingly, when either miR-205-binding site was mutated, we observed a relief of silencing of the reporter gene.}
{As expected, decreased reporter activity was observed in the cells expressing the luciferase shRNA (positive control).Accordingly, when either miR-205-binding site was mutated, we observed a relief of silencing of the reporter gene.}
{Statistically significant repression of luciferase activity was observed in 293T cells cotransfectedwith Pre-miR-181c and a reporter vector containing the NOTCH4 or KRAS 3'-UTR target site (Figure 5E).}
{Statistically significant repression of luciferase activity was observed in 293T cells cotransfectedwith Pre-miR-181c and a reporter vector containing the NOTCH4 or KRAS 3'-UTR target site (Figure 5E).}
{"Cotransfection of KYSE140 cells with pIS0-KLF4‚Äì3' -UTR and pcDNA3.0-miR-10b caused a 30% decrease in the luciferase activity compared with the negative control. This suppression was rescued by the four-nucleotide substitution in the core binding sites(Fig. 3C)."}
{In addition, our results indicate that both of the binding sites could be involved in miR-30d regulation, as mutation of the two sites separately could restore luciferase activity when this construct was cotransfected with miR-30d (Fig. 3E).}
{In addition, our results indicate that both of the binding sites could be involved in miR-30d regulation, as mutation of the two sites separately could restore luciferase activity when this construct was cotransfected with miR-30d (Fig. 3E).}
{The normalized luciferase activity increased by 60% (P¬º0.0017) in pIS0-TIMP3-3'-UTR transfected cells but not in pIS0-TIMP3-3'-UTR‚ñ≥ expressing cells (Figure 2d(ii)).}
{"Cytomeg-alovirus-driven miR-15b, -16, -195, and -424 resulted in a decrease in luciferase activity, whereas similaramounts of miR-214 had no effect (Fig. 2C). Moreover, miR-15b, -16, -195, and -424 did not decrease the luciferase activities of mutated Arl2‚Äì3 -UTR constructs."}
{"Cytomeg-alovirus-driven miR-15b, -16, -195, and -424 resulted in a decrease in luciferase activity, whereas similaramounts of miR-214 had no effect (Fig. 2C). Moreover, miR-15b, -16, -195, and -424 did not decrease the luciferase activities of mutated Arl2‚Äì3 -UTR constructs."}
{"Cytomeg-alovirus-driven miR-15b, -16, -195, and -424 resulted in a decrease in luciferase activity, whereas similaramounts of miR-214 had no effect (Fig. 2C). Moreover, miR-15b, -16, -195, and -424 did not decrease the luciferase activities of mutated Arl2‚Äì3 -UTR constructs."}
{"Cytomeg-alovirus-driven miR-15b, -16, -195, and -424 resulted in a decrease in luciferase activity, whereas similaramounts of miR-214 had no effect (Fig. 2C). Moreover, miR-15b, -16, -195, and -424 did not decrease the luciferase activities of mutated Arl2‚Äì3 -UTR constructs."}
{"As shown in Fig. 2A, miR-145 suppressed more than 55% activity for Luc-MUC1-UTR compared with vector control;neither miR-206 nor miR-224 suppressed Luc-MUC1-UTR. Furthermore, the mutant miR-145 had no suppressive effect."}
{Luciferase activity of the 39 UTR ERK-Luc construct was decreased in the presence of miR-143, whereas luciferase activity of mutated -Luc construct was not affected.}
{"A 60% repression of the RhoB 3'UTR reporter was observed, Mutated seed sequences in both target sites in the RhoB 30UTR abolished the repression."}
{"A 60% repression of the RhoB 3'UTR reporter was observed, Mutated seed sequences in both target sites in the RhoB 30UTR abolished the repression."}
{Transfection of let-7c to HuH7 cells significantly inhibited the level of Luciferase/Renilla ratio when compared to pre-miR negative control in the presence of the wt 59 and 39 sites, as well as the long 39UTR fragment (Figure 6F). Mutations of either of these two sites considerably reduced luciferase repression by let-7c. }
{Transfection of let-7c to HuH7 cells significantly inhibited the level of Luciferase/Renilla ratio when compared to pre-miR negative control in the presence of the wt 59 and 39 sites, as well as the long 39UTR fragment (Figure 6F). Mutations of either of these two sites considerably reduced luciferase repression by let-7c. }
{Transient transfec- tion of SW620 cells (low endogenous miR-141 expression) with pGL3-SIP1 reporter construct (not pGL3-SIP1-mut) and pre-miR-141 led to a signiÔ¨Åcant decrease of reporter activity as compared with the control .}
{Transient transfec- tion of SW620 cells (low endogenous miR-141 expression) with pGL3-SIP1 reporter construct (not pGL3-SIP1-mut) and pre-miR-141 led to a signiÔ¨Åcant decrease of reporter activity as compared with the control .}
{Indeed, Renilla luciferase activity decreased by 50% compared with non-targeting control transfected cells. In addition, site-directed mutagenesis of the seed region abolished the inhibitory effect ofmiR-27b on Renilla luciferase activity .}
{Co-transfection of H293 cells with miR-182 and psiCHECK2 containing the complete RARG 3'-UTR was signiÔ¨Åcantly decreased compared to cells co-transfected either with miR-182 and psiCHECK2 containing RARG 3'-UTR in which the region containing the predicted miR-182 target sequence was deleted.}
{The single mutants both still were responsive to miR-155 but at slightly higher concentrations than those of the wild-type 3 UTR (Fig. 4C). The double mutant was completely unresponsive to miR-155, indicating that both sites in the JARID2 3 ' UTR are direct targets of miR-155 (Fig. 4C).}
{The single mutants both still were responsive to miR-155 but at slightly higher concentrations than those of the wild-type 3 UTR (Fig. 4C). The double mutant was completely unresponsive to miR-155, indicating that both sites in the JARID2 3 ' UTR are direct targets of miR-155 (Fig. 4C).}
{Cotransfection of the catenin-wt construct and pre-miR-200a resulted in significantly decreased luciferase activity compared to transfection with pre-control 1, whereas luciferase activity was not significantly reduced in cells cotransfected with  catenin-mt and pre-miR-200a.}
{As a result, the intensity of EGFP fluorescence in pcDNA3B‚ÅÑ pri-miR-9-treated cells is significantly lower than that in the control vector group (Fig. 3B). Similarly, we constructed another EGFP reporter vector containing the mutational NF-jB1 3'-UTR (Fig. 3A). It was shown that overexpression of miR-9 could not affect the intensity of EGFP fluorescence in this 3'-UTR mutant vector.}
{It demonstrated that miR-155 still efficiently inhibited luciferase activity after abrogation of the first seed while it had no inhibitory effect on the activity of MUT2.}
{It demonstrated that miR-155 still efficiently inhibited luciferase activity after abrogation of the first seed while it had no inhibitory effect on the activity of MUT2.}
{In agreement with previous data, miR-155 significantly inhibited the luciferase activity of the 2 constructs, the duplicated site construct being even more sensitive than the wild type 3'UTR KGF construct.}
{In agreement with previous data, miR-155 significantly inhibited the luciferase activity of the 2 constructs, the duplicated site construct being even more sensitive than the wild type 3'UTR KGF construct.}
{The results indicated that let-7e suppressed while as-let-7e enhanced luciferase expression and activity, indicating a direct interaction between let-7e and the 3‚Ä≤UTR of TLR4 (Figure 4C). Mutation at the seed sequence of the TLR4-3‚Ä≤UTR where let-7e was predicted to bind (mutTLR4-UTR-luc) abolished suppression by let-7e (Figure 4C).}
{The results indicated that miR-155 suppressed while as-miR-155 enhanced luciferase expression and activity while mutation of the seed sequence for miR-155 at the 3‚Ä≤UTR of SOCS1.}
{Because cotransfection with either miR-200c (Fig. 6E), miR-200b, or miR-429 (data not shown) and wild-type 3‚Ä≤-UTR/ERRFI-1 significantly reduced the luciferase activity in UMUC3 and T24 and because the double-mutant constructs were able to rescue this activity, we concluded that ERRFI-1 is a direct target of the miR-200 in these cells (Fig. 6E).}
{Because cotransfection with either miR-200c (Fig. 6E), miR-200b, or miR-429 (data not shown) and wild-type 3‚Ä≤-UTR/ERRFI-1 significantly reduced the luciferase activity in UMUC3 and T24 and because the double-mutant constructs were able to rescue this activity, we concluded that ERRFI-1 is a direct target of the miR-200 in these cells (Fig. 6E).}
{Because cotransfection with either miR-200c (Fig. 6E), miR-200b, or miR-429 (data not shown) and wild-type 3‚Ä≤-UTR/ERRFI-1 significantly reduced the luciferase activity in UMUC3 and T24 and because the double-mutant constructs were able to rescue this activity, we concluded that ERRFI-1 is a direct target of the miR-200 in these cells (Fig. 6E).}
{luciferase expression from reporter constructs derived from BCL2 was significantly suppressed by synthetic miRNA and increased by anti-miR transfections (mutant constructs were unresponsive).}
{luciferase expression from reporter constructs derived from BCL2 was significantly suppressed by synthetic miRNA and increased by anti-miR transfections (mutant constructs were unresponsive).}
{The MEF2D wildtype constructs produced significant suppression by each miRNA and anti-miR transfection also resulted in a significant increase in luciferase expression. }
{The MEF2D wildtype constructs produced significant suppression by each miRNA and anti-miR transfection also resulted in a significant increase in luciferase expression. }
{the wildtype MAP3K12 MREs displayed significant luciferase suppression by the synthetic miRNA and was increased by anti-miR transfections (with the exception of the combined anti-miR). Mutant MREs were all unresponsive except miR-20a.}
{the wildtype MAP3K12 MREs displayed significant luciferase suppression by the synthetic miRNA and was increased by anti-miR transfections (with the exception of the combined anti-miR). Mutant MREs were all unresponsive except miR-20a.}
{As shown in Figure 4B, co-transfection with miR-210 but not miR-185 specifically decreased luciferase levels from the reporter. This effect requires the miR-210 seed region as the miR-210 mutant duplex is much less effective in reducing reporter activity.}
{As shown in Figure 5B, we observed a marked reduction in luciferase activity in cells transfected with miR-155 mimics and luciferase report vectors (P <.05). In contrast, no change of luciferase was observed in cells transfected with the mutant 3 -UTR constructs or miRcontrol.}
{As shown in Figure 5B, we observed a marked reduction in luciferase activity in cells transfected with miR-155 mimics and luciferase report vectors (P <.05). In contrast, no change of luciferase was observed in cells transfected with the mutant 3 -UTR constructs or miRcontrol.}
{As shown in Figure 5B, we observed a marked reduction in luciferase activity in cells transfected with miR-155 mimics and luciferase report vectors (P <.05). In contrast, no change of luciferase was observed in cells transfected with the mutant 3 -UTR constructs or miRcontrol.}
{Transfection of 293T cells with Œ≤TrCP1Œ¥miR-183 resulted in higher levels of expression of Œ≤TrCP1 mRNA and protein, as compared with the wild-type.}
{The relative luciferase activity of the WT construct of DNMT3A 30UTR in both the colon cancer cells was significantly reduced in the presence of miR-143.whereas such a suppressive effect of miR-143 on luciferase activity was not observed in both cells with the MUT construct of DNMT3A 3'UTR.}
{cotransfected with premiR-7 into HEK293T cells. miR-7 significantly decreased luciferase activity from  Œ±-Syn-3 -UTR construct in a dose-dependent manner}
{"Similarly, the Bmi-1 3‚Ä≤UTR (OV-202 and CP-70) luciferase activity was also significantly lower in the miR-15a‚Äì and miR-16‚Äìtransfectedcells compared with the control (Fig. 4).Mutation of the single miR-16 site rescued the luciferase activity, thus confirming a directinteraction of miR-16 with the 3‚Ä≤UTR of Bmi-1 mRNA."}
{Similarly, the Bmi-1 3‚Ä≤UTR (OV-202 and CP-70) luciferase activity was also significantly lower in the miR-15a‚Äì and miR-16‚Äìtransfected cells compared with the control (Fig. 4) However, mutation of the twomiR-15a sites independently rescued the luciferase activity partially (Fig. 4).}
{Similarly, the Bmi-1 3‚Ä≤UTR (OV-202 and CP-70) luciferase activity was also significantly lower in the miR-15a‚Äì and miR-16‚Äìtransfected cells compared with the control (Fig. 4) However, mutation of the twomiR-15a sites independently rescued the luciferase activity partially (Fig. 4).}
{"miR-125a reduced the activity of a luciferase reporter fused to the wild-type HuR 3'UTR by 50% (Fig. 6B and HuR3'UTR-WT). Moreover,deletion of the 7-nt sequence in the HuR 3'UTR complementary to the miR-125a seed sequence restored the luciferase activity of miR-125a transfected cells from 50% to 87% (Fig. 6B and HuR3'UTR-mut)."}
{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, the observed luciferase/Renilla reduction was abrogated when we cotransfected a mutated luciferase reporter vector containing the 3 UTR of MCL-1, CXXC6, and CDK6 with 4 deleted nucleotides at the site of interactions with miR-29 (Figure 5E-G).}
{Furthermore, the observed luciferase/Renilla reduction was abrogated when we cotransfected a mutated luciferase reporter vector containing the 3 UTR of MCL-1, CXXC6, and CDK6 with 4 deleted nucleotides at the site of interactions with miR-29 (Figure 5E-G).}
{Furthermore, the observed luciferase/Renilla reduction was abrogated when we cotransfected a mutated luciferase reporter vector containing the 3 UTR of MCL-1, CXXC6, and CDK6 with 4 deleted nucleotides at the site of interactions with miR-29 (Figure 5E-G).}
{Furthermore, the observed luciferase/Renilla reduction was abrogated when we cotransfected a mutated luciferase reporter vector containing the 3 UTR of MCL-1, CXXC6, and CDK6 with 4 deleted nucleotides at the site of interactions with miR-29 (Figure 5E-G).}
{Furthermore, the observed luciferase/Renilla reduction was abrogated when we cotransfected a mutated luciferase reporter vector containing the 3 UTR of MCL-1, CXXC6, and CDK6 with 4 deleted nucleotides at the site of interactions with miR-29 (Figure 5E-G).}
{Furthermore, the observed luciferase/Renilla reduction was abrogated when we cotransfected a mutated luciferase reporter vector containing the 3 UTR of MCL-1, CXXC6, and CDK6 with 4 deleted nucleotides at the site of interactions with miR-29 (Figure 5E-G).}
{Furthermore, the observed luciferase/Renilla reduction was abrogated when we cotransfected a mutated luciferase reporter vector containing the 3 UTR of MCL-1, CXXC6, and CDK6 with 4 deleted nucleotides at the site of interactions with miR-29 (Figure 5E-G).}
{As expected the normalized luciferase activity of Luc-reg-15/16 was reduced significantly by 58.99% ¬± 6.12 and 66.73% ¬± 4.44 upon addition of miR-16-1 and miR-15a precursors respectively whereas the non-cognate control miR-95 did not alter the luc expression. Moreover, the Luc-reg16mut plasmid was insensitive to miR-15/16 introduction confirming the specificity of the microRNA::target interaction (Fig. 3B, left).}
{As expected the normalized luciferase activity of Luc-reg-15/16 was reduced significantly by 58.99% ¬± 6.12 and 66.73% ¬± 4.44 upon addition of miR-16-1 and miR-15a precursors respectively whereas the non-cognate control miR-95 did not alter the luc expression. Moreover, the Luc-reg16mut plasmid was insensitive to miR-15/16 introduction confirming the specificity of the microRNA::target interaction (Fig. 3B, left).}
{The introduction of a region containing a highly conserved miR-17/20a binding site (Luc-reg-17/20a) led to a significant reduction of luciferase activity [-21.46% ¬± 5.44 in cells co-transfected with the CMV-miR-17 cluster plasmid (a kind gift from Joshua Mendell)]. This reduction was completely rescued by the mutation of the putative ‚Äòseed‚Äô sequence in the miRNA binding sites (Fig. 3B, right).}
{The introduction of a region containing a highly conserved miR-17/20a binding site (Luc-reg-17/20a) led to a significant reduction of luciferase activity [-21.46% ¬± 5.44 in cells co-transfected with the CMV-miR-17 cluster plasmid (a kind gift from Joshua Mendell)]. This reduction was completely rescued by the mutation of the putative ‚Äòseed‚Äô sequence in the miRNA binding sites (Fig. 3B, right).}
{The 5 UTR of RIP140 mRNA significantly elevated reporter activity 6-fold, relative to the control reporter (Figure 2A).}
{we measured changes after miR-24 cotransfection in luciferase activity from a MYC 30UTR reporter. miR-24 reduced luciferase activity 2.2-fold (Figure 4D).}
{we measured changes after miR-24 cotransfection in luciferase activity from a MYC 30UTR reporter. miR-24 reduced luciferase activity 2.2-fold (Figure 4D).}
{we measured changes after miR-24 cotransfection in luciferase activity from a MYC 30UTR reporter. miR-24 reduced luciferase activity 2.2-fold (Figure 4D).}
{we measured changes after miR-24 cotransfection in luciferase activity from a MYC 30UTR reporter. miR-24 reduced luciferase activity 2.2-fold (Figure 4D).}
{we measured changes after miR-24 cotransfection in luciferase activity from a MYC 30UTR reporter. miR-24 reduced luciferase activity 2.2-fold (Figure 4D).}
{we measured changes after miR-24 cotransfection in luciferase activity from a MYC 30UTR reporter. miR-24 reduced luciferase activity 2.2-fold (Figure 4D).}
{miR-24 significantly suppressed luciferase activity of a reporter gene containing the E2F2 MRE1.}
{"We selected eight genes (AURKB, BRCA1, CCNA2, CHEK1, CDC2, CDK4, FEN1, and PCNA) that play important roles in cell-cycle progressionand cloned their entire 3'UTRs into the luciferase reporter. The 30UTR of six out of eight genes (AURKB, BRCA1, CCNA2, CDC2, CDK4, and FEN1, but not CHEK1 or PCNA) was significantly repressed by miR-24, suggesting that these genes may be direct targets (Figure 6C)."}
{"We selected eight genes (AURKB, BRCA1, CCNA2, CHEK1, CDC2, CDK4, FEN1, and PCNA) that play important roles in cell-cycle progressionand cloned their entire 3'UTRs into the luciferase reporter. The 30UTR of six out of eight genes (AURKB, BRCA1, CCNA2, CDC2, CDK4, and FEN1, but not CHEK1 or PCNA) was significantly repressed by miR-24, suggesting that these genes may be direct targets (Figure 6C)."}
{"We selected eight genes (AURKB, BRCA1, CCNA2, CHEK1, CDC2, CDK4, FEN1, and PCNA) that play important roles in cell-cycle progressionand cloned their entire 3'UTRs into the luciferase reporter. The 30UTR of six out of eight genes (AURKB, BRCA1, CCNA2, CDC2, CDK4, and FEN1, but not CHEK1 or PCNA) was significantly repressed by miR-24, suggesting that these genes may be direct targets (Figure 6C)."}
{"We selected eight genes (AURKB, BRCA1, CCNA2, CHEK1, CDC2, CDK4, FEN1, and PCNA) that play important roles in cell-cycle progressionand cloned their entire 3'UTRs into the luciferase reporter. The 30UTR of six out of eight genes (AURKB, BRCA1, CCNA2, CDC2, CDK4, and FEN1, but not CHEK1 or PCNA) was significantly repressed by miR-24, suggesting that these genes may be direct targets (Figure 6C)."}
{"We selected eight genes (AURKB, BRCA1, CCNA2, CHEK1, CDC2, CDK4, FEN1, and PCNA) that play important roles in cell-cycle progressionand cloned their entire 3'UTRs into the luciferase reporter. The 30UTR of six out of eight genes (AURKB, BRCA1, CCNA2, CDC2, CDK4, and FEN1, but not CHEK1 or PCNA) was significantly repressed by miR-24, suggesting that these genes may be direct targets (Figure 6C)."}
{"We selected eight genes (AURKB, BRCA1, CCNA2, CHEK1, CDC2, CDK4, FEN1, and PCNA) that play important roles in cell-cycle progressionand cloned their entire 3'UTRs into the luciferase reporter. The 30UTR of six out of eight genes (AURKB, BRCA1, CCNA2, CDC2, CDK4, and FEN1, but not CHEK1 or PCNA) was significantly repressed by miR-24, suggesting that these genes may be direct targets (Figure 6C)."}
{However, miR-206 dose-dependently decreased the luciferase activity when cotransfected with the mouse notch3 3 -UTR containing the miR- 206 target site.}
{"As expected, miR-206 showed a strong inhibitory effect on normal (Fig. 2A, left) but not deleted (Fig. 2A, right) or mutated (supplemental Fig.S2B) human notch3 3 -UTR-luciferase activity."}
{with the wild-type reporter construct to gether with plasmid DpA-miR-128 led to a significant decrease of luciferase activity ( 40%) as compared to the control (Fig. 4A).}
{the presence of the wild-type sites was sufficient to significantly decrease luciferase activity (57%), while the mutated sites were inactive (Fig. 4B).}
{As indicated, introduction of miR-1 or miR-206 in HEK293 cells with the wild-type 3 -UTR (pLuc-MET 3UTR) construct had significant inhibition of luciferase activity as opposed to negative control (Fig. 4D).}
{As indicated, introduction of miR-1 or miR-206 in HEK293 cells with the wild-type 3 -UTR (pLuc-MET 3UTR) construct had significant inhibition of luciferase activity as opposed to negative control (Fig. 4D).}
{The luciferase activities of this reporter in miR-21 knockout cells were f60% higher than that in parental RKO cells but were suppressed by premiR-21 transfection (Fig. 4B).We then mutated the miR-21 binding site in the reporter construct Luc-Cdc25A-Mut-UTRand found its activities were similar in parental and miR-21 knockout RKO cells (Fig. 4).}
{As indicated, introduction of miR-1 or miR-206 in HEK293 cells with the wild-type 3 -UTR (pLuc-MET 3UTR) construct had significant inhibition of luciferase activity as opposed to negative control (Fig. 4D).}
{As indicated, introduction of miR-1 or miR-206 in HEK293 cells with the wild-type 3 -UTR (pLuc-MET 3UTR) construct had significant inhibition of luciferase activity as opposed to negative control (Fig. 4D).}
{Transient transfection of T47D cells, with a high endogenous miR-193b level, with the wild-type 30UTR-uPA-luc reporter and anti-193b inhibitor, led to a significant increase in reporter activity compared with control oligo (Figure 3b).However, miR-193b could no longer decrease the luciferase activity of a mutant construct‚Äîwith substitutions of four nucleotides in the miR-193b-binding site (Figure 3d).}
{When placed downstream of a luci-ferase reporter gene,this target sequence,but not its mutant version, was indeed repressed by miR-133b(Fig. 5A).}
{cotransfection with miR-181a significantly suppressed activity of the PLAG1-3 UTRluciferase construct by 43.6% and in miR-181b by 48.4% . }
{cotransfection with miR-181a significantly suppressed activity of the PLAG1-3 UTRluciferase construct by 43.6% and in miR-181b by 48.4% . }
{PLAG1-3 UTR-reporter construct was also significantly suppressed, with 28.6% activity reduction by miR-107 and 42.7% activity reduction by miR-424 .}
{PLAG1-3 UTR-reporter construct was also significantly suppressed, with 28.6% activity reduction by miR-107 and 42.7% activity reduction by miR-424 .}
{"we observed that miR-153 repressed the reporter activities based on the 30UTR regions of both Bcl-2 and Mcl-1 moderately but significantly (Figs. 4a and 4b). MiR-153 did not significantly alter the reporter activities based on the mutant 30UTR regions of these respective Bcl-2 and Mcl-1 fragments (Figs. 4a and 4b)."}
{"we observed that miR-153 repressed the reporter activities based on the 30UTR regions of both Bcl-2 and Mcl-1 moderately but significantly (Figs. 4a and 4b). MiR-153 did not significantly alter the reporter activities based on the mutant 30UTR regions of these respective Bcl-2 andMcl-1 fragments (Figs. 4a and 4b)."}
{PC-3 cells transiently transfected with the WT-3'-UTR-reporter construct and pre-miR-330 led to a significant decrease of reporter activity when compared with the control.}
{We found that forced expression of miR-181s resulted in decreased luciferase activity when the wild-type (WT) sequences were present (Fig 5D). This effect was significantly reduced when the corresponding miR-181 binding sites (five nucleotides within the complementary seed sequences) were mutated (MT) (Fig 5D).}
{We found that forced expression of miR-181s resulted in decreased luciferase activity when the wild-type (WT) sequences were present (Fig 5D). This effect was significantly reduced when the corresponding miR-181 binding sites (five nucleotides within the complementary seed sequences) were mutated (MT) (Fig 5D).}
{We found that forced expression of miR-181s resulted in decreased luciferase activity when the wild-type (WT) sequences were present (Fig 5D). This effect was significantly reduced when the corresponding miR-181 binding sites (five nucleotides within the complementary seed sequences) were mutated (MT) (Fig 5D).}
{We found that forced expression of miR-181s resulted in decreased luciferase activity when the wild-type (WT) sequences were present (Fig 5D). This effect was significantly reduced when the corresponding miR-181 binding sites (five nucleotides within the complementary seed sequences) were mutated (MT) (Fig 5D).}
{We found that forced expression of miR-181s resulted in decreased luciferase activity when the wild-type (WT) sequences were present (Fig 5D). This effect was significantly reduced when the corresponding miR-181 binding sites (five nucleotides within the complementary seed sequences) were mutated (MT) (Fig 5D).}
{We found that forced expression of miR-181s resulted in decreased luciferase activity when the wild-type (WT) sequences were present (Fig 5D). This effect was significantly reduced when the corresponding miR-181 binding sites (five nucleotides within the complementary seed sequences) were mutated (MT) (Fig 5D).}
{We found that forced expression of miR-181s resulted in decreased luciferase activity when the wild-type (WT) sequences were present (Fig 5D). This effect was significantly reduced when the corresponding miR-181 binding sites (five nucleotides within the complementary seed sequences) were mutated (MT) (Fig 5D).}
{We found that forced expression of miR-181s resulted in decreased luciferase activity when the wild-type (WT) sequences were present (Fig 5D). This effect was significantly reduced when the corresponding miR-181 binding sites (five nucleotides within the complementary seed sequences) were mutated (MT) (Fig 5D).}
{We found that forced expression of miR-181s resulted in decreased luciferase activity when the wild-type (WT) sequences were present (Fig 5D). This effect was significantly reduced when the corresponding miR-181 binding sites (five nucleotides within the complementary seed sequences) were mutated (MT) (Fig 5D).}
{We found that forced expression of miR-181s resulted in decreased luciferase activity when the wild-type (WT) sequences were present (Fig 5D). This effect was significantly reduced when the corresponding miR-181 binding sites (five nucleotides within the complementary seed sequences) were mutated (MT) (Fig 5D).}
{We found that forced expression of miR-181s resulted in decreased luciferase activity when the wild-type (WT) sequences were present (Fig 5D). This effect was significantly reduced when the corresponding miR-181 binding sites (five nucleotides within the complementary seed sequences) were mutated (MT) (Fig 5D).}
{We found that forced expression of miR-181s resulted in decreased luciferase activity when the wild-type (WT) sequences were present (Fig 5D). This effect was significantly reduced when the corresponding miR-181 binding sites (five nucleotides within the complementary seed sequences) were mutated (MT) (Fig 5D).}
{miR-34a overexpression reduced luciferase activity by 45%.Deletion of MRE2 or mutation of its seed in the context of the full-length 3 '-UTR significantly increased reporter luciferase activity (supplemental Figure 3B) but did not restore it to control levels.}
{miR-34a overexpression reduced luciferase activity by 45%.Deletion of MRE2 or mutation of its seed in the context of the full-length 3 '-UTR significantly increased reporter luciferase activity (supplemental Figure 3B) but did not restore it to control levels.}
{miR-34a overexpression reduced luciferase activity by 45%.Deletion of MRE2 or mutation of its seed in the context of the full-length 3 '-UTR significantly increased reporter luciferase activity (supplemental Figure 3B) but did not restore it to control levels.}
{However, cotransfection of B7-H3-WT with miR-29a significantly reduced luciferase activity, reducing by > 60% when compared to control level. This repression was reversed by a single base mutation in the binding site (B7-H3-mt).}
{Similarly, with the full-length ERBB-2 3‚Äô-UTR, the miR-331-3p precursor induced a significant decrease in reporter activity.In 22RV1 cells transfected with mutant A and B site reporters, the effect of miR-331-3p was eliminated, consistent with miR-331-3p binding directly to each of the two target sites (Figure 4B).}
{Similarly, with the full-length ERBB-2 3‚Äô-UTR, the miR-331-3p precursor induced a significant decrease in reporter activity.In 22RV1 cells transfected with mutant A and B site reporters, the effect of miR-331-3p was eliminated, consistent with miR-331-3p binding directly to each of the two target sites (Figure 4B).}
{As expected, the anti-miR-130a 29-O-methyl oligonucleotide had no effect on the translational efficiency of the reporter with a mutation of the A site (compare columns 4 & 5, Fig. 3E). In contrast, translational repression of the reporter construct was relieved in a dosedependent fashion with the addition of increasing amounts of antimiR-130a 29-O-methyl oligonucleotide, resulting in luciferase levels that were equivalent to those seen with the mutated A site in the FOG-2 UTR.}
{It was found that miR-21 decreased the luciferase activity of the reporter vector containing either the miR-21 response elements B12 or B34 [Fig. 3(A,B)].}
{It was found that miR-21 decreased the luciferase activity of the reporter vector containing either the miR-21 response elements B12 or B34 [Fig. 3(A,B)].}
{It was found that miR-21 decreased the luciferase activity of the reporter vector containing either the miR-21 response elements B12 or B34 [Fig. 3(A,B)].}
{It was found that miR-21 decreased the luciferase activity of the reporter vector containing either the miR-21 response elements B12 or B34 [Fig. 3(A,B)].}
{The results showed that the expression level of EGFP was significantly increased when endogenous miR-21 was blocked by miR-21 ASO (Fig. 3D). However, when the reporter vector containing a mutational miR-21 binding site was used, the increase in the level of EGFP was aborted.}
{We found that the expression of EGFP was strongly increased when miR-21 function was inhibited by transfecting cells with an antisense oligonucleotide (Figure 4B),The EGFP expression from the mutated vector, pcDNA3/EGFP-BTG2MUT, was not affected by miR-21, regardless of its expression levels (Figure 4B and 4C).}
{we transfected 4175 cells with the pMIR-ST14-wt along with the antisense miR-27b and assayed for luciferase activity. The cells showed enhancement in luciferase activity compared with control cells (without the oligomer) or to cells transfected with the control oligomer.}
{miR-216a or miR-217 mimics significantly inhibited Pten 3‚Ä≤UTR Luc activity relative to negative controls, while Pten 3‚Ä≤UTR containing mutations at miR-216a and/or miR-217 sites lost this response (Fig. 2k and Supplementary Fig. S4a).}
{miR-216a or miR-217 mimics significantly inhibited Pten 3‚Ä≤UTR Luc activity relative to negative controls, while Pten 3‚Ä≤UTR containing mutations at miR-216a and/or miR-217 sites lost this response (Fig. 2k and Supplementary Fig. S4a).}
{Moreover, pmiR-145, but not pDNR-CMV or pmiR-31 inhibited luciferase activity (Figure 6B). In the mutated control groups, the inhibitory effect of pmiR-145 on luciferase activity disappeared (Figure 6B).}
{As shown in Figure 3C, cotransfection of T98G, MDA-MB-468 and H1299 cells with 50 nM or 100 nM of the miR-30a mimic resulted in a 40‚Äì80% reduction in the activity of the reporter gene vector containing the wild-type miR-30a targeting sequences (psiCHECKTM2-WT-BECN-3'-UTR), in comparison to that of the vector with the deletion mutant (psiCHECKTM2-MTBECN-3'-UTR).}
{Our results demonstrate a decrease of the relative luciferase activity following ectopic expression of hsa-miR-127, thus suggesting that BLIMP-1 expression is directly regulated by this miRNA (Fig. 7a). As a further confirmation, no increase of the relative luciferase activity was observed when sequence mutants in the 30-UTR region of BLIMP-1 were cotransfected with the synthetic miRNA (Fig. 7b).}
{Reporter assays using miR-31-expressing 231 cells revealed that miR-31 repressed six of the UTRs: frizzled3 (Fzd3), integrin Œ±5 (ITGA5), myosin phosphatase-Rho interacting protein (M-RIP), matrix metallopeptidase 16 (MMP16), radixin (RDX), and RhoA (Figure 4A). Mutation of the putative miR-31 site(s) in these six 3‚Äô UTRs (Table S3) abrogated responsiveness to miR-31 (Figure 4B).}
{Reporter assays using miR-31-expressing 231 cells revealed that miR-31 repressed six of the UTRs: frizzled3 (Fzd3), integrin Œ±5 (ITGA5), myosin phosphatase-Rho interacting protein (M-RIP), matrix metallopeptidase 16 (MMP16), radixin (RDX), and RhoA (Figure 4A). Mutation of the putative miR-31 site(s) in these six 3‚Äô UTRs (Table S3) abrogated responsiveness to miR-31 (Figure 4B).}
{Reporter assays using miR-31-expressing 231 cells revealed that miR-31 repressed six of the UTRs: frizzled3 (Fzd3), integrin Œ±5 (ITGA5), myosin phosphatase-Rho interacting protein (M-RIP), matrix metallopeptidase 16 (MMP16), radixin (RDX), and RhoA (Figure 4A). Mutation of the putative miR-31 site(s) in these six 3‚Äô UTRs (Table S3) abrogated responsiveness to miR-31 (Figure 4B).}
{Reporter assays using miR-31-expressing 231 cells revealed that miR-31 repressed six of the UTRs: frizzled3 (Fzd3), integrin Œ±5 (ITGA5), myosin phosphatase-Rho interacting protein (M-RIP), matrix metallopeptidase 16 (MMP16), radixin (RDX), and RhoA (Figure 4A). Mutation of the putative miR-31 site(s) in these six 3‚Äô UTRs (Table S3) abrogated responsiveness to miR-31 (Figure 4B).}
{Reporter assays using miR-31-expressing 231 cells revealed that miR-31 repressed six of the UTRs: frizzled3 (Fzd3), integrin Œ±5 (ITGA5), myosin phosphatase-Rho interacting protein (M-RIP), matrix metallopeptidase 16 (MMP16), radixin (RDX), and RhoA (Figure 4A). Mutation of the putative miR-31 site(s) in these six 3‚Äô UTRs (Table S3) abrogated responsiveness to miR-31 (Figure 4B).}
{Reporter assays using miR-31-expressing 231 cells revealed that miR-31 repressed six of the UTRs: frizzled3 (Fzd3), integrin Œ±5 (ITGA5), myosin phosphatase-Rho interacting protein (M-RIP), matrix metallopeptidase 16 (MMP16), radixin (RDX), and RhoA (Figure 4A). Mutation of the putative miR-31 site(s) in these six 3‚Äô UTRs (Table S3) abrogated responsiveness to miR-31 (Figure 4B).}
{The relative luciferase activity was reduced by 50% following cotransfection with miR-210 compared with transfection with control RNA (Fig. 3B).When the targeted sequence of AcvR1b was mutated, the reduction of the luciferase activity by miR-210 was impaired (Fig. 3C).}
{Co-transfection of HEK293 cells with Luciferase construct (pMIR-TLR2) and a vector overexpressing miR-105 precursor (pMIF-miR-105) inhibited Luciferase activity (Fig. 7A, panel C). The mutant Luciferase vector (pMIRmutTLR2) co-transfected with pMIF-miR-105 retained luciferase activity (Fig. 7A, panelD).}
{GALNT1 and SOX4 both showed significant reduction in luciferase activity on transfection with pre-miR-129 compared with GALNT1 and SOX4 3¬∂-UTRs with mutated miR-129 target sites.}
{Interestingly, the relative luciferase activity was markedly diminished in cells cotransfected with the pMIR/618/ 5'-3'construct and miR-548d-3p, miR-559 or miR-548d-3p plus miR-559, especially miR-548d-3p plus miR-559 (50 nM final concentration, Fig. 3A).}
{Interestingly, the relative luciferase activity was markedly diminished in cells cotransfected with the pMIR/618/ 5'-3'construct and miR-548d-3p, miR-559 or miR-548d-3p plus miR-559, especially miR-548d-3p plus miR-559 (50 nM final concentration, Fig. 3A).}
{Indeed, miR-92a overexpression reduced luciferase activity (Fig.4C).Mutation of the target sequence prevented down-regulation of luciferase activity by pre-miR-92a (Fig. 4C).}
{The levels of luciferase activity in pGL3-Dlx5/miR-141 and -200a sensor-B or -D, which contain the 1072‚Äì1091 region of the 3 -UTR of Dlx5, were lower than those in the nonspecific control sensor vector (Fig. 5B).Onthe other hand, the levels of luciferase activity in pGL3-Dlx5/miR-141 and -200a sensor-A or -C, which lacks the 1072‚Äì1091 region of the 3 -UTR of Dlx5, were unchanged compared with those in the nonspecific control sensor vector (p   0.1). We further attempted to confirm these observations by using three different mutants of pGL3-Dlx5/miR-141 and -200a sensor-B. The levels of luciferase activity in cells transfected with the pGL3-Dlx5/miR-141 and -200a sensor-B (wild type) were significantly reduced, although in cells transfected with the mutants of seed region (Fig. 5C) the decrease in luciferase activities was canceled (Fig. 5D).}
{The levels of luciferase activity in pGL3-Dlx5/miR-141 and -200a sensor-B or -D, which contain the 1072‚Äì1091 region of the 3 -UTR of Dlx5, were lower than those in the nonspecific control sensor vector (Fig. 5B).Onthe other hand, the levels of luciferase activity in pGL3-Dlx5/miR-141 and -200a sensor-A or -C, which lacks the 1072‚Äì1091 region of the 3 -UTR of Dlx5, were unchanged compared with those in the nonspecific control sensor vector (p   0.1). We further attempted to confirm these observations by using three different mutants of pGL3-Dlx5/miR-141 and -200a sensor-B. The levels of luciferase activity in cells transfected with the pGL3-Dlx5/miR-141 and -200a sensor-B (wild type) were significantly reduced, although in cells transfected with the mutants of seed region (Fig. 5C) the decrease in luciferase activities was canceled (Fig. 5D).}
{Coexpression of pc3-miR-195 significantly suppressed firefly luciferase activity of the reporter with wildtype 3  UTR but not that of the mutant reporter (Fig.4A).}
{Coexpression of pc3-miR-195 significantly suppressed firefly luciferase activity of the reporter with wildtype 3  UTR but not that of the mutant reporter (Fig.4A).}
{Coexpression of pc3-miR-195 significantly suppressed firefly luciferase activity of the reporter with wildtype 3  UTR but not that of the mutant reporter (Fig.4A).}
{Coexpression of pc3-miR-195 significantly suppressed firefly luciferase activity of the reporter with wildtype 3  UTR but not that of the mutant reporter (Fig.4A).}
{Coexpression of pc3-miR-195 significantly suppressed firefly luciferase activity of the reporter with wildtype 3  UTR but not that of the mutant reporter (Fig.4A).}
{Co-expression of either miR-221 or miR-222 significantly (P < 0.05) suppressed the reporter activity compared to the control (Fig. 5C, wt). Mutations introduced into the predicted binding site almost eliminated this suppression.}
{Co-expression of either miR-221 or miR-222 significantly (P < 0.05) suppressed the reporter activity compared to the control (Fig. 5C, wt). Mutations introduced into the predicted binding site almost eliminated this suppression.}
{"Furthermore, co-transfection of HeLa cells with psiCHECK-UTRwt and miR-31 showed a significant decrease (48%) in reporter luciferase activitycompared with psiCHECK-UTRwt alone (Fig. 6A), while psiCHECK-UTRwt activity was not affected by co-transfection with miR-Ctrl."}
{As shown in Figure 4D, miR-124a selectively suppressed the luciferase activity driven by the wild-type 3 -UTR, but not the mutant form.}
{Subsequent luciferase assays revealed that miR-124a specifically suppressed the luciferase activity driven by the 3 '-UTR of MCP-1 mRNA (Figure 5B).}
{In the case of the WNT1 3 -UTR vector, miR-34a and let-7e, individually or in combination, but not the negative nontargeting miRNA, suppressed the luciferase activity by approximately 30% to 50% (Figure 4B).}
{In the case of the WNT1 3 -UTR vector, miR-34a and let-7e, individually or in combination, but not the negative nontargeting miRNA, suppressed the luciferase activity by approximately 30% to 50% (Figure 4B).}
{In the JAG1 3 '-UTR vector, both miR-21 and miR-34a, alone or in combination, suppressed luciferase activity by a modest but significant amount compared with the negative nontargeting miRNA.}
{It was found that co-transfection of the plasmid expressing miR-155 reduced the activity of the wild type 3 -UTR of the PU.1 reporter by  85%. The reporters that did not contain the seeding sequence for miR-155, both control and mutant exhibited no significant reduction in their Renilla luciferase activity, when co-transfected with the miR-155 expressing vector (Fig. 2A).}
{Lefties wild-type 3'UTRs (Lefty1-WT and Lefty2-WT) were specifically repressed by a synthetic miR-302, whereas this negative effect was absent when reporters lacking the seed-complementary sequence (Lefty1-Mut and Lefty2-Mut) were used (Figure 2C).}
{Lefties wild-type 3'UTRs (Lefty1-WT and Lefty2-WT) were specifically repressed by a synthetic miR-302, whereas this negative effect was absent when reporters lacking the seed-complementary sequence (Lefty1-Mut and Lefty2-Mut) were used (Figure 2C).}
{Co-transfection of miR-320 in H9c2 cells strongly inhibited the luciferase activity from the reporter construct containing the 3‚Ä≤UTR segment of Hsp20, whereas no effect was observed with a construct containing a mutated segment of Hsp20 3‚Ä≤UTR (seed sequence CAGCUUU was mutated to GACACAA, Fig. 5H).}
{This reporter system showed that the translational level of all five luciferase-UTR constructs was controlled by human miR-96, and that both mutations led to reduced silencing of luciferase expression compared to wild-type miR-96.}
{This reporter system showed that the translational level of all five luciferase-UTR constructs was controlled by human miR-96, and that both mutations led to reduced silencing of luciferase expression compared to wild-type miR-96.}
{This reporter system showed that the translational level of all five luciferase-UTR constructs was controlled by human miR-96, and that both mutations led to reduced silencing of luciferase expression compared to wild-type miR-96.}
{This reporter system showed that the translational level of all five luciferase-UTR constructs was controlled by human miR-96, and that both mutations led to reduced silencing of luciferase expression compared to wild-type miR-96.}
{This reporter system showed that the translational level of all five luciferase-UTR constructs was controlled by human miR-96, and that both mutations led to reduced silencing of luciferase expression compared to wild-type miR-96.}
{We used a luciferase assay with siRNAs mimicking the wildtype and mutant miR-96. The siRNAs were co-transfected into NIH 3T3 cells with a construct containing the 3‚Ä≤UTR of each candidate gene, either with the putative binding sites intact, or with the sites replaced by an EcoR1 site to disrupt binding. Five genes were validated as targets of miR-96: Aqp5, Celsr2, Myrip, Odf2 and Ryk (Fig. S3a,b).}
{We used a luciferase assay with siRNAs mimicking the wildtype and mutant miR-96. The siRNAs were co-transfected into NIH 3T3 cells with a construct containing the 3‚Ä≤UTR of each candidate gene, either with the putative binding sites intact, or with the sites replaced by an EcoR1 site to disrupt binding. Five genes were validated as targets of miR-96: Aqp5, Celsr2, Myrip, Odf2 and Ryk (Fig. S3a,b).}
{We used a luciferase assay with siRNAs mimicking the wildtype and mutant miR-96. The siRNAs were co-transfected into NIH 3T3 cells with a construct containing the 3‚Ä≤UTR of each candidate gene, either with the putative binding sites intact, or with the sites replaced by an EcoR1 site to disrupt binding. Five genes were validated as targets of miR-96: Aqp5, Celsr2, Myrip, Odf2 and Ryk (Fig. S3a,b).}
{We used a luciferase assay with siRNAs mimicking the wildtype and mutant miR-96. The siRNAs were co-transfected into NIH 3T3 cells with a construct containing the 3‚Ä≤UTR of each candidate gene, either with the putative binding sites intact, or with the sites replaced by an EcoR1 site to disrupt binding. Five genes were validated as targets of miR-96: Aqp5, Celsr2, Myrip, Odf2 and Ryk (Fig. S3a,b).}
{We used a luciferase assay with siRNAs mimicking the wildtype and mutant miR-96. The siRNAs were co-transfected into NIH 3T3 cells with a construct containing the 3‚Ä≤UTR of each candidate gene, either with the putative binding sites intact, or with the sites replaced by an EcoR1 site to disrupt binding. Five genes were validated as targets of miR-96: Aqp5, Celsr2, Myrip, Odf2 and Ryk (Fig. S3a,b).}
{Co-transfection of MCF-7 cells with a wild-type RTKN-3'UTR (RTKN-wt) reporter and the miR-145-overexpressing plasmid resulted in a significant decrease in luminescence, whereas mutation of the RTKN miR-145 seed region (RTKN-mu) rendered the reporter construct insensitive to inhibition by miR-145 (Fig. 3C).}
{Cotransfection with the miR-302 expression plasmid and subsequent luciferase assays revealed that both putative target sites indeed contribute to the suppressive effect of miR-302 (Fig. 3B).}
{Cotransfection with the miR-302 expression plasmid and subsequent luciferase assays revealed that both putative target sites indeed contribute to the suppressive effect of miR-302 (Fig. 3B).}
{We found a direct effect of miR-92-1 on VHL 3' UTR with significant repression of luciferase activity (approximately 60%) by miR-92-1 compared with control vectors, a mutated target mRNAsequence, or a miRNAthat has no interaction site with VHL (miR-16; Figure 3C).}
{we blocked endogenous miR-34a using inhibitor antisense RNA oligo. 5ULuc3U expression was greater than that of 5ULuc or Luc3U, suggesting that the 5'-UTR of AXIN2 together with the 3'-UTR are functional target sites for miR-34a in the cells (Fig. 2D).}
{we blocked endogenous miR-34a using inhibitor antisense RNA oligo. 5ULuc3U expression was greater than that of 5ULuc or Luc3U, suggesting that the 5'-UTR of AXIN2 together with the 3'-UTR are functional target sites for miR-34a in the cells (Fig. 2D).}
{"We found that ectopic expression of miR-451 inhibited the expression of the reporter vector containing MIF 3'UTR but not the reporter vectorcontaining the mutation of the seed‚ÄìmiR-451 binding site (Fig. 5D)."}
{Transient transfection of HEK-293T cells, with a low endogenous miR-21 expression, with the wildtype-3'-UTR reporter plasmid and miR-21 mimics led to a significant decrease in reporter activity as compared to the control (Fig. 4A).}
{The fact that expression from the 3 UTR reporter corresponding to the parental ADAM17 sequences (but not from one with a mutated binding sequences, ADAM17-M) had significantly suppressed luciferase activity in miR-122 transfected 293T cells confirmed the authenticity of this binding site (Fig. 4B).}
{Aggregate data (Figure 5A left) reveal an 86% increase in NFAT1 protein expression when the cells were treated with antisense to miR-184.}
{the miR-497 mimic administered at a concentration of 50 nM decreases luciferase activity of the reporter vector containing miR-497 binding sequences (Fig. 4). As expected the miR-497 mimic has no significant effect on the reporter vector with mutated miR-497 binding sequences.}
{Co-transfections of miR-10a mimic with the HDAC4‚Äì3 -UTR reporter resulted in the repression of luciferase activity when compared with the mimic negative control (p 0.05) (Fig. 2B). On the other hand, the HDAC4-mut-3 -UTR reporter was not repressed by miR-10a mimic}
{As indicated in Fig. 2B, the introduction of miR-133a mimic decreased luciferase activity 40% compared with the cells treated with a control mimic (P   0.05). In a parallel experiment, the inhibitory effect of miR-133a in cells transfected with the mutant reporter vector (the two putative targeting sites were mutated) was completely abolished,}
{"Luciferase analysis revealed that the activities of three wild type 3'UTR reporters were significantly suppressed by miR-129, but not by miR-410(Fig. 4B). Suppression by miR-129 depends on the wild type miR-129 complementary sites, and was no longer observed in reporters in which miR-129 complementary sites were mutated (Fig. 4B)."}
{Introduction of miR-148a precursor into PC3 cells decreased relative luciferase activity of the reporter gene containing the MSK1 3 -UTR WT compared with a control precursor, while that of the reporter gene containing the MSK1 3 -UTRMTwas not affected by miR-148a (Fig. 5A).}
{miR-17/20 repressed IL-8 luciferase reporter gene activity, and mutation of themiR-17/20 binding site of the IL-8 3‚Ä≤ UTR reduced the repression (Fig. 5 B and C).}
{Indeed, mutant Peli1 reporter levels in cultured cells did not decrease upon pre-miR-21 cotransfections(Fig. 4)}
{As indicated in Fig. 2B, the introduction of miR-133a mimic decreased luciferase activity 40% compared with the cells treated with a control mimic (P   0.05). In a parallel experiment, the inhibitory effect of miR-133a in cells transfected with the mutant reporter vector (the two putative targeting sites were mutated) was completely abolished,}
{As indicated in Fig. 2B, the introduction of miR-133a mimic decreased luciferase activity 40% compared with the cells treated with a control mimic (P   0.05). In a parallel experiment, the inhibitory effect of miR-133a in cells transfected with the mutant reporter vector (the two putative targeting sites were mutated) was completely abolished,}
{"The recipient cell line Caco-2 (derived from colon carcinoma) endogenously expressed all three groups of miRNAs (Figure 2). Luciferase activity was strongly suppressed when the reporter gene was linked to the wild-type RECK 30UTR (30RECK-wt) (Figure 1e, bar 2). This effect was relievedwhen the predicted miRNA-target sites were mutated (Figure 1e and Supplementary Figure 1),"}
{"The recipient cell line Caco-2 (derived from colon carcinoma) endogenously expressed all three groups of miRNAs (Figure 2). Luciferase activity was strongly suppressed when the reporter gene was linked to the wild-type RECK 30UTR (30RECK-wt) (Figure 1e, bar 2). This effect was relievedwhen the predicted miRNA-target sites were mutated (Figure 1e and Supplementary Figure 1),"}
{"The recipient cell line Caco-2 (derived from colon carcinoma) endogenously expressed all three groups of miRNAs (Figure 2). Luciferase activity was strongly suppressed when the reporter gene was linked to the wild-type RECK 30UTR (30RECK-wt) (Figure 1e, bar 2). This effect was relievedwhen the predicted miRNA-target sites were mutated (Figure 1e and Supplementary Figure 1),"}
{"The recipient cell line Caco-2 (derived from colon carcinoma) endogenously expressed all three groups of miRNAs (Figure 2). Luciferase activity was strongly suppressed when the reporter gene was linked to the wild-type RECK 30UTR (30RECK-wt) (Figure 1e, bar 2). This effect was relievedwhen the predicted miRNA-target sites were mutated (Figure 1e and Supplementary Figure 1),"}
{"The recipient cell line Caco-2 (derived from colon carcinoma) endogenously expressed all three groups of miRNAs (Figure 2). Luciferase activity was strongly suppressed when the reporter gene was linked to the wild-type RECK 30UTR (30RECK-wt) (Figure 1e, bar 2). This effect was relievedwhen the predicted miRNA-target sites were mutated (Figure 1e and Supplementary Figure 1),"}
{Cotransfection of a plasmid expressing miR-33 resulted in a 50‚Äì60% decrease in luciferase activity when the reporter plasmid contained the human/mouse box 1 sequence from ABCA1 or the mouse ABCG1 sequence(Fig. 2D, lanes 5, 6, 9, and 10). The specificity of this effect issupported by the finding of no repression when the reporter gene contained either sequences corresponding to box 2 of ABCA1 (Fig. 2D), or mutant box 1 or mutant mouse ABCG1 sequences}
{Cotransfection of a plasmid expressing miR-33 resulted in a 50‚Äì60% decrease in luciferase activity when the reporter plasmid contained the human/mouse box 1 sequence from ABCA1 or the mouse ABCG1 sequence(Fig. 2D, lanes 5, 6, 9, and 10). The specificity of this effect issupported by the finding of no repression when the reporter gene contained either sequences corresponding to box 2 of ABCA1 (Fig. 2D), or mutant box 1 or mutant mouse ABCG1 sequences}
{Cotransfection of a plasmid expressing miR-33 resulted in a 50‚Äì60% decrease in luciferase activity when the reporter plasmid contained the human/mouse box 1 sequence from ABCA1 or the mouse ABCG1 sequence(Fig. 2D, lanes 5, 6, 9, and 10). The specificity of this effect issupported by the finding of no repression when the reporter gene contained either sequences corresponding to box 2 of ABCA1 (Fig. 2D), or mutant box 1 or mutant mouse ABCG1 sequences}
{The relative luciferase activity of the WT construct of the KRAS 3#-UTR in PANC-1 cells was significantly reduced in the presence of pre-miR-217 (P , 0.05); the relative luciferase activity of the WT construct of the KRAS 3#-UTR in MIAPaCa-2 cells was significantly increased in the presence of anti-miR-217 (P , 0.05). However, this effect was not observed in either cell line carrying the MUT construct of the KRAS 3#-UTR, highlighting the direct and specific interaction of miR-217 with the KRAS 3#-UTR (Figure 4D).}
{found that wild-type CTLA-4 39-UTR luciferase activity was significantly (P < .01) suppressed by cotransfected miR-155 (premiR-155) in comparison with nontargeting miRNA (control;Fig 5, B). Moreover, significantly (P < .01) lower luciferase activitywas detected in the cells transfected by the wild-type CTLA-4 construct relative to the constructs containing the mutated miR-155 seed region, indicating that the CTLA-4 transcript is a direct target of miR-155 (Fig 5, B).}
{"Cells cotransfected with pcDNA3-miR-34a and luciferase linked to the FOXP1-30UTR showed repression of luciferase compared to control (Figure 3B). When the conserved site in the FOXP1 30UTR was mutated (as depicted in Figure 3A), luciferase expressionwas derepressed (Figure 3B; FOXP1mt)."}
{Similarly, co-transfection of miR-451 resulted in greater reduction of the luciferase activity, compared to miR controls (Fig. 4E).}
{Similarly, co-transfection of miR-451 resulted in greater reduction of the luciferase activity, compared to miR controls (Fig. 4E).}
{"When Faslg-luc was cotransfected with miR-21 mimic into 293T-HEK cells, the luciferase activity was attenuated by 43 ¬± 13% compared to cellscotransfected with miR-67 mimic (n=4; P<0.02; Fig. 3C).To confirm the putative miR-21 binding site, we repeated the above experiment with a mutated Faslg 3‚Ä≤UTR. The sequence differed from the Faslg-luc in that the 9-mer putative binding site was scrambled (Mut-luc; see methods for sequence). After transfection with Mut-luc, there was no statistically significant difference in any experimental group"}
{"Luciferase activity of cells transfected with miR-122a was decreased by 19% as compared with NC (Fig. 4B);To test whether miR-122a regulated luciferase activity of50-UTR of PRDXII through its binding to the predictedtarget site, we generated the mutated reporter vector inwhich the predicted target sites were mutated in PRDXII50-UTR. We found that mutation in the predicted target sitesabolished the repressive effects of miR-122a on luciferaseactivity (Fig. 4C),"}
{"When the WT-p53 reporter was transfected along withmiR-380 into human embryonic kidney cells, luciferase activity wasdecreased ~30% compared to cells transfected with a control vector(Fig. 3d). Suppression of luciferase activity was not observed whenthe MUT-p53 reporter was transfected with miR-380"}
{Using the luciferase reporter construct containing the FBXW7 3‚Ä≤-UTR, we confirmed that expression of miR-25, miR-27a, and miR-223 repressed reporter activity in HCT116 cells, We confirmed both the predicted binding sites for miR-25 and miR-223 within the FBXW7 3‚Ä≤-UTR by showing that mutation of each of these sites relieved repression of reporter activity by the corresponding miRNA but not the other (Fig. 2, A and B). Thus, miR-25 and miR-223 are able to bind sites within the FBXW7 3‚Ä≤-UTR.}
{Using the luciferase reporter construct containing the FBXW7 3‚Ä≤-UTR, we confirmed that expression of miR-25, miR-27a, and miR-223 repressed reporter activity in HCT116 cells, We confirmed both the predicted binding sites for miR-25 and miR-223 within the FBXW7 3‚Ä≤-UTR by showing that mutation of each of these sites relieved repression of reporter activity by the corresponding miRNA but not the other (Fig. 2, A and B). Thus, miR-25 and miR-223 are able to bind sites within the FBXW7 3‚Ä≤-UTR.}
{Reporter assay revealed reduction of miR-221/222 led to a marked increase of luciferase activity of pGL3-WT-PUMA-3'UTR plasmid, without change in luciferase activity of pGL3-MUT-PUMA-3'UTR plasmid (Fig. ‚Äã(Fig.2D).2D). }
{Reporter assay revealed reduction of miR-221/222 led to a marked increase of luciferase activity of pGL3-WT-PUMA-3'UTR plasmid, without change in luciferase activity of pGL3-MUT-PUMA-3'UTR plasmid (Fig. ‚Äã(Fig.2D).2D). }
{We normalised luciferase activity, taking the value for MDVT-150mut cells as 100%, as mutated gga-miR-150 did not repress activity in MDVT-150mut cells. The activity of the MYB WT reporter was specifically repressed by gga-miR-150 in PA9 and MSB-1T-150 cells (by up to about 80%) (Fig. ‚Äã(Fig.2B).2B). The same reporter activity patterns were observed in both systems, demonstrating that the observed pattern of target site usage resulted from intrinsic characteristics of chicken c-myb 3'UTR. We found that pmiR-150 did not repress the activity of the MYB mt1234 reporter or the MYB mt2 construct whereas the activities of MYB mt1, mt3 and mt4 were significantly repressed (by about 40 to 60%) (Fig. ‚Äã(Fig.3B).3B).}
{Cells cotransfected with MT-fLuc-dpt-3‚Ä≤UTR and control mir-92b vectors strongly expressed reporter luciferase (Fig. 3). Luciferase expression was markedly repressed in cells cotransfected with MT-fLuc-dpt-3‚Ä≤UTR and let-7 vectors. To verify that the identified let-7 sequence of the diptericin 3‚Ä≤UTR was responsible for its repression, we generated a MT-fLuc-dpt-3‚Ä≤UTRmutant where 20 nucleotides including the candidate let-7 binding site were deleted. This mutant luciferase reporter was not repressed in cells cotransfected with the let-7 vector }
{the relative luciferase activity was reduced by 50% following cotransfection with miR-155 mimics compared with transfection with miR-155 control. In contrast, no change of luciferase was observed in cells transfected with 3‚Ä≤MUT constructs or miR-155 inhibitors. }
{Results showed that luciferase activity was greatly decreased in the embryos injected with I300 RNA and TK-Rluc-dkk3 3‚Ä≤ UTR, compared to that of embryos injected with I300 RNA with a plasmid in which the luciferase cDNA did not contain 3‚Ä≤ UTR of dkk3 (TK-Rluc) (Figure 5C). If embryos received injection with I300 RNA using a plasmid in which the luciferase cDNA contained mutated sequences of dkk3 3‚Ä≤ UTR (TK-Rluc-mdkk3 3‚Ä≤ UTR) (Figure 5B), the luciferase activity was not responsive to miR-In300 }
{The luciferase signals for wild-type 3‚Ä≤-UTR sequences of both LATS2 and PPP2R2A were repressed by cotransfection of pre‚ÄìmiR-31 in ED-1 cells and increased by cotransfection of anti‚ÄìmiR-31. In contrast, engineered mutations of the miR-31‚Äìbinding site antagonized these effects (Figure ‚Äã(Figure6,6, C and D). These results indicated that miR-31 can directly bind to the 3‚Ä≤-UTR sequences of LATS2 and PPP2R2A. }
{The luciferase signals for wild-type 3‚Ä≤-UTR sequences of both LATS2 and PPP2R2A were repressed by cotransfection of pre‚ÄìmiR-31 in ED-1 cells and increased by cotransfection of anti‚ÄìmiR-31. In contrast, engineered mutations of the miR-31‚Äìbinding site antagonized these effects (Figure ‚Äã(Figure6,6, C and D). These results indicated that miR-31 can directly bind to the 3‚Ä≤-UTR sequences of LATS2 and PPP2R2A. }
{Our results showed that overexpression of miR-1 significantly inhibited the activity of Pim-1 3‚Ä≤ UTR luciferase reporter gene (Fig. 5E). More specifically, mutations introduce into the miR-1 seed sequences resulted in loss of inhibition by miR-1 (Fig. 5E). }
{We found that co-transfection with miR-31 did not affect the luciferase activity of the original empty vector control, while coexpression of miR-31 with the reporter containing SATB2 3‚Ä≤UTR reduced the luciferase activity (Fig. 5B). To identify whether both predicted binding sites are functional, we cloned each of them separately into the same reporter vector. To prove the specificity of the miR-31 effect, we also deleted several base pairs in the seed regions of the miR-31 binding sites. The results show that each of the wild-type binding sites (but not the mutated sequence) is capable of reducing luciferase activity when co-transfected with miR-31 (Fig. 5B). These data confirm that SATB2 is a direct target of miR-31.}
{This was further confirmed by the luciferase reporter assay wherein miR-29a significantly inhibited the luciferase activity of the reporter vector containing the PI3Kp85Œ± wild type 3‚Ä≤UTR (Fig. 6C). This inhibition was not observed with the reporter containing the PI3Kp85Œ± mutated 3‚Ä≤UTR that indicates the specificity of the miR-29a-PI3Kp85Œ± 3‚Ä≤UTR interaction.}
{We compared the ASF-UTR GFP activity in HEK293 recipient cells with equivalent constructs where either the miR-28 (mut-28) or the miR-505 (mut-505) seed sites were mutated. Inactivation of either miR-28 or miR-505 binding sites in the ASF-UTR significantly increases GFP expression, indicating a direct binding of these miRNAs on ASF/SF2 3‚Ä≤-UTR (Fig. 3d).}
{We compared the ASF-UTR GFP activity in HEK293 recipient cells with equivalent constructs where either the miR-28 (mut-28) or the miR-505 (mut-505) seed sites were mutated. Inactivation of either miR-28 or miR-505 binding sites in the ASF-UTR significantly increases GFP expression, indicating a direct binding of these miRNAs on ASF/SF2 3‚Ä≤-UTR (Fig. 3d).}
{As shown in Fig. 6 B, transfected miR-125b duplex drastically inhibited the reporter activity, whereas it had no effect on the control reporter without the 3‚Ä≤ UTR (vector). Most importantly, when the predicted miR-125b seed region in the 3‚Ä≤ UTR was mutated, the mutant reporter no longer responded to miR-125b (Fig. 6 B), strongly suggesting that the predicted site is a bona fide target of miR-125b and it is solely responsible for miR-125b targeting of the Igf2 3‚Ä≤ UTR.}
{miR-98 significantly reduced the activity of D2-3UTR compared to that of D2-3UTRm in cardiomyocytes, whereas the control vector did not (Fig. 7I). }
{Co-transfection of the pAck1 3‚ÄôUTR-wt construct (453‚Äì840 nt) and pre-miR-7 resulted in significantly decreased luciferase activity compared to transfection with pre-control 1 (Fig. 4D). We next investigated the relative contribution of each putative miR-7 target site in the Ack1 3‚ÄôUTR. miR-7 reduced the expression of all three reporters carrying the different putative target sites A and B, and C, but not of the corresponding mutant (mt) reporters (Fig. 4E). }
{The reporter assay showed that miR-15a was able to significantly repress luciferase expression (Fig. 4C), and mutation of 4 nt in the miR-15a target sequence led to complete abrogation the suppressive effect (Fig. 4C). }
{In Drosophila S2 cultured cells, overexpression of miR-310 cluster caused a significant repression of a luciferase sensor carrying the entire Khc-73 3‚Ä≤-UTR (p=0.00002), indicating that Khc-73 is sensitive to the activity of the miR-310 cluster (Figure 3B). We tested the importance of the predicted conserved miR-310 cluster sites on Khc-73 3‚Ä≤-UTR by mutating all three conserved sites (see Experimental Procedures); these mutations abolished the sensitivity of the luciferase sensor to the miR-310 cluster (Figure 3B). }
{In Drosophila S2 cultured cells, overexpression of miR-310 cluster caused a significant repression of a luciferase sensor carrying the entire Khc-73 3‚Ä≤-UTR (p=0.00002), indicating that Khc-73 is sensitive to the activity of the miR-310 cluster (Figure 3B). We tested the importance of the predicted conserved miR-310 cluster sites on Khc-73 3‚Ä≤-UTR by mutating all three conserved sites (see Experimental Procedures); these mutations abolished the sensitivity of the luciferase sensor to the miR-310 cluster (Figure 3B). }
{In Drosophila S2 cultured cells, overexpression of miR-310 cluster caused a significant repression of a luciferase sensor carrying the entire Khc-73 3‚Ä≤-UTR (p=0.00002), indicating that Khc-73 is sensitive to the activity of the miR-310 cluster (Figure 3B). We tested the importance of the predicted conserved miR-310 cluster sites on Khc-73 3‚Ä≤-UTR by mutating all three conserved sites (see Experimental Procedures); these mutations abolished the sensitivity of the luciferase sensor to the miR-310 cluster (Figure 3B). }
{In Drosophila S2 cultured cells, overexpression of miR-310 cluster caused a significant repression of a luciferase sensor carrying the entire Khc-73 3‚Ä≤-UTR (p=0.00002), indicating that Khc-73 is sensitive to the activity of the miR-310 cluster (Figure 3B). We tested the importance of the predicted conserved miR-310 cluster sites on Khc-73 3‚Ä≤-UTR by mutating all three conserved sites (see Experimental Procedures); these mutations abolished the sensitivity of the luciferase sensor to the miR-310 cluster (Figure 3B). }
{In this case, co-transfection of the miR-132 mimic, but not the microRNA Negative Control, caused a subtle (~5‚Äì10%) reduction in luciferase activity, which we attributed as a non-sequence-specific effect of miR-132 overexpression in vitro. Most importantly, the wild-type 3‚Ä≤UTRs of all genes examined were significantly repressed by the miR-132 mimic, with a range of repression between 28 and 50% (Fig. 1D). In all cases except for Jarid1a, the effects of the miR-132 mimic were abolished by the mutation of the seed site (Fig. 1D). For Jarid1a, which bears two putative miR-132 MREs within its 3‚Ä≤UTR, mutation of the first MRE (site 1), but not the second (site 2), abolished the repressive effects of the miR-132 mimic (Fig. 1D).}
{In this case, co-transfection of the miR-132 mimic, but not the microRNA Negative Control, caused a subtle (~5‚Äì10%) reduction in luciferase activity, which we attributed as a non-sequence-specific effect of miR-132 overexpression in vitro. Most importantly, the wild-type 3‚Ä≤UTRs of all genes examined were significantly repressed by the miR-132 mimic, with a range of repression between 28 and 50% (Fig. 1D). In all cases except for Jarid1a, the effects of the miR-132 mimic were abolished by the mutation of the seed site (Fig. 1D). For Jarid1a, which bears two putative miR-132 MREs within its 3‚Ä≤UTR, mutation of the first MRE (site 1), but not the second (site 2), abolished the repressive effects of the miR-132 mimic (Fig. 1D).}
{In this case, co-transfection of the miR-132 mimic, but not the microRNA Negative Control, caused a subtle (~5‚Äì10%) reduction in luciferase activity, which we attributed as a non-sequence-specific effect of miR-132 overexpression in vitro. Most importantly, the wild-type 3‚Ä≤UTRs of all genes examined were significantly repressed by the miR-132 mimic, with a range of repression between 28 and 50% (Fig. 1D). In all cases except for Jarid1a, the effects of the miR-132 mimic were abolished by the mutation of the seed site (Fig. 1D). For Jarid1a, which bears two putative miR-132 MREs within its 3‚Ä≤UTR, mutation of the first MRE (site 1), but not the second (site 2), abolished the repressive effects of the miR-132 mimic (Fig. 1D).}
{In this case, co-transfection of the miR-132 mimic, but not the microRNA Negative Control, caused a subtle (~5‚Äì10%) reduction in luciferase activity, which we attributed as a non-sequence-specific effect of miR-132 overexpression in vitro. Most importantly, the wild-type 3‚Ä≤UTRs of all genes examined were significantly repressed by the miR-132 mimic, with a range of repression between 28 and 50% (Fig. 1D). In all cases except for Jarid1a, the effects of the miR-132 mimic were abolished by the mutation of the seed site (Fig. 1D). For Jarid1a, which bears two putative miR-132 MREs within its 3‚Ä≤UTR, mutation of the first MRE (site 1), but not the second (site 2), abolished the repressive effects of the miR-132 mimic (Fig. 1D).}
{In this case, co-transfection of the miR-132 mimic, but not the microRNA Negative Control, caused a subtle (~5‚Äì10%) reduction in luciferase activity, which we attributed as a non-sequence-specific effect of miR-132 overexpression in vitro. Most importantly, the wild-type 3‚Ä≤UTRs of all genes examined were significantly repressed by the miR-132 mimic, with a range of repression between 28 and 50% (Fig. 1D). In all cases except for Jarid1a, the effects of the miR-132 mimic were abolished by the mutation of the seed site (Fig. 1D). For Jarid1a, which bears two putative miR-132 MREs within its 3‚Ä≤UTR, mutation of the first MRE (site 1), but not the second (site 2), abolished the repressive effects of the miR-132 mimic (Fig. 1D).}
{In this case, co-transfection of the miR-132 mimic, but not the microRNA Negative Control, caused a subtle (~5‚Äì10%) reduction in luciferase activity, which we attributed as a non-sequence-specific effect of miR-132 overexpression in vitro. Most importantly, the wild-type 3‚Ä≤UTRs of all genes examined were significantly repressed by the miR-132 mimic, with a range of repression between 28 and 50% (Fig. 1D). In all cases except for Jarid1a, the effects of the miR-132 mimic were abolished by the mutation of the seed site (Fig. 1D). For Jarid1a, which bears two putative miR-132 MREs within its 3‚Ä≤UTR, mutation of the first MRE (site 1), but not the second (site 2), abolished the repressive effects of the miR-132 mimic (Fig. 1D).}
{Relative to the effect of scrambled control oligomers, miR-200b and 200c suppressed the activity of the Flt1 3‚Ä≤-UTR reporter by 50% to 60%, which was similar to the effect of these oligomers on the Zeb1 3‚Ä≤-UTR reporter, and had no effect on the control luciferase reporter (Fig. 1B). This suppressive effect was abrogated by site-directed mutagenesis of the 5‚Ä≤ putative miR-200 binding site but not the 3‚Ä≤ binding site (Fig. 1B). Furthermore, other miR-200 family members (200a and 429) had no effect on Flt1 3‚Ä≤-UTR reporter activity (data not shown), suggesting that Flt1 regulation is specific to miR-200b and 200c.}
{Relative to the effect of scrambled control oligomers, miR-200b and 200c suppressed the activity of the Flt1 3‚Ä≤-UTR reporter by 50% to 60%, which was similar to the effect of these oligomers on the Zeb1 3‚Ä≤-UTR reporter, and had no effect on the control luciferase reporter (Fig. 1B). This suppressive effect was abrogated by site-directed mutagenesis of the 5‚Ä≤ putative miR-200 binding site but not the 3‚Ä≤ binding site (Fig. 1B). Furthermore, other miR-200 family members (200a and 429) had no effect on Flt1 3‚Ä≤-UTR reporter activity (data not shown), suggesting that Flt1 regulation is specific to miR-200b and 200c.}
{we used a luciferase 3‚Ä≤-UTR reporter assay (Fig. 4A) to measure the inhibitory effects of different miR-302 concentrations on the target sites of CDK2, cyclin D1/D2, and BMI1 polycomb ring finger oncogene (BMI-1). In the presence of 10 Œºmol/L Dox, miR-302 effectively bound to these target sites and successfully silenced >80% of the reporter luciferase expression in all targets (Fig. 4B).}
{we used a luciferase 3‚Ä≤-UTR reporter assay (Fig. 4A) to measure the inhibitory effects of different miR-302 concentrations on the target sites of CDK2, cyclin D1/D2, and BMI1 polycomb ring finger oncogene (BMI-1). In the presence of 10 Œºmol/L Dox, miR-302 effectively bound to these target sites and successfully silenced >80% of the reporter luciferase expression in all targets (Fig. 4B).}
{we used a luciferase 3‚Ä≤-UTR reporter assay (Fig. 4A) to measure the inhibitory effects of different miR-302 concentrations on the target sites of CDK2, cyclin D1/D2, and BMI1 polycomb ring finger oncogene (BMI-1). In the presence of 10 Œºmol/L Dox, miR-302 effectively bound to these target sites and successfully silenced >80% of the reporter luciferase expression in all targets (Fig. 4B).}
{we used a luciferase 3‚Ä≤-UTR reporter assay (Fig. 4A) to measure the inhibitory effects of different miR-302 concentrations on the target sites of CDK2, cyclin D1/D2, and BMI1 polycomb ring finger oncogene (BMI-1). In the presence of 10 Œºmol/L Dox, miR-302 effectively bound to these target sites and successfully silenced >80% of the reporter luciferase expression in all targets (Fig. 4B).}
{we used a luciferase 3‚Ä≤-UTR reporter assay (Fig. 4A) to measure the inhibitory effects of different miR-302 concentrations on the target sites of CDK2, cyclin D1/D2, and BMI1 polycomb ring finger oncogene (BMI-1). In the presence of 10 Œºmol/L Dox, miR-302 effectively bound to these target sites and successfully silenced >80% of the reporter luciferase expression in all targets (Fig. 4B).}
{we used a luciferase 3‚Ä≤-UTR reporter assay (Fig. 4A) to measure the inhibitory effects of different miR-302 concentrations on the target sites of CDK2, cyclin D1/D2, and BMI1 polycomb ring finger oncogene (BMI-1). In the presence of 10 Œºmol/L Dox, miR-302 effectively bound to these target sites and successfully silenced >80% of the reporter luciferase expression in all targets (Fig. 4B).}
{we used a luciferase 3‚Ä≤-UTR reporter assay (Fig. 4A) to measure the inhibitory effects of different miR-302 concentrations on the target sites of CDK2, cyclin D1/D2, and BMI1 polycomb ring finger oncogene (BMI-1). In the presence of 10 Œºmol/L Dox, miR-302 effectively bound to these target sites and successfully silenced >80% of the reporter luciferase expression in all targets (Fig. 4B).}
{we used a luciferase 3‚Ä≤-UTR reporter assay (Fig. 4A) to measure the inhibitory effects of different miR-302 concentrations on the target sites of CDK2, cyclin D1/D2, and BMI1 polycomb ring finger oncogene (BMI-1). In the presence of 10 Œºmol/L Dox, miR-302 effectively bound to these target sites and successfully silenced >80% of the reporter luciferase expression in all targets (Fig. 4B).}
{"Rat ST15A cells were transfected with the indicated miRNA mimics and, as opposed to the negative control and the unrelated miR-494, only miR-18 prominently inhibited the luciferase activity (Fig. 3C). In order to define the site of interaction, we mutated the reporter construct in the putative binding region, which abolished the effect of miR-18 on the luciferase activity (Fig. 3D). These results confirmed Hsf2 as a direct target of miR-18 and identified the site of interaction in the 3‚Ä≤UTR of Hsf2 mRNA. "}
{we find that in mir-52 rescued embryos, marked by DLG-1mCHERRY (Figure 4B), and in otherwise wild-type embryos (Figure S2), a col-10gfpcdh-3 transgene under the control of the wild-type cdh-3 3‚Ä≤ UTR (WT, Figure 4C) is downregulated. Importantly, a similar transgene but with a 4-bp mutation in the seed region of the predicted miR-52 binding site (Figure S3, MUT) does not show this effect (Figure 4F). In contrast, both WT and MUT transgenes are expressed in mir-51 family mutant embryos (Figure 4, I and L). A second transgene with an unrelated 3‚Ä≤ UTR (myo-2mcherryunc-54) is not regulated in a miR-52-dependent manner (Figure 4, B, E, H, and K). Taken together, these data show that miR-52 directly regulates the CDH-3 3‚Ä≤ UTR in vivo.}
{"The result showed that, co-transfecting with the pre-miRNAs expression constructs, the MT1 (co-transfecting with the construct expressing miR-19b), MT2 and MT4 mutation constructs resulted in a significantly increase in luciferase activity comparing to that of the wild-type construct (Fig. 4), suggesting that the miRNAs, miR-19b, miR-302b* and miR-323-3p, could repress gene expression in HEK-293 cells."}
{"The result showed that, co-transfecting with the pre-miRNAs expression constructs, the MT1 (co-transfecting with the construct expressing miR-19b), MT2 and MT4 mutation constructs resulted in a significantly increase in luciferase activity comparing to that of the wild-type construct (Fig. 4), suggesting that the miRNAs, miR-19b, miR-302b* and miR-323-3p, could repress gene expression in HEK-293 cells."}
{"The result showed that, co-transfecting with the pre-miRNAs expression constructs, the MT1 (co-transfecting with the construct expressing miR-19b), MT2 and MT4 mutation constructs resulted in a significantly increase in luciferase activity comparing to that of the wild-type construct (Fig. 4), suggesting that the miRNAs, miR-19b, miR-302b* and miR-323-3p, could repress gene expression in HEK-293 cells."}
{To do this, Luc constructs containing the 3‚Ä≤ UTR of the three mRNAs or the corresponding antisense fragments were generated. In all cases Luc expression was down-regulated by pre-miR transfection, while the Luc-antisense constructs were unaffected (Fig. 2B‚áì ). Furthermore, we generated three Luc constructs bearing the 3‚Ä≤UTR of the three mRNAs, each mutated at the putative target site of the cognate miRNA (see Supplemental Fig. 3). Also, in this case, the constructs bearing mutated UTRs were unaffected by miR transfection, thus demonstrating that Sirt1, Smarca5, and Jarid1b are direct targets of miRs 34a, 100, and 137, respectively (Fig. 2B‚áì ). }
{To do this, Luc constructs containing the 3‚Ä≤ UTR of the three mRNAs or the corresponding antisense fragments were generated. In all cases Luc expression was down-regulated by pre-miR transfection, while the Luc-antisense constructs were unaffected (Fig. 2B‚áì ). Furthermore, we generated three Luc constructs bearing the 3‚Ä≤UTR of the three mRNAs, each mutated at the putative target site of the cognate miRNA (see Supplemental Fig. 3). Also, in this case, the constructs bearing mutated UTRs were unaffected by miR transfection, thus demonstrating that Sirt1, Smarca5, and Jarid1b are direct targets of miRs 34a, 100, and 137, respectively (Fig. 2B‚áì ). }
{Transient cotransfection of miR-93 mimics and luciferase expression plasmids in HeLa cells resulted in significant repression of the basal level of the vegfa transcript, whereas transfection of cells with miR-690 mimics, a control microRNA that was not predicted to target VEGF, did not have any effect on the expression of luciferase (Fig. 5C). Importantly, suppression of the vegfa 3‚Ä≤-UTR by miR-93 mimics was abrogated when cells were transfected with miR-93 mutant (Fig. 5C), }
{We found that overexpression of miR-137 suppressed over 50% of the R-luc activity in primary neurons at 48 hours post-transfection (n = 7, p < 0.001) (Figure 5C). On the other hand, transfected anti-miR-137 led to a 28% increase in R-luc activity compared with the anti-miR control (anti-miR-Con, n = 3, p < 0.05) (Figure 5D). To further validate the interaction between miR-137 and its target Mib1 3‚Ä≤-UTR, we mutated the seed sequence of miR-137 located within the Mib1-3‚Ä≤-UTR reporter (Figure 5,B lower panel). This mutation substantially alleviated the miR-137-mediated suppression of luciferase activity, suggesting that the action of miR-137 is specific to the miR-137 seed region within the Mib1-3‚Ä≤-UTR (n = 5, p < 0.001) (Figure 5E).}
{Relative to the virgin luciferase reporter, annexation of the N-ras 3‚Ä≤-UTR resulted in an ~5-fold reduction in the luciferase activity (Fig. 5D). Deletion and point mutation analyses showed that the proximal miR-214 recognition site had a stronger role in attenuating the expression, whereas mutating both sequences severely curtailed the transcriptional repression activity of N-ras 3‚Ä≤-UTR (Fig. 5, C and D). }
{Cells transfected with premiR-214 expressed significantly more miR-214 when compared with cells transfected with control plasmid. Pre-miR214 activity was then examined based on luciferase activity. Expression of pre-miR-214 significantly decreased the level of luciferase activity in cells transfected with MMP-LUC-3'-PTEN (Fig. 5B). A reduction in luciferase activity was not observed in cells transfected with or MMP-LUC-MUT-3'-PTEN (Fig. 5B). }
{These experiments demonstrated that miR-802 can regulate luciferase activity in a concentration-dependent manner (maximal inhibition 10 nM), with a significant decrease in r-luc activity as low as 2 nM miR-802. In contrast, transfection with control nontargeting miRNA (i.e., scramble) had no effect on luciferase activity.The mutant construct, psiCHECK/hAT1R/mut-802, was transfected into CHO cells with increasing concentrations of miR-802 or control nontargeting miRNA, and luciferase activity was compared with that of the nonaltered construct (Fig. 4C). miR-802 could no longer diminish r-luc activity in the altered construct, even at a concentration of 25 nM. Transfection of control nontargeting miRNA (i.e., scramble) with either construct had no effect on luciferase activity.}
{However, no such effect was observed in cells expressing rhodopsin encoded by a mutant mRNA in which the putative miR-708 seed binding site was replaced with a scrambled sequence (Fig. 4 D and not depicted). Together, our results suggest that miR-708 targets rhodopsin mRNA, resulting in its decreased expression in mammalian cells.}
{Using HCT-116 cells cotransfected with pMIR-CC reporter constructs and the pre-miR miRNA precursor of miR-379, we found that the pMIR-CC luminescent signal was about 55% that of the control (P = 0.015, Figure 7C). There was no significant difference between the signal from the cells cotransfected with the pMIR-TT reporter constructs and those cotransfected with the pre-miR miRNA precursor of miR-379 (P = 0.86, see Figure 7C).}
{ we cloned the two predicted binding sites in PODXL 3‚Ä≤-UTR and linked them to firefly luciferase vectors. When these luciferase vectors were co-transfected with miR-199a-5p mimics in NT2 cells, luciferase activity of the vector carrying the conserved binding site was significantly suppressed. However, miR-199a-5p did not suppress the vector carrying a poorly conserved binding site. To show that the suppression of luciferase activity is due to binding of the miRNA to the seed sequence, we generated the mutant constructs by mutating the seed sequence. As expected, miR-199a-5p had little effect on the mutant constructs (Figure 5e). These data show that miR-199a-5p regulates PODXL through a conserved binding site in its 3‚Ä≤-UTR.}
{"To assess whether the regulation of Smad3 is direct, we fused the 3‚Ä≤UTR region of Smad3 to a luciferase reporter to validate the effects of miR-23b mimics on wild type and mutant plasmids (Fig. 3C). Mutations in seed complementary sites fully rescued repression of Smad3 (Fig. 3D)."}
{We found that ectopic expression of miR-3960 significantly repressed luciferase activity of the wild-type Hoxa2 CDS construct (Fig. 4B). Mutation of three nucleotides within the putative target site in the Hoxa2 CDS (MUT-pGL3-Hoxa2) abolished the repression (Fig. 4B). }
{Transient co-transfection of miR-29c mimics with luciferase expression plasmids resulted in a significant repression of luciferase reporter gene in podocytes, whereas transfection of cells with miRNA control mimics (negative control) did not have any effect on the expression of luciferase (Fig. 3C). However, mutations within the seed sequence binding site of Spry1 abrogated the effect of miR-29c mimics (Fig. 3C). }
{For Tgfbr2, luciferase assay also showed ~50% decrease of GL activity while miR-93 mutant did not have such effect (Supplementary Figure S18a and b).}
{For Tgfbr2, luciferase assay also showed ~50% decrease of GL activity while miR-93 mutant did not have such effect (Supplementary Figure S18a and b).}
{we constructed a luciferase reporter with p21 3‚Ä≤UTR sequence inserted downstream of the firefly luciferase coding sequence. We observed consistent ~40% repression of luciferase activity following transfection of miR-93 and miR-106b mimics into co-transfected Hela cells, a repression lost when mutations were introduced into the seed region of conserved p21 3‚Ä≤UTR target sites (Supplementary Figure S17a and b).}
{we constructed a luciferase reporter with p21 3‚Ä≤UTR sequence inserted downstream of the firefly luciferase coding sequence. We observed consistent ~40% repression of luciferase activity following transfection of miR-93 and miR-106b mimics into co-transfected Hela cells, a repression lost when mutations were introduced into the seed region of conserved p21 3‚Ä≤UTR target sites (Supplementary Figure S17a and b).}
{we conducted a series of luciferase reporter assays by co-transfection of miR-1258 (wild type/mutant) and luciferase constructs containing HPSE 3‚Ä≤-UTR (wild type/mutant). Luciferase activity was decreased by miR-1258 approximately 70% compared to scrambled control, and neither a miR-1258 mutant or an alternate microRNA (miR-100) decreased activity in the Luc-HPSE 3‚Ä≤-UTR constructs (Fig. 1B). Moreover, co-transduction of miR-1258 with a luciferase construct containing the 5‚Ä≤-CCU-3‚Ä≤ deletion mutant (Luc-HPSE 3‚Ä≤-UTR-del) failed to decrease luciferase activity (Fig. 1C).}
{To test if mir-363 is capable of regulating Hand2 and Tbx3 expression through binding to the predicted sites, we inserted the Hand2 or Tbx3 3‚Ä≤ UTR sequence downstream of luciferase and measured the level of luciferase activity in the presence or absence of mir-363 transfection in HeLa cells. We found that addition of mir-363 led to a statistically significant downregulation of luciferase activity with either Hand2 or Tbx3 3‚Ä≤ UTR (Fig. 8C). Furthermore, this downregulation is abolished by mutating the sequence of the predicted site in either of the 3‚Ä≤ UTRs. }
{To test if mir-363 is capable of regulating Hand2 and Tbx3 expression through binding to the predicted sites, we inserted the Hand2 or Tbx3 3‚Ä≤ UTR sequence downstream of luciferase and measured the level of luciferase activity in the presence or absence of mir-363 transfection in HeLa cells. We found that addition of mir-363 led to a statistically significant downregulation of luciferase activity with either Hand2 or Tbx3 3‚Ä≤ UTR (Fig. 8C). Furthermore, this downregulation is abolished by mutating the sequence of the predicted site in either of the 3‚Ä≤ UTRs. }
{We cloned the entire 3‚Ä≤-UTR sequences for both mouse and chick FoxP1 into a luciferase construct. While control miRNAs (miR-17 and miR-128) had no effect on luciferase activity, miR-9-1 and miR-9-2, but not miR-9-Mut, significantly reduced luciferase activity of the constructs containing the 3‚Ä≤-UTR for FoxP1, suggesting that miR-9 directly targets FoxP1}
{We cloned the entire 3‚Ä≤-UTR sequences for both mouse and chick FoxP1 into a luciferase construct. While control miRNAs (miR-17 and miR-128) had no effect on luciferase activity, miR-9-1 and miR-9-2, but not miR-9-Mut, significantly reduced luciferase activity of the constructs containing the 3‚Ä≤-UTR for FoxP1, suggesting that miR-9 directly targets FoxP1}
{co-transfection of the luciferase reporter and miR-34a oligos into three PCa cell types produced lower luciferase activity than cells co-transfected with the NC oligos but mutation of the seed sequence in either site, especially the distal site, partially abrogated the suppressive effect of miR-34a (Fig. 4d; Supplementary Fig. 11f,g). These results suggest that miR-34a regulates CD44 expression via two binding sites located in the 3‚Äô-UTR of the CD44 gene.}
{co-transfection of the luciferase reporter and miR-34a oligos into three PCa cell types produced lower luciferase activity than cells co-transfected with the NC oligos but mutation of the seed sequence in either site, especially the distal site, partially abrogated the suppressive effect of miR-34a (Fig. 4d; Supplementary Fig. 11f,g). These results suggest that miR-34a regulates CD44 expression via two binding sites located in the 3‚Äô-UTR of the CD44 gene.}
{The overexpression of mir-200c and mir-141 in PC-3 cells decreased luciferase activity from the JAG1 3‚Ä≤UTR reporter by 25% but expression of luciferase was unaltered in the mutant sensor construct (Figure 4D). }
{The overexpression of mir-200c and mir-141 in PC-3 cells decreased luciferase activity from the JAG1 3‚Ä≤UTR reporter by 25% but expression of luciferase was unaltered in the mutant sensor construct (Figure 4D). }
{For a subset of target genes including MET, GMNN, CCNE2, SIRT1 and HDAC1, we confirmed direct interaction of miR-449 with the target gene 3' UTR using luciferase assay (figure ‚Äã(figure3d3d).}
{For a subset of target genes including MET, GMNN, CCNE2, SIRT1 and HDAC1, we confirmed direct interaction of miR-449 with the target gene 3' UTR using luciferase assay (figure ‚Äã(figure3d3d).}
{For a subset of target genes including MET, GMNN, CCNE2, SIRT1 and HDAC1, we confirmed direct interaction of miR-449 with the target gene 3' UTR using luciferase assay (figure ‚Äã(figure3d3d).}
{For a subset of target genes including MET, GMNN, CCNE2, SIRT1 and HDAC1, we confirmed direct interaction of miR-449 with the target gene 3' UTR using luciferase assay (figure ‚Äã(figure3d3d).}
{We found that overexpression of miR-137 suppressed over 50% of the R-luc activity in primary neurons at 48 hours post-transfection (n = 7, p < 0.001) (Figure 5C). On the other hand, transfected anti-miR-137 led to a 28% increase in R-luc activity compared with the anti-miR control (anti-miR-Con, n = 3, p < 0.05) (Figure 5D). To further validate the interaction between miR-137 and its target Mib1 3‚Ä≤-UTR, we mutated the seed sequence of miR-137 located within the Mib1-3‚Ä≤-UTR reporter (Figure 5,B lower panel). This mutation substantially alleviated the miR-137-mediated suppression of luciferase activity, suggesting that the action of miR-137 is specific to the miR-137 seed region within the Mib1-3‚Ä≤-UTR (n = 5, p < 0.001) (Figure 5E).}
{Relative to the virgin luciferase reporter, annexation of the N-ras 3‚Ä≤-UTR resulted in an ~5-fold reduction in the luciferase activity (Fig. 5D). Deletion and point mutation analyses showed that the proximal miR-214 recognition site had a stronger role in attenuating the expression, whereas mutating both sequences severely curtailed the transcriptional repression activity of N-ras 3‚Ä≤-UTR (Fig. 5, C and D). }
{Relative to the virgin luciferase reporter, annexation of the N-ras 3‚Ä≤-UTR resulted in an ~5-fold reduction in the luciferase activity (Fig. 5D). Deletion and point mutation analyses showed that the proximal miR-214 recognition site had a stronger role in attenuating the expression, whereas mutating both sequences severely curtailed the transcriptional repression activity of N-ras 3‚Ä≤-UTR (Fig. 5, C and D). }
{Interestingly, brain cancer cells transfected with the hsa-miR-381 mimics, consistently had decreased luciferase activity relative to cells transfected with the chimeric plasmid without hsa-miR-381 (Student's t test, p < 0.01; Fig. 4B). However, when these chimeric plasmids were transfected with or without hsa-miR-381 in other (non-brain) cell lines, no significant differences were observed, indicating that hsa-miR-381 specifically targets LRRC4 in brain cancer cells. Co-application of the chimeric plasmid and the hsa-miR-381 antagomir eliminated the effect (Student's t test, p < 0.01; Fig. 4B). The luciferase's reporter vector containing the mutated 3‚Ä≤ UTR of LRRC4 was not responsive to the hsa-miR-381 mimics, }
{"The luciferase activityof cells transfected with miR-375 precursor was significantlydecreased compared with the negative control (P< 0.01) (Figure 4C and 4D). Moreover, mutation of the putative miR-375-binding sites clearly abrogated therepression of luciferase activity caused by miR-375 overexpression(Figure 4C and 4D). These data suggest thatmiR-375 may inhibit JAK2 protein expression through3‚Ä≤-UTR at the posttranscriptional level."}
{"The luciferase activityof cells transfected with miR-375 precursor was significantlydecreased compared with the negative control (P< 0.01) (Figure 4C and 4D). Moreover, mutation of the putative miR-375-binding sites clearly abrogated therepression of luciferase activity caused by miR-375 overexpression(Figure 4C and 4D). These data suggest thatmiR-375 may inhibit JAK2 protein expression through3‚Ä≤-UTR at the posttranscriptional level."}
{"The relative luciferase activityof the reporter containing wild-type E2F3 30-UTR was significantlysuppressed when miR-125b was cotransfected. In contrast,the luciferase activity of the reporter containing themutant miR-125b-binding site was unaffected, indicating thatmiR-125b may suppress gene expression through miR-125bbindingsequences at the 30-UTR of E2F3 gene (Fig. 5c)."}
{"Overexpressionof miR-448 resulted in a reduction of luciferaseactivity (Fig. 4B and Supplemental Fig. 3A). To furtherconfirm this specificity, we mutated the candidate miR-448 target sites (Fig. 4A), which resulted in the loss ofmiR-448-mediated repression (Fig. 4B)."}
{"We tested if miR-27b is able to regulate Mef2c by generatingluciferase reporter constructs with wild-type and miR-27 ¬¥seed¬¥ sequence mutated Mef2c3¬¥UTRs. As illustrated in Figure 6A, miR-27b significantly decreased luciferase activity inthe context of the wild-type Mef2c 3¬¥UTR, while mutation of miR-27b target sites (onepredicted by TargetScan, one non-canonical seed sequence; Supplementary Figure 5)returned luciferase levels to that of the control oligonucleotide."}
{"As shown in Figure 2B, cotransfectionof pMIR-Bin3-3‚Ä≤ UTR with the mousemiR-184 mimic down-regulates luciferase activity by57%, and co-transfection of pMIR-Bin3-3‚Ä≤ UTR-mutwith the mouse miR-184 mimic has no effect on theluciferase activity."}
{"The 3'-UTR segments of each target were synthesizedand subcloned downstream of the Renilla luciferasein the psiCHECK-2 dual luciferase reporter vector(Fig. 3A), and reporter assays were performed as indicated.Surprisingly, as shown in Fig. 3B, 11 reporterswere significantly repressed by miR-122 to differentdegrees (30%-70% reduction), including the twoknown targets. MSN (moesin) and serum response factor(SRF) were not significant in this group. Thesedata indicate that most candidate genes could bedirectly repressed by miR-122. To further confirm thishypothesis, we performed mutational analyses on eachpredicted site. As expected, all mutant sites lost theirresponse to miR-122 (Fig. 3C), indicating the specificityof the repression."}
{"The 3'-UTR segments of each target were synthesizedand subcloned downstream of the Renilla luciferasein the psiCHECK-2 dual luciferase reporter vector(Fig. 3A), and reporter assays were performed as indicated.Surprisingly, as shown in Fig. 3B, 11 reporterswere significantly repressed by miR-122 to differentdegrees (30%-70% reduction), including the twoknown targets. MSN (moesin) and serum response factor(SRF) were not significant in this group. Thesedata indicate that most candidate genes could bedirectly repressed by miR-122. To further confirm thishypothesis, we performed mutational analyses on eachpredicted site. As expected, all mutant sites lost theirresponse to miR-122 (Fig. 3C), indicating the specificityof the repression."}
{"The 3'-UTR segments of each target were synthesizedand subcloned downstream of the Renilla luciferasein the psiCHECK-2 dual luciferase reporter vector(Fig. 3A), and reporter assays were performed as indicated.Surprisingly, as shown in Fig. 3B, 11 reporterswere significantly repressed by miR-122 to differentdegrees (30%-70% reduction), including the twoknown targets. MSN (moesin) and serum response factor(SRF) were not significant in this group. Thesedata indicate that most candidate genes could bedirectly repressed by miR-122. To further confirm thishypothesis, we performed mutational analyses on eachpredicted site. As expected, all mutant sites lost theirresponse to miR-122 (Fig. 3C), indicating the specificityof the repression."}
{"The 3'-UTR segments of each target were synthesizedand subcloned downstream of the Renilla luciferasein the psiCHECK-2 dual luciferase reporter vector(Fig. 3A), and reporter assays were performed as indicated.Surprisingly, as shown in Fig. 3B, 11 reporterswere significantly repressed by miR-122 to differentdegrees (30%-70% reduction), including the twoknown targets. MSN (moesin) and serum response factor(SRF) were not significant in this group. Thesedata indicate that most candidate genes could bedirectly repressed by miR-122. To further confirm thishypothesis, we performed mutational analyses on eachpredicted site. As expected, all mutant sites lost theirresponse to miR-122 (Fig. 3C), indicating the specificityof the repression."}
{"The 3'-UTR segments of each target were synthesizedand subcloned downstream of the Renilla luciferasein the psiCHECK-2 dual luciferase reporter vector(Fig. 3A), and reporter assays were performed as indicated.Surprisingly, as shown in Fig. 3B, 11 reporterswere significantly repressed by miR-122 to differentdegrees (30%-70% reduction), including the twoknown targets. MSN (moesin) and serum response factor(SRF) were not significant in this group. Thesedata indicate that most candidate genes could bedirectly repressed by miR-122. To further confirm thishypothesis, we performed mutational analyses on eachpredicted site. As expected, all mutant sites lost theirresponse to miR-122 (Fig. 3C), indicating the specificityof the repression."}
{"The 3'-UTR segments of each target were synthesizedand subcloned downstream of the Renilla luciferasein the psiCHECK-2 dual luciferase reporter vector(Fig. 3A), and reporter assays were performed as indicated.Surprisingly, as shown in Fig. 3B, 11 reporterswere significantly repressed by miR-122 to differentdegrees (30%-70% reduction), including the twoknown targets. MSN (moesin) and serum response factor(SRF) were not significant in this group. Thesedata indicate that most candidate genes could bedirectly repressed by miR-122. To further confirm thishypothesis, we performed mutational analyses on eachpredicted site. As expected, all mutant sites lost theirresponse to miR-122 (Fig. 3C), indicating the specificityof the repression."}
{"The 3'-UTR segments of each target were synthesizedand subcloned downstream of the Renilla luciferasein the psiCHECK-2 dual luciferase reporter vector(Fig. 3A), and reporter assays were performed as indicated.Surprisingly, as shown in Fig. 3B, 11 reporterswere significantly repressed by miR-122 to differentdegrees (30%-70% reduction), including the twoknown targets. MSN (moesin) and serum response factor(SRF) were not significant in this group. Thesedata indicate that most candidate genes could bedirectly repressed by miR-122. To further confirm thishypothesis, we performed mutational analyses on eachpredicted site. As expected, all mutant sites lost theirresponse to miR-122 (Fig. 3C), indicating the specificityof the repression."}
{"The 3'-UTR segments of each target were synthesizedand subcloned downstream of the Renilla luciferasein the psiCHECK-2 dual luciferase reporter vector(Fig. 3A), and reporter assays were performed as indicated.Surprisingly, as shown in Fig. 3B, 11 reporterswere significantly repressed by miR-122 to differentdegrees (30%-70% reduction), including the twoknown targets. MSN (moesin) and serum response factor(SRF) were not significant in this group. Thesedata indicate that most candidate genes could bedirectly repressed by miR-122. To further confirm thishypothesis, we performed mutational analyses on eachpredicted site. As expected, all mutant sites lost theirresponse to miR-122 (Fig. 3C), indicating the specificityof the repression."}
{"The 3'-UTR segments of each target were synthesizedand subcloned downstream of the Renilla luciferasein the psiCHECK-2 dual luciferase reporter vector(Fig. 3A), and reporter assays were performed as indicated.Surprisingly, as shown in Fig. 3B, 11 reporterswere significantly repressed by miR-122 to differentdegrees (30%-70% reduction), including the twoknown targets. MSN (moesin) and serum response factor(SRF) were not significant in this group. Thesedata indicate that most candidate genes could bedirectly repressed by miR-122. To further confirm thishypothesis, we performed mutational analyses on eachpredicted site. As expected, all mutant sites lost theirresponse to miR-122 (Fig. 3C), indicating the specificityof the repression."}
{ The LNA-let-7 inhibitor (1, 5, or 25 pmol) or scrambled control (25 pmol) was co-transfected into the HEK239 cells together with the psiCHECK/MOR1UTR or the psiCHECK/MOR1UTRmut. The LNA-let-7 inhibitor increased the reporter gene activity in a dose-dependant manner. Co-transfection with 25 pmol LNA-let-7 inhibitor completely restored the luciferase repression by psiCHECK/MOR1UTR. In contrast, the LNA-let-7 inhibitor did not change the luciferase signal when co-transfected with the psiCHECK/MOR1UTRmut that lacks the let-7 binding sequence (Fig. 1C). }
{A significant decrease in luciferase activity was observed in the construct bearing an intact miR-21 binding site when compared with the control (Fig. 8B; P < 0.05). The decrease in luciferase activity was lost on mutation of the putative binding site for miR-21 by site-directed mutagenesis (Fig. 8C; P = not significant). }
{To verify whether the Ybx1 3‚Ä≤-UTR is a direct target of miR-216a, Luc reporter vectors containing the Ybx1 3‚Ä≤-UTR were constructed (Fig. 2E). miR-216a mimic significantly inhibited Luc activity of Ybx1 3‚Ä≤-UTR vector by almost 70% relative to a negative control mimic (NC Mimic) but not in the two control constructs, namely control without 3‚Ä≤-UTR (first pair of bars) or that with Ybx1 3‚Ä≤-UTR that has base substitutions in the miR-216a site (third pair of bars) (Fig. 2F). }
{"Our results showed that pcDNA-3.1-miR-107 produced a 60%decrease in relative luciferase activity compared with pcDNA3.1-transfected cells, when the full-length wild-type 3 -UTR of DICER1was present (Fig. 3C). However, the relative luciferase activity didnot drop as sharply in UTRs that contained mutant binding sitesas in those that contained wild-type binding sites, which indicatesthat this reduction was sequence-specific (Fig. 3C)."}
{"In the dual-luciferase assay, the relative luciferaseactivity of the reporter that contained the RB1 3#-UTR wasmarkedly decreased compared with that seen with the parentplasmid containing the mutant sequence or the NC mimicgroup and the 16HBE-T blank group (Fig. 7E)."}
{"We cloned the3‚Ä≤ UTR of MMP-9 downstream of the firefly luciferase gene and foundthat the normalized luciferase activity was substantially reduced uponcotransfectionwith either miR-212 or miR-132 or with both microRNAstogether (‚ÄòMMP-9‚Äô in Supplementary Fig. 8). However, this decrease inluciferase activity was abolished when the binding sites of miR-212 andmiR-132 on the 3‚Ä≤ UTR of MMP-9 were mutated in the luciferase construct(‚ÄòMMP-9-mut.‚Äô in Supplementary Fig. 8). These results indicatethat MMP-9 is a direct target of both miR-212 and miR-132."}
{"We cloned the3‚Ä≤ UTR of MMP-9 downstream of the firefly luciferase gene and foundthat the normalized luciferase activity was substantially reduced uponcotransfectionwith either miR-212 or miR-132 or with both microRNAstogether (‚ÄòMMP-9‚Äô in Supplementary Fig. 8). However, this decrease inluciferase activity was abolished when the binding sites of miR-212 andmiR-132 on the 3‚Ä≤ UTR of MMP-9 were mutated in the luciferase construct(‚ÄòMMP-9-mut.‚Äô in Supplementary Fig. 8). These results indicatethat MMP-9 is a direct target of both miR-212 and miR-132."}
{"we generatedmiRNA expression reporters, where firefly luciferase was placed underpost‚Äëtranslational control of the native 3Õ¥ UTR of Dyrk1a or a Dyrk1a3Õ¥UTR harbouring a mutated miR‚Äë199b seed region. Co‚Äëtransfection ofsynthetic miR‚Äë199b decreased Dyrk1a 3Õ¥UTR reporter activity (Fig. 2i),whereas no sensitivity was observed for co‚Äëexpression of a control micro‚ÄëRNA (Fig. 2i) or a scrambled precursor microRNA (Supplementary Information, Fig. S2c, d). In contrast, a pMiR‚ÄëREPORT‚Äë3Õ¥UTR Dyrk1awith a mutated seed region showed no sensitivity to miR‚Äë199b expression(Fig. 2i)."}
{"To confirm whether mkp-1 is a direct target of miR-101, wegenerated luciferase reporter constructs by cloning either the wildtypeor a mutated portion of the 39 UTR of mkp-1 into the 39 UTRof a pGL-3M-Luc vector. The mutated 39 UTR of mkp-1 had threebases changed, from GUACUGUA to GGACGGGU, at the putativemiR-101 binding sites (Fig. 2D). We transfected these vectorswith miR-101 mimics into 293 cells and analyzed the lysates48 h later. Transfection with miR-101 mimics markedly inhibitedthe luciferase activity for the wild-type 39 UTR of mkp-1 butshowed no repression effect for the mutated 39 UTR of mkp-1when compared with that for the control dsRNA (Fig. 2E), suggestingthat miR-101 may repress MKP-1 expression by bindingto the 39 UTR of mkp-1 in a direct and sequence-specific manner."}
{"The relative luciferase activity was diminishedin cells co-transfected with miR-126-pSuper and VEGFAor PIK3R2-30 UTR-PGL3, and others remained unchanged(Fig. 4a). In addition, co-transfection of ‚Äò‚Äòseed sequence‚Äô‚Äôbased mutation of miR-126 binding site of pGL3-VEGFA-30 UTR(mut) restored the luciferase activity (Fig. 4b)."}
{"The relative luciferase activity was diminishedin cells co-transfected with miR-126-pSuper and VEGFAor PIK3R2-30 UTR-PGL3, and others remained unchanged(Fig. 4a). In addition, co-transfection of ‚Äò‚Äòseed sequence‚Äô‚Äôbased mutation of miR-126 binding site of pGL3-VEGFA-30 UTR(mut) restored the luciferase activity (Fig. 4b)."}
{In fact, the expression of a luciferase reporter gene containing the DDX6 3‚Ä≤UTR was decreased by miR-130a and -130b (Figure 3B, left), but a DDX6 3‚Ä≤UTR mutant at the miR-130a- and -130b-binding site (Figure 3A) showed no change in luciferase activity (Figure 3B, right). }
{The wild type of mouse Big-h3 mRNA 3'UTR carries a putative miR-21 binding site while the 429-431 mutated mouse Big-h3 3' UTR failed to bind miR-21 (Figure ‚Äã(Figure4.A).4.A). To validate the miRNA-target interactions, the wild type and the 429-431 mutated Big-h3 complementary sites were cloned into the 3‚Ä≤UTR of the firefly luciferase gene and co-transfected with miR-21 mimics or negative control RNA (NC) in NIH3T3 cells. As shown in Figure ‚ÄãFigure4B,4B, miR-21 significantly reduced the luciferase activity of the wild type Big-h3 3'UTR with respect to the negative control. The miR-21 mimics at a final concentration of 50 nM and 100 nM both reduced the luciferase activity, but there were no significant difference between the two groups (Data not shown). Therefore, miRNA at a final concentration of 50 nM was transfected into cells in the following experiment. However, miR-21 has no effect on the luciferase activity of the 429-431 mutated Big-h3 3‚Ä≤UTR (Figure ‚Äã(Figure4B).4B). These data indicated that miR-21 may target Big-h3 in a 3‚Ä≤UTR dependent manner.}
{The luciferase reporter assay indicated that the activity of the reporter containing the 3‚Ä≤-UTR of the RB1 gene was decreased following treatment with miR-192 mimics, whereas the reporters containing the 3‚Ä≤-UTRs of the other genes were not obviously altered (Figure 3A). We performed site-directed mutagenesis of the reporter containing the 3‚Ä≤-UTR of the RB1 gene as described in Figure 3B and the luciferase reporter assay was performed with the mutated construct. The results demonstrate that the inhibitory effect of miR-192 mimics was abolished in the mutant reporter (Figure 3B).}
{To show a direct interaction between miR-622 and the 3‚ÄôUTR of ING1, we cloned the 3‚ÄôUTR region that is predicted to interact with miR-622 into a luciferase reporter vector. We then assessed luciferase activity by co-transfection the luciferase reporter vector bearing the 3‚ÄôUTR of ING1 with the miR-622 precursor or the miR-622 inhibitor or with control plasmids. Luciferase activity was markedly diminished in the cells transfected with the miR-622 precursor and wild-type 3‚ÄôUTR reporter plasmid, compared with the cells transfected with the miR-622 precursor and mutant 3‚ÄôUTR reporter plasmids (Figure ‚Äã(Figure4C,4C, P < 0.05). }
{Furthermore, luciferase reporter assays were performed to validate specificity of the miR-196a regulation of NOBOX through the predicted miR-196a recognition sequence in the 3' UTR of NOBOX. NOBOX 3' UTR sequence was inserted downstream of the firefly luciferase coding region. Mutations in the predicted MRE in the 3' UTR of the NOBOX for miR-196a were created such that interaction between miR-196a and NOBOX is compromised (Figure ‚Äã(Figure4A).4A). Ectopic expression of miR-196a by transfection of miR-196a duplex into the HeLa cells suppressed activity of a chimeric luciferase construct containing the miR-196a MRE of NOBOX at its 3' end (Figure ‚Äã(Figure4B).4B). Luciferase activity was restored when a four-base mismatch mutation was introduced into the seed region of the miRNA-196a recognition sequence in the NOBOX 3' UTR (Figure ‚Äã(Figure4B).4B). }
{ To determine whether miR-148a could directly regulate p27, p27 mRNA 3'UTR encompassing the predicted binding site with or without mutation that would disrupt miRNA interaction (underlined in Fig. ‚ÄãFig.3A),3A), were inserted to the downstream of luciferase open reading frame in pGL3-CM reporter vector. When introduced into SGC-7901 cells, the wild type p27 3'UTR reporter showed a 39% reduction in luciferase activity in the miR-148a-transfected cells compared with that in scrambled-miRNA-transfected cells (Fig. ‚Äã(Fig.3B,3B, p<0.01). Mutation of miR-148a binding site abrogated the repression of luciferase activity caused by miR-148a overexpression (Fig. ‚Äã(Fig.3B).3B). }
{ To detect whether the regulation of INHBB was direct, we fused the 3‚Ä≤-UTR region of INHBB to a luciferase system. As shown in Figure 3C,D, miR-34a remarkably repressed the expression of luciferase containing an original miR-34a binding site (INHBB-UTR) but not a mutant binding site (INHBB-Mu-UTR). And mutations in seed complementary sites of the 3‚Ä≤-UTR region of INHBB could restore the luciferase expression.}
{The results showed that compared with the negative control, the luciferase activity of miR-29a and miR-29b groups decreased by approximately 70% and 60% (Fig. 5C). Furthermore, the decrease of luciferase activity was rescued by introducing mutations at the seed site in pGl3-Col2a1M (Fig. 5C). }
{The results showed that compared with the negative control, the luciferase activity of miR-29a and miR-29b groups decreased by approximately 70% and 60% (Fig. 5C). Furthermore, the decrease of luciferase activity was rescued by introducing mutations at the seed site in pGl3-Col2a1M (Fig. 5C). }
{To test whether miR-1 is capable of directly repressing Cdc42 via this site, the Cdc42 3‚Ä≤-UTR was inserted into the 3‚Ä≤-UTR of a luciferase reporter, downstream of the coding region. Transfection of the Cdc42 3‚Ä≤-UTR luciferase reporter with miR-1 mimic into HL-1 cells results in a decrease in luciferase activity, whereas cotransfection with miR-1 inhibitor increases luciferase activity (Fig. 5 G). A gradient of miR-1 was used to confirm the luciferase activity was dose responsive (Fig. S4 A). Mutations of the miR-1 binding site in the Cdc42 3‚Ä≤-UTR abolished miR-1 responsiveness (Fig. 5 G), suggesting that miR-1 represses Cdc42 by physically binding to its 3‚Ä≤-UTR.}
{Function of predicted miRNA responsive elements (MREs) was tested by luciferase assays on constructs containing either the full-length 3‚Ä≤UTR or the 3‚Ä≤-end of SV2A 3‚Ä≤UTR. Overexpression of a miR-485 mimic (miR-M; a small chemically modified dsRNA designed to mimic the endogenous miRNA) significantly reduced luciferase activity of both constructs (n = 6; P < 0.001) (Fig. 2A), and this was reversed when both MREs were mutated within the 5‚Ä≤-seed region (Fig. S2B)}
{We generated sensors containing chicken Pax3 3‚Ä≤-UTR fragments downstream of firefly luciferase. Pax3 target sites were tested independently (TS1 or TS2) and in a sensor containing both sites (TS1 + TS2). Dual-luciferase assays showed that miR-1 and miR-206 directly target the Pax3 3‚Ä≤-UTR through both predicted target sites (Fig. 2B). TS1 was strongly down-regulated by miR-206, less so by miR-1, whereas TS2 was equally well regulated by both miR-1 and miR-206. Sensors containing both sites also were strongly down-regulated by both miRNAs, with miR-1 benefiting more from the presence of two sites. An unrelated miRNA not predicted to target Pax3, miR-140, had no effect on Pax3 sensors (Fig. 2B). Finally, mutant sensors containing point mutations within putative target sites (TS1m and TS2m) demonstrated that the sites were required for miRNA-mediated repression.}
{We generated sensors containing chicken Pax3 3‚Ä≤-UTR fragments downstream of firefly luciferase. Pax3 target sites were tested independently (TS1 or TS2) and in a sensor containing both sites (TS1 + TS2). Dual-luciferase assays showed that miR-1 and miR-206 directly target the Pax3 3‚Ä≤-UTR through both predicted target sites (Fig. 2B). TS1 was strongly down-regulated by miR-206, less so by miR-1, whereas TS2 was equally well regulated by both miR-1 and miR-206. Sensors containing both sites also were strongly down-regulated by both miRNAs, with miR-1 benefiting more from the presence of two sites. An unrelated miRNA not predicted to target Pax3, miR-140, had no effect on Pax3 sensors (Fig. 2B). Finally, mutant sensors containing point mutations within putative target sites (TS1m and TS2m) demonstrated that the sites were required for miRNA-mediated repression.}
{We generated sensors containing chicken Pax3 3‚Ä≤-UTR fragments downstream of firefly luciferase. Pax3 target sites were tested independently (TS1 or TS2) and in a sensor containing both sites (TS1 + TS2). Dual-luciferase assays showed that miR-1 and miR-206 directly target the Pax3 3‚Ä≤-UTR through both predicted target sites (Fig. 2B). TS1 was strongly down-regulated by miR-206, less so by miR-1, whereas TS2 was equally well regulated by both miR-1 and miR-206. Sensors containing both sites also were strongly down-regulated by both miRNAs, with miR-1 benefiting more from the presence of two sites. An unrelated miRNA not predicted to target Pax3, miR-140, had no effect on Pax3 sensors (Fig. 2B). Finally, mutant sensors containing point mutations within putative target sites (TS1m and TS2m) demonstrated that the sites were required for miRNA-mediated repression.}
{We generated sensors containing chicken Pax3 3‚Ä≤-UTR fragments downstream of firefly luciferase. Pax3 target sites were tested independently (TS1 or TS2) and in a sensor containing both sites (TS1 + TS2). Dual-luciferase assays showed that miR-1 and miR-206 directly target the Pax3 3‚Ä≤-UTR through both predicted target sites (Fig. 2B). TS1 was strongly down-regulated by miR-206, less so by miR-1, whereas TS2 was equally well regulated by both miR-1 and miR-206. Sensors containing both sites also were strongly down-regulated by both miRNAs, with miR-1 benefiting more from the presence of two sites. An unrelated miRNA not predicted to target Pax3, miR-140, had no effect on Pax3 sensors (Fig. 2B). Finally, mutant sensors containing point mutations within putative target sites (TS1m and TS2m) demonstrated that the sites were required for miRNA-mediated repression.}
{The luciferase activity in COS7 cells was significantly reduced (by 34 to 57%) when the reporter was cotransfected with increasing amount of plasmids expressing wild-type miR-146a. Such reduction is specific since a control plasmid expressing seed region-mutated miR-146a (Ctrl) has little effect on reporter activity. Moreover, wild-type miR-146a did not change the activity of a luciferase reporter when the binding site for miR-146a in 3‚Ä≤UTR of Notch1 was mutated (Fig. 5B). }
{Mutation assays were performed as indicated (Fig. 2E and F). In contrast to the negative control, the luciferase activity of the wild-type group decreased significantly (P < 0.01) in the presence of a miR-15a mimic, whereas the mutagenised groups restored luciferase activity to varying degrees. }
{"To test the effect of miR-19b on the NeuroD1 expression, a fragment of NeuroD1 3‚Ä≤ UTR containing the three putative target sites was cloned into the downstream of the firefly luciferase stop codon of pGL3 control expression vector. After the recombinant vector was co-transfected with synthetic miR-19b into 3T3 cells, the luciferase activity was significantly inhibited than that co-transfected with the control miRNA (Fig. 2C).Then we mutated the targeting sites of NeuroD1 3‚Ä≤ UTR in the pGL3-control vector (Fig. 2B). Mutational analysis showed that the first and second site in the fragment were important in mediating the inhibition of miR-19b (Fig. 2C). When the first site was mutated, luciferase activity was less efficiently repressed by miR-19b than that in the wild-type construct. This result indicates that the first site is functional. In addition, the mutation of the second site also modified miR-19b-induced repression of luciferase, which indicates that the second site is also necessary for miR-19b activity. However, the mutation of the third site did not rescue the luciferase activity. Furthermore, when the first site and the second site were both mutated, the rescue of luciferase activity was stronger than either mutation of them, these results demonstrate that the two target sites exert cooperative functions."}
{"As shown in Figure 3C, using luciferaseassay, we validated cdc25A and CCNE1 as direct target genes ofmiR-503. Significant inhibition of the luciferase activity wasobserved in cells transfected with the constructs bearing an intactmiR-503 binding site when compared with pLUC. Mutation ofcdc25A target sequence prevented downregulation of luciferaseactivity by miR-503. For CCNE1, mutation of each conservedtarget sequence had only a partial effect, and mutation of bothsequences fully rescued CCNE1 repression (Figure 3C)."}
{"As shown in Figure 3C, using luciferaseassay, we validated cdc25A and CCNE1 as direct target genes ofmiR-503. Significant inhibition of the luciferase activity wasobserved in cells transfected with the constructs bearing an intactmiR-503 binding site when compared with pLUC. Mutation ofcdc25A target sequence prevented downregulation of luciferaseactivity by miR-503. For CCNE1, mutation of each conservedtarget sequence had only a partial effect, and mutation of bothsequences fully rescued CCNE1 repression (Figure 3C)."}
{"As shown in Figure 3C, using luciferaseassay, we validated cdc25A and CCNE1 as direct target genes ofmiR-503. Significant inhibition of the luciferase activity wasobserved in cells transfected with the constructs bearing an intactmiR-503 binding site when compared with pLUC. Mutation ofcdc25A target sequence prevented downregulation of luciferaseactivity by miR-503. For CCNE1, mutation of each conservedtarget sequence had only a partial effect, and mutation of bothsequences fully rescued CCNE1 repression (Figure 3C)."}
{"Both xhairy1 30UTR (xHairy1-WT)and mHes1 30UTR were significantly repressed by syntheticmiR-9 precursors, whereas this effect was absent when a mutantreporter lacking the seed-complementary sequence (xHairy1_Mut) was used."}
{As shown in Fig. 7B, the transfection of TNF-Œ± 3'UTR-wt together with miR-19a AS, but not with miR-19a SC, led to an increase in luciferase activity in EC9706 cells, due to the inhibition of the endogenous miR-19a. In contrast, miR-19a AS did not increase the luciferase activity of TNF-Œ± 3'UTR-mut, thus demonstrating that the deletion of the miR-19a binding site in the TNF-Œ± 3'UTR abolished the inhibitory effect of miR-19a in regulating its expression (Fig. 6B).}
{"In the presence of miR-33mimic, the wild type, but not the mutated, reporteractivity was significantly lowered as compared to thecontrol reporter activity (Fig. 4B). Further, miR-33mimic repressed the reporter activity in a dose-dependentmanner (Fig. 4C)."}
{"To demonstratedirect regulation by miR-125b, luciferase reporter containingwild-type Stat3 3 UTR sequences or their mutant-derivateswith deletion of the putative miR-125b-binding sites weretransfected into NIH3T3 cells stably over-expressing miR-125b(NIH3T3/miR-125b; Figure 4B and supplemental Figure 3A).As shown in Figure 4C, miR-125b represses luciferase activityby approximately 40% depending on the presence of miR-125bbinding in the Stat3 3 UTR. Finally,"}
{"To demonstratedirect regulation by miR-125b, luciferase reporter containingwild-type Stat3 3 UTR sequences or their mutant-derivateswith deletion of the putative miR-125b-binding sites weretransfected into NIH3T3 cells stably over-expressing miR-125b(NIH3T3/miR-125b; Figure 4B and supplemental Figure 3A).As shown in Figure 4C, miR-125b represses luciferase activityby approximately 40% depending on the presence of miR-125bbinding in the Stat3 3 UTR. Finally,"}
{"The complete 3 UTRs of mouse and human c-Fos mRNA wereinserted into reporter vectors downstream of the Renilla luciferasegene. As controls we used vectors in which the seed regions of the2 predicted miR155-binding sites within the c-Fos 3 UTRs weremutated. These constructs were transfected into 293T cells with orwithout the corresponding human or mouse miR155 precursors.Cotransfection of the nonmutated construct with the miR155precursors resulted in a significant reduction in luciferase activity(Figure 3D). In contrast, no reduction was observed when themutated constructs were cotransfected with the miR155 precursors.These results confirmed that the predicted miR155-binding sites inthe 3 UTR of c-Fos mRNAwere indeed targeted directly by miR155."}
{"The complete 3 UTRs of mouse and human c-Fos mRNA wereinserted into reporter vectors downstream of the Renilla luciferasegene. As controls we used vectors in which the seed regions of the2 predicted miR155-binding sites within the c-Fos 3 UTRs weremutated. These constructs were transfected into 293T cells with orwithout the corresponding human or mouse miR155 precursors.Cotransfection of the nonmutated construct with the miR155precursors resulted in a significant reduction in luciferase activity(Figure 3D). In contrast, no reduction was observed when themutated constructs were cotransfected with the miR155 precursors.These results confirmed that the predicted miR155-binding sites inthe 3 UTR of c-Fos mRNAwere indeed targeted directly by miR155."}
{"The complete 3 UTRs of mouse and human c-Fos mRNA wereinserted into reporter vectors downstream of the Renilla luciferasegene. As controls we used vectors in which the seed regions of the2 predicted miR155-binding sites within the c-Fos 3 UTRs weremutated. These constructs were transfected into 293T cells with orwithout the corresponding human or mouse miR155 precursors.Cotransfection of the nonmutated construct with the miR155precursors resulted in a significant reduction in luciferase activity(Figure 3D). In contrast, no reduction was observed when themutated constructs were cotransfected with the miR155 precursors.These results confirmed that the predicted miR155-binding sites inthe 3 UTR of c-Fos mRNAwere indeed targeted directly by miR155."}
{"The complete 3 UTRs of mouse and human c-Fos mRNA wereinserted into reporter vectors downstream of the Renilla luciferasegene. As controls we used vectors in which the seed regions of the2 predicted miR155-binding sites within the c-Fos 3 UTRs weremutated. These constructs were transfected into 293T cells with orwithout the corresponding human or mouse miR155 precursors.Cotransfection of the nonmutated construct with the miR155precursors resulted in a significant reduction in luciferase activity(Figure 3D). In contrast, no reduction was observed when themutated constructs were cotransfected with the miR155 precursors.These results confirmed that the predicted miR155-binding sites inthe 3 UTR of c-Fos mRNAwere indeed targeted directly by miR155."}
{"The expression level of GFP in GFPRhoAmRNA-injected embryos was decreased when co-injected withmiR-133b compared to those without added miR-133b (Fig. 4B).However, GFP expression levels from either control GFP mRNA- orfrom GFP-mtRhoA mRNA-injected embryos were not suppressed byexogenous miR-133b. Inhibition of the GFP signal only in thepresence of the RhoA RE demonstrates the sequence-specific targetingof RhoA mRNA by miR-133b."}
{"The expression level of GFP in GFPRhoAmRNA-injected embryos was decreased when co-injected withmiR-133b compared to those without added miR-133b (Fig. 4B).However, GFP expression levels from either control GFP mRNA- orfrom GFP-mtRhoA mRNA-injected embryos were not suppressed byexogenous miR-133b. Inhibition of the GFP signal only in thepresence of the RhoA RE demonstrates the sequence-specific targetingof RhoA mRNA by miR-133b."}
{"To ensure the specificity of this interaction,three mutants of the Fbxw7 mRNA in 30-UTR(named Mut-1, Mut-2 or Mut-1√æMut-2) were constructedthat contained a 4-nt change in seed region ofthe target sequence (Figure 6a). As we expected, mutatedboth miR-27a-binding sites (Mut-1√æMut-2) at the 30-UTR of Fbxw7 mRNA completely abrogated the effectson inhibition of luciferase activity (Figure 6b). Notably,each of the miR-27a-binding sites tested in the 30-UTR ofFbxw7 mRNA appeared to be effective, as demonstratedby a decreased in luciferase activity of 30% in Mut-1 and40% in Mut-2. These observations indicate that Fbxw7is also an important target of miR-27a."}
{"To ensure the specificity of this interaction,three mutants of the Fbxw7 mRNA in 30-UTR(named Mut-1, Mut-2 or Mut-1√æMut-2) were constructedthat contained a 4-nt change in seed region ofthe target sequence (Figure 6a). As we expected, mutatedboth miR-27a-binding sites (Mut-1√æMut-2) at the 30-UTR of Fbxw7 mRNA completely abrogated the effectson inhibition of luciferase activity (Figure 6b). Notably,each of the miR-27a-binding sites tested in the 30-UTR ofFbxw7 mRNA appeared to be effective, as demonstratedby a decreased in luciferase activity of 30% in Mut-1 and40% in Mut-2. These observations indicate that Fbxw7is also an important target of miR-27a."}
{"Next, to further confirm the specificity of the regulation of IL-63 UTR by miR-142-3p, we first knocked-down endogenous miR-142-3p or miR-223 as a control with LNA-knockdown probes. ThemiR-142-3p and miR-223 were efficiently knocked down (supplementalFigure 3A-B). The suppression of miR-142-3p but notmiR-223 increased the ratio by   2-fold, P   .01 (Figure 2F)."}
{"To further examine whether miR-9 and miR-181b targetNRP1 directly, the luciferase reporter containing the NRP1 30-UTR fragment with the miR-9 or miR-181b binding sites wastransfected into EA.hy926 cells. When miR-9 or miR-181b werecotransfected with the NRP1 reporter constructs, luciferaseactivity was significantly suppressed compared withcotransfection with positive and negative controls of pre-miRmiRNA precursors (Fig. 3A). However, when miR-9 or miR-181b were cotransfected with the deleted seed sequence of the30-UTR, the luciferase activity suppression was completelyabolished. Taken together, these results suggest that miR-9 andmiR-181b suppressed NRP1 through a specific 30-UTR bindingsite."}
{"To further examine whether miR-9 and miR-181b targetNRP1 directly, the luciferase reporter containing the NRP1 30-UTR fragment with the miR-9 or miR-181b binding sites wastransfected into EA.hy926 cells. When miR-9 or miR-181b werecotransfected with the NRP1 reporter constructs, luciferaseactivity was significantly suppressed compared withcotransfection with positive and negative controls of pre-miRmiRNA precursors (Fig. 3A). However, when miR-9 or miR-181b were cotransfected with the deleted seed sequence of the30-UTR, the luciferase activity suppression was completelyabolished. Taken together, these results suggest that miR-9 andmiR-181b suppressed NRP1 through a specific 30-UTR bindingsite."}
{"To test whether the BD sequencecould mediate the miR-140‚ÄìDnpep interaction, a luciferasereporter plasmid carrying the BD sequence was constructed.Cotransfection with a miR-140 miRNA precursor reduced theluciferase activity of the reporter construct with the BD but nota mutated BD sequence, suggesting that miR-140 suppressesDnpep expression by binding to BD (Fig. 7C). Furthermore,transfection of the miR-140 precursor into chondrogenicATDC5 cells reduced Dnpep expression (Fig. 7D)"}
{"To test whether the BD sequencecould mediate the miR-140‚ÄìDnpep interaction, a luciferasereporter plasmid carrying the BD sequence was constructed.Cotransfection with a miR-140 miRNA precursor reduced theluciferase activity of the reporter construct with the BD but nota mutated BD sequence, suggesting that miR-140 suppressesDnpep expression by binding to BD (Fig. 7C). Furthermore,transfection of the miR-140 precursor into chondrogenicATDC5 cells reduced Dnpep expression (Fig. 7D)"}
{"To test whether the BD sequencecould mediate the miR-140‚ÄìDnpep interaction, a luciferasereporter plasmid carrying the BD sequence was constructed.Cotransfection with a miR-140 miRNA precursor reduced theluciferase activity of the reporter construct with the BD but nota mutated BD sequence, suggesting that miR-140 suppressesDnpep expression by binding to BD (Fig. 7C). Furthermore,transfection of the miR-140 precursor into chondrogenicATDC5 cells reduced Dnpep expression (Fig. 7D)"}
{"To determine whether the altered expression of EPB41L3 in metastatic cells is, in part, mediatedthrough its 3‚Äô UTR, we cloned the 3‚Äô UTRs of EPB41L3 downstream of a luciferase gene as areporter, and assayed EPB41L3 expression in NUGC-3 cells (low miR-223) and XGC-9811L cells(high miR-223). Transient transfection of NUGC-3 cells, with a low endogenous miR-223 expression,with the EPB41L3-3‚Äô-UTR-reporter construct and miR-223, led to a significant decrease of reporteractivity as compared to the control (Fig.4B). However, the activity of the reporter construct mutatedat the specific miR-223 target site at nt 89-95 was unaffected by a simultaneous transfection withmiR-223 (Fig.4B)."}
{"Conversely,antagonism of miR-296-5p by LNA transfection increasedluciferase activity ( Fig. 3A ). As a control, modulation ofmiR-296 levels using the miR-296 expression vector or theLNA for miR-296-5p did not signifi cantly modify luciferaseactivity when Huh-7 cells were transfected with pMIRempty vector ( Fig. 3A ). Functional derepression of luciferaseactivity was observed when critical residues of oneor both miR-296-5p binding sites within PUMA 3 ‚Ä≤ UTR(BS1 and BS2) were mutated (pMIR- PUMA -BS1/BS2mut)( Figs. 1 and 3B ). These results indicate that miR-296-5pfunctionally binds to both PUMA 3 ‚Ä≤ UTR binding sites."}
{"Conversely,antagonism of miR-296-5p by LNA transfection increasedluciferase activity ( Fig. 3A ). As a control, modulation ofmiR-296 levels using the miR-296 expression vector or theLNA for miR-296-5p did not signifi cantly modify luciferaseactivity when Huh-7 cells were transfected with pMIRempty vector ( Fig. 3A ). Functional derepression of luciferaseactivity was observed when critical residues of oneor both miR-296-5p binding sites within PUMA 3 ‚Ä≤ UTR(BS1 and BS2) were mutated (pMIR- PUMA -BS1/BS2mut)( Figs. 1 and 3B ). These results indicate that miR-296-5pfunctionally binds to both PUMA 3 ‚Ä≤ UTR binding sites."}
{"Measurements of luciferase activity inHEK 293 cells transfected with reporter plasmids containing the39UTRs of mCav1or hCAV1 showed reduced expression of theseconstructs in the presence of miR-103 (Fig. 4c). By mutating the conservedseed, we could fully reverse the miR-103-induced decrease inluciferase activity in both mCav1 and hCAV1 constructs (Fig. 4c)."}
{"Measurements of luciferase activity inHEK 293 cells transfected with reporter plasmids containing the39UTRs of mCav1or hCAV1 showed reduced expression of theseconstructs in the presence of miR-103 (Fig. 4c). By mutating the conservedseed, we could fully reverse the miR-103-induced decrease inluciferase activity in both mCav1 and hCAV1 constructs (Fig. 4c)."}
{"To verify whether or not ROCK2 and EZH2 are direct targets of miR-124, ROCK2 and EZH2 3‚Ä≤-UTRs (figure 2A) and two mutants containing the miR-124 binding sites were cloned downstream of the luciferase open reading frame. Thesereporter constructs were used to cotransfect HCC LM9 and HepG2 cells. Increased expression of miR-124 upon infection, significantly affected the luciferase expression, measured by the luciferase activity. Conversely, when we performedluciferase assays using a plasmid harbouring the 3‚Ä≤-UTR of ROCK2 and EZH2 mRNAs, in which the binding sites for miR-124 were inactivated by site-directed mutant genesis, the luciferase activity of mutant reporters were unaffected by thesimultaneous infection of miR-124 (figure 2B and supplementary figure S1)."}
{"To verify whether or not ROCK2 and EZH2 are direct targets of miR-124, ROCK2 and EZH2 3‚Ä≤-UTRs (figure 2A) and two mutants containing the miR-124 binding sites were cloned downstream of the luciferase open reading frame. Thesereporter constructs were used to cotransfect HCC LM9 and HepG2 cells. Increased expression of miR-124 upon infection, significantly affected the luciferase expression, measured by the luciferase activity. Conversely, when we performedluciferase assays using a plasmid harbouring the 3‚Ä≤-UTR of ROCK2 and EZH2 mRNAs, in which the binding sites for miR-124 were inactivated by site-directed mutant genesis, the luciferase activity of mutant reporters were unaffected by thesimultaneous infection of miR-124 (figure 2B and supplementary figure S1)."}
{"However, co-transfection with miR-200b or miR-15bmimics significantly reduced the luciferase activity inCAL27-res cells (P¬º0.01 or Po0.001, respectively,Figure 4a). However, when the miRNA targetingsequence was mutated in the reporter plasmids, transfectionwith mimics of miR-200b or miR-15b did notinfluence the relative luciferase activity (Figure 4a),"}
{"However, co-transfection with miR-200b or miR-15bmimics significantly reduced the luciferase activity inCAL27-res cells (P¬º0.01 or Po0.001, respectively,Figure 4a). However, when the miRNA targetingsequence was mutated in the reporter plasmids, transfectionwith mimics of miR-200b or miR-15b did notinfluence the relative luciferase activity (Figure 4a),"}
{"To confirm that Trp53inp1 is a direct target gene of miR-125b,the wild-type 30UTR of Trp53inp1 or the mutated 30UTR wascloned to downstream of the Renilla luciferase open readingframe (Figure 3a). We performed luciferase assays and foundthat miR-125b directly represses the expression of the reportergene with the Trp53inp1-30UTR but not that with mutated30UTR of Trp53inp1 (Figure 3a)."}
{"Each construct wasthen cotransfected with pre-miR-296-5p or a randomlygenerated negative control Pre-miR into 293A or HeLacells. In addition, in HeLa cells, each construct wascotransfected with anti-miR-296-5p. Cotransfection of thepGL3-IKBKE-39UTR construct containing the potentialtarget site and pre-miR-296-5p into either 293A or HeLacells resulted in decreased luciferase production of thereporter construct compared with the randomly generatednegative control (highly significant in 293A cells, P < 0.01)(Fig. 3D,E)."}
{"To test whether or not the regulation of miR-208 on p21is via a direct or indirect mechanism, we constructedluciferase reporters with entire wild-type p21 30UTR(pGL3-p21‚Äì30 UTR-wild-type) or a mutant p21 30UTRUTR (pGL3- p21‚Äì30 UTR-mutant-type), which wereboth confirmed by sequencing and then co-transfectedinto HEK293 cells with miR-208 mimics or mimicscontrol. We found that neither the mimics control northe mutant p21 30UTR had any effect on the luciferaseactivity in transfected HEK293 cells (Fig. 6a and b). Incontrast, miR-208 mimics decreased luciferase activitytransfected with pGL3-p21‚Äì30 UTR-wild-type (Fig. 6b)."}
{"We then transfected cultured DCs witheach reporter construct, as well as let-7i mimic or inhibitor. Luciferaseactivity was assessed at 24 h after transfection. As shownin Fig. 5, the translational level of the luciferase reporter wassignificantly decreased by let-7i mimic, but markedly increased bylet-7i inhibitor. No changes in luciferase were observed in cellstransfected with the mutant SOCS1 39-UTR construct whencombined with let-7i mimic or inhibitor."}
{"Figure 1 (Part B) shows miR-133a inhibition of Col1A1 30-UTRluciferasereporter construct signal. Cotransfection of miR-133a with the luciferase reporter gene linked to the wild-type30-UTR of Col1A1 resulted in a significant decrease of luciferaseactivity ( P<0.01). In contrast, no effect was observed whenmiR-133a was cotransfected with a construct containingmutated 30-UTR seed sequences."}
{"Cotransfection of miR-29b with theluciferase reporter gene linked to the wild-type 30-UTR ofCol1A1 resulted in a significant decrease of luciferase activity( P<0.01). In contrast, no effect was observed when miR-29bwas cotransfected with a construct containing mutated 30-UTRseed sequences."}
{"We amplified3 UTR regions surrounding these miR-23 sites and placedthem each individually downstream of an eGFP codingsequence for use in a GFP silencing assay. We only observedstrong silencing of the GFP signal with miR-23 mimics whenthe Has2, icat, and tmem2 3 UTR sequences were present(Figure 6B through G, Online Figure V, and Online Table I).Silencing of these GFP-3 UTR mRNAs was miR-23‚Äìdependent,because no silencing was observed after coinjectingmutated miR-23 mimics (Figure 6H through J) or after mutating2 nucleotides within the seed sequence of miR-23 target sites(Online Figure V). Together, these data reveal that miR-23targets several transcripts expressed in the embryonic heart."}
{"We amplified3 UTR regions surrounding these miR-23 sites and placedthem each individually downstream of an eGFP codingsequence for use in a GFP silencing assay. We only observedstrong silencing of the GFP signal with miR-23 mimics whenthe Has2, icat, and tmem2 3 UTR sequences were present(Figure 6B through G, Online Figure V, and Online Table I).Silencing of these GFP-3 UTR mRNAs was miR-23‚Äìdependent,because no silencing was observed after coinjectingmutated miR-23 mimics (Figure 6H through J) or after mutating2 nucleotides within the seed sequence of miR-23 target sites(Online Figure V). Together, these data reveal that miR-23targets several transcripts expressed in the embryonic heart."}
{"We amplified3 UTR regions surrounding these miR-23 sites and placedthem each individually downstream of an eGFP codingsequence for use in a GFP silencing assay. We only observedstrong silencing of the GFP signal with miR-23 mimics whenthe Has2, icat, and tmem2 3 UTR sequences were present(Figure 6B through G, Online Figure V, and Online Table I).Silencing of these GFP-3 UTR mRNAs was miR-23‚Äìdependent,because no silencing was observed after coinjectingmutated miR-23 mimics (Figure 6H through J) or after mutating2 nucleotides within the seed sequence of miR-23 target sites(Online Figure V). Together, these data reveal that miR-23targets several transcripts expressed in the embryonic heart."}
{"We next conducted 3 UTR luciferase reporter assays todetermine whether miR-195 could directly target Chek1 forrepression. A plasmid consisting of the Chek1 3 UTR linkedto a constitutively active luciferase reporter was transfectedinto COS cells along with increasing amounts of miR-195mimic. The Chek1 3 UTR luciferase reporter displayed adose-dependent repression in activity by miR-195 (Figure4C). Mutation of the miR-195 target site abolished repressionby miR-195, which demonstrates that this effect was dependenton binding of miR-195 to its target site within the 3 UTRof Chek1 (Figure 4C)."}
{Firstly, we constructed a reporter vector containing a consensus miR-145-binding site within the 3‚Ä≤-UTR (pMIR-PT, Table 1) as a positive control. After we co-transfected this reporter plasmid into HEK293 cells with pre-miR-145 or its control pre-miR, we found that luciferase expression significantly decreased in the HEK293 cells transfected with pre-miR-145 (Figure 2B).Co-transfection of pre-miR-145 with wild-type pMIR-MRE resulted in a suppression of luciferase gene expression, but co-transfection of pre-miR-145 and mutant pMIR-MUT did not }
{To demonstrate that IRS1 is a direct target of miR-144, we constructed independent reporter vectors that contained the different combinations of binding sites of miR-144 at the 3‚Ä≤UTR of IRS1 (Table S12) downstream of a firefly luciferase reporter gene. These constructs were then co-transfected with anti- or pre-miR-144 into HeLa cells. Cells transfected with pre-miR-144 exhibited a significant reduction in the luciferase activity which was reduced further when both miR-144 binding sites are present (Fig. 10). Likewise, transfection with anti miR-144 showed an increasing pattern of luciferase activity from constructs where only one binding site is present to that where both binding sites are included (Fig. 10). Site-directed mutagenesis of the miR-144 binding sites abolished interactions between miR-144 and IRS1 accordingly. These findings indicate IRS1 forms a direct target of miR-144.}
{The luciferase reporter pGL3-Sp1-UTR, which harboured miR-140 binding sites, was repressed by the co-transfection of pEGFP-Pri-miR-140 compared to the control in 293T cells (Fig. 5D). Endogenous Sp1 protein expression was also suppressed by the overexpression of miR-140, while its mRNA levels remained unchanged (Fig. 5E and F). This suppression could be released by mutations in miR-140 binding sites in the Sp1 3‚Ä≤ UTR domain (Fig. 5G). In addition}
{The luciferase reporter pGL3-Sp1-UTR, which harboured miR-140 binding sites, was repressed by the co-transfection of pEGFP-Pri-miR-140 compared to the control in 293T cells (Fig. 5D). Endogenous Sp1 protein expression was also suppressed by the overexpression of miR-140, while its mRNA levels remained unchanged (Fig. 5E and F). This suppression could be released by mutations in miR-140 binding sites in the Sp1 3‚Ä≤ UTR domain (Fig. 5G). In addition}
{The three programs all predicted that the binding sequence in 3‚Ä≤UTR of Osx was a very good match for the miR-637 seed. To verify this prediction, the target sequence (nt 387‚Äì713) of Osx 3‚Ä≤UTR (Wt) or the site-mutated sequence (Mu; the base that is underlined in Figure 5A) was cloned into the luciferase reporter vector pMIR (Figure 5A). Cos-7 cells were then cotransfected with WT vector and miRNA mimics, and the results showed a significant decrease in luciferase activity in miR-637‚Äìtransfected cells compared with NC (Figure 5B; p <0.01). The activity of the Mu vector was unaffected by a simultaneous transfection with miR-637 (Figure 5B). }
{To further validate this computational finding that miR-124a may negatively regulate JAG1, we generated a luciferase construct harboring the 3‚Ä≤-UTR fragment of JAG1 containing a broadly conserved binding site of miR-124a (Luc-JAG1, Fig. 5D) and a mutant luciferase construct with deletion of the binding site (Luc-JAG1-mu, Fig. 5D). Luciferase assay showed that miR-124a significantly repressed the luciferase activity in the 3T3 cell line transiently transfected with Luc-JAG1, compared with cells transfected with Luc-JAG1-mu (Fig. 5E), which is consistent with previous findings that JAG1 is a putative target of miR-124a [14].}
{Short luciferase targets corresponding to the strongest predicted miR-877 3‚Ä≤-UTR target sites for each of these genes where generated and expressed alongside either mirt-miR-877 or the pre-miR-877 control in HEK-293 cells. Normalization to non-specific control constructs demonstrated that six of these targets were silenced in response to mirt-miR-877 and pre-miR-877 despite highly similar seed sequences across all targets (Figure 5A). }
{"Dual-luciferase reporter analysis showedthat coexpression of miR-29b significantly inhibited theactivity of firefly luciferase that carried wildtype but notmutant 30-UTR of MMP-2 (Fig. 3A,B), indicating thatmiR-29b may suppress gene expression through itsbinding sequence at 30-UTR of MMP-2."}
{"Weinvestigated whether the 30-UTR of ZEB2 and BMI1 are functionaltargets of miR-708 in renal cancer. Transient transfectionof human A498 cancer cells with the respective 30-UTR plasmidsalong with different concentrations of miR-708 precursorled to a significant decrease in promoter activity when comparedwith the control vector (Fig. 5B and C) suggesting thatmiR-708 directly represses these genes. The luciferase activityof the reporter vectors containing a mutated 30-UTR of the respective genes was unaffected by miR-708."}
{"Weinvestigated whether the 30-UTR of ZEB2 and BMI1 are functionaltargets of miR-708 in renal cancer. Transient transfectionof human A498 cancer cells with the respective 30-UTR plasmidsalong with different concentrations of miR-708 precursorled to a significant decrease in promoter activity when comparedwith the control vector (Fig. 5B and C) suggesting thatmiR-708 directly represses these genes. The luciferase activityof the reporter vectors containing a mutated 30-UTR of the respective genes was unaffected by miR-708."}
{"To identify the miR-365 target region in the HDAC4mRNA, we mutated 3 nucleotides from the putativeseed region in the HDAC4 3 -UTR (Supplemental Fig.S2C). While transfection of miR-365 mimic suppressedthe luciferase activity of the wild-type HDAC4 3 -UTRreporter, this suppression was abolished in the mutatedHDAC4 3 -UTR reporter (Fig. 5E)."}
{"The CAMTA1 30-UTRwas fused to firefly luciferase and co-transfected togetherwith inhibitors against miR-9/9* (Figure 4B, panels 1 and 2),miR-106b (panel 3) or miR-17-5p (panel 3) into primaryglioblastoma cells. In all cases, firefly expression waselevated upon miRNA inhibition. Increased firefly activitywas not observed, when reporters with mutated miR-9 ormiR-9* binding sites were transfected."}
{"The CAMTA1 30-UTRwas fused to firefly luciferase and co-transfected togetherwith inhibitors against miR-9/9* (Figure 4B, panels 1 and 2),miR-106b (panel 3) or miR-17-5p (panel 3) into primaryglioblastoma cells. In all cases, firefly expression waselevated upon miRNA inhibition. Increased firefly activitywas not observed, when reporters with mutated miR-9 ormiR-9* binding sites were transfected."}
{"HEK 293T cells transfected with miR-124a expression plasmids produced significant amounts ofmiR-124a (P < 0.001; Supplementary Fig. 8a,b). The luciferase activity of the native Lhx2 3‚Ä≤-UTR plasmid was significantly reduced by all of the pre-miR-124a expression plasmids (P < 0.01; Fig. 5b). However, luciferase activity of the mutated Lhx2 3‚Ä≤-UTR plasmid was not attenuated by miR-124a (Fig. 5b), and the luciferase mRNA levels of both native and mutated Lhx2 3‚Ä≤-UTR plasmids were not significantly reduced(P < 0.004; Supplementary Fig. 8c,d)."}
{"To assess the effects ofmiR-758 on the 3 UTR of human Abca1, we used luciferasereporter constructs. miR-758 markedly repressed the activityof the Abca1 3 UTR reporter construct (Figure 2C). Specificsite-directed mutations in site 2, which is widely conservedamong several species, abolishes the miR-758 repression ofAbca1 3 -UTR activity, suggesting that this site is the mostimportant in the posttranscriptional repression of Abca1 bymiR-758 (Figure 2D)."}
{"To assess the effects ofmiR-758 on the 3 UTR of human Abca1, we used luciferasereporter constructs. miR-758 markedly repressed the activityof the Abca1 3 UTR reporter construct (Figure 2C). Specificsite-directed mutations in site 2, which is widely conservedamong several species, abolishes the miR-758 repression ofAbca1 3 -UTR activity, suggesting that this site is the mostimportant in the posttranscriptional repression of Abca1 bymiR-758 (Figure 2D)."}
{"Toassess miRNA binding to the 3 UTR, we constructed luciferasereporters with the full-length pten 3 UTR. The reporter was testedin NIH3T3 cell lines stably overexpressing miR-19b, miR-19a, ormiR-17.We observed significant suppression of luciferase productionby miR-19b, but not miR-17 or miR-19a (Figure 5B). Inaddition, site-directed mutagenesis of the miR-19‚Äìbinding motifswithin the 3 UTR of pten26 completely abolished this suppression(Figure 5B)"}
{"Toassess miRNA binding to the 3 UTR, we constructed luciferasereporters with the full-length pten 3 UTR. The reporter was testedin NIH3T3 cell lines stably overexpressing miR-19b, miR-19a, ormiR-17.We observed significant suppression of luciferase productionby miR-19b, but not miR-17 or miR-19a (Figure 5B). Inaddition, site-directed mutagenesis of the miR-19‚Äìbinding motifswithin the 3 UTR of pten26 completely abolished this suppression(Figure 5B)"}
{"Luciferase activity was measured after 24h of transfection (Figure 2B). Our data demonstrated that relativeluciferase unit was decreased (.70%) in wild-type 3# UTR transfectedPC cells that were cotransfected with miR-150 mimic as comparedwith that cotransfected with miR-NC. Furthermore, cellstransfected with MUT 3# UTR were resistant to the suppressor activityof miR-150 (Figure 2B). Thus, our data strongly suggest that miR-150 negatively regulates the expression of MUC4 by directly targetingthe 3# UTR of MUC4 transcript."}
{"pISO-TIMP3-Luc plasmid harboring 3 -UTR of TIMP3 (12).Luciferase expression was reduced significantly ( 50%, Fig.1D) when transfected with miR-221, -222, or -181b whencompared with the negative control miRs (Fig. 1E). Conversely,deletion or mutation of the miR seed sequencesblocked the inhibitory effects of all three miRs, and luciferaseactivity was completely recovered."}
{"pISO-TIMP3-Luc plasmid harboring 3 -UTR of TIMP3 (12).Luciferase expression was reduced significantly ( 50%, Fig.1D) when transfected with miR-221, -222, or -181b whencompared with the negative control miRs (Fig. 1E). Conversely,deletion or mutation of the miR seed sequencesblocked the inhibitory effects of all three miRs, and luciferaseactivity was completely recovered."}
{"pISO-TIMP3-Luc plasmid harboring 3 -UTR of TIMP3 (12).Luciferase expression was reduced significantly ( 50%, Fig.1D) when transfected with miR-221, -222, or -181b whencompared with the negative control miRs (Fig. 1E). Conversely,deletion or mutation of the miR seed sequencesblocked the inhibitory effects of all three miRs, and luciferaseactivity was completely recovered."}
{"pISO-TIMP3-Luc plasmid harboring 3 -UTR of TIMP3 (12).Luciferase expression was reduced significantly ( 50%, Fig.1D) when transfected with miR-221, -222, or -181b whencompared with the negative control miRs (Fig. 1E). Conversely,deletion or mutation of the miR seed sequencesblocked the inhibitory effects of all three miRs, and luciferaseactivity was completely recovered."}
{"Suppression by pre-miR 206 was  50% for bothreporters harboring the first two miR-206 sites (1412‚Äì1889)and all three miR-206 sites (1412‚Äì2766) (p < 0.01) (Fig. 3c).These 3¬¢UTR reporter assays suggested that the first twotarget sites are required for regulation by miR-206 to occur.We next set out to determine which of the first two miR-206 target sites are functional. The two miR-206 seedsequences were individually mutated in the context of the1412‚Äì1889 vector (MUT 1 and MUT 2). Neither of thesemutations in isolation abrogated the repressive effect of miR-206 (Fig. 3d). We thus created a construct that had bothtarget sites mutated (MUT 1&2) and found that thiscompletely attenuated the repressive effects of miR-206(p > 0.01) (Fig. 3e). Thus, the first two miR-206 sites arerequired for regulation by miR-206. We noticed that the thirdmiR-206 target site also had supplementary base-pairing withmiR-206, suggesting that this site might provide a strongerinteraction with the microRNA compared with the othertarget sites (Fig. 2b)."}
{"Suppression by pre-miR 206 was  50% for bothreporters harboring the first two miR-206 sites (1412‚Äì1889)and all three miR-206 sites (1412‚Äì2766) (p < 0.01) (Fig. 3c).These 3¬¢UTR reporter assays suggested that the first twotarget sites are required for regulation by miR-206 to occur.We next set out to determine which of the first two miR-206 target sites are functional. The two miR-206 seedsequences were individually mutated in the context of the1412‚Äì1889 vector (MUT 1 and MUT 2). Neither of thesemutations in isolation abrogated the repressive effect of miR-206 (Fig. 3d). We thus created a construct that had bothtarget sites mutated (MUT 1&2) and found that thiscompletely attenuated the repressive effects of miR-206(p > 0.01) (Fig. 3e). Thus, the first two miR-206 sites arerequired for regulation by miR-206. We noticed that the thirdmiR-206 target site also had supplementary base-pairing withmiR-206, suggesting that this site might provide a strongerinteraction with the microRNA compared with the othertarget sites (Fig. 2b)."}
{"In contrast, injection of miR-26b strongly repressed GFPexpression from the wild-type reporter, but not fromreporters containing the antisense or point-mutated39 UTRs (Fig. 1B,C; Supplemental Fig. S2)."}
{"To further investigate the interaction between miR-143 and the target site of miR-143 in the coding region of PTN, we generated a PTN mutant with 2 bp mutations in seed region of miR-143 binding site (PTN site-m) (Fig. 3A). The mutation eliminated the repressive effect of miR-143 on PTN protein expression (Figs. 4B and C). These results indicate that miR-143 may directly target the coding region of PTN."}
{we constructed a plasmid with a luciferase transcript and the 3‚Ä≤ UTR of NT-3. MiR21 but not a scrambled microRNA suppressed luciferase activity (Fig. 3, CTL mean 1 ¬± 0.04 SEM; miR21 mean 0.4 ¬± 0.05 SEM; scrambled miR mean 1.4 ¬± 0.3 SEM; Kruskal‚ÄìWallis Dunn's Multiple comparison P *** < 0.001). To confirm direct suppression by miR21 on translation of the NT-3 3‚Ä≤ UTR construct, we mutated the putative miR21-binding site in the NT-3 3‚Ä≤ UTR. Suppression of luciferase activity by miR21 was lost with co-transfection of the mutated NT-3 3‚Ä≤ UTR (Fig. 3, }
{The luciferase activity of the ‚ÄòPerfect‚Äô A-clone was lower than the ‚ÄòScr‚Äô B-clone (p<0.0001)(Fig. 6B). The B-construct had increased activity compared with pGL4.13, which could be due to destruction of a negative regulatory signal by the cloned insertion. Mutation of the miR-21 target site of Srebf1 led to an increased luciferase activity (Fig. 6B, ‚ÄòC‚Äô vs. ‚ÄòD‚Äô, p<0.0001). }
{" As expected, the luciferase activity of wild-type 3‚Ä≤-UTR (pMIR-luc-sirt1-3‚Ä≤-UTR) was dramatically inhibited by the introduction of miR-199a, but not by negative control, while mutant of the binding site completely abolished the ability of miR-199a to inhibit luciferase expression (Fig. 4C). These results demonstrated that sirt1 is a potential target of miR-199a."}
{When tested using the pEGFP-C3 and pGL3 dual luciferase reporter vectors containing a complete wild-type IŒ∫BŒ± 3‚Ä≤-UTR, miR-30e* robustly inhibited the expression of GFP, but not the GFP‚ÄìŒ≥-tubulin control, in glioma as well as 293FT cells (Figure ‚Äã(Figure4C).4C). }
{"We alsomisexpressed our Macho-1 UTR sensor specifically in PNS tissueusing a gelsolin promoter, to assay regulation by endogenous miR-124. Again, Macho-1 expression was almost completely abolished(93.9% downregulation, PÔÄΩ2.9 10‚Äì37) (Fig. 5C). To determine ifthis effect was mediated specifically by miR-124, we repeated theassay using Macho-1 mutant 3 UTRs with scrambled miR-124 seedsites (Fig. 5A). Mutating the seed sites restored target expressionlevels to ~75% of that of control embryos expressing a neutral3 UTR reporter (Fig. 5C,D), showing that Macho-1 downregulationis predominately regulated by miR-124."}
{"The reporter plasmids were cotransfectedwith miR-622 mimic or miR-622 inhibitor. The wild-typereporter wt-K-Ras plasmids cotransfected with miR-622 mimic exhibitedinhibited luciferase activity compared with the vector controlgroup or the NC mimic group, whereas plasmids cotransfected withmiR-622 inhibitor exhibited increased luciferase activity comparedwith the vector control group or NC inhibitor group. In contrast, theluciferase activity was not affected by mutant type mt-K-Ras plasmidcotransfection with miR-622 mimic and miR-622 inhibitor, potentiallybecause miR-622 was unable to recognize the complete mutantbinding sites of K-Ras mRNA, thereby disrupting the interactionbetween miR-622 and K-Ras (Figure 4F). These data indicate thatK-Ras is targeted directly by miR-622."}
{"The reporter plasmids were cotransfectedwith miR-622 mimic or miR-622 inhibitor. The wild-typereporter wt-K-Ras plasmids cotransfected with miR-622 mimic exhibitedinhibited luciferase activity compared with the vector controlgroup or the NC mimic group, whereas plasmids cotransfected withmiR-622 inhibitor exhibited increased luciferase activity comparedwith the vector control group or NC inhibitor group. In contrast, theluciferase activity was not affected by mutant type mt-K-Ras plasmidcotransfection with miR-622 mimic and miR-622 inhibitor, potentiallybecause miR-622 was unable to recognize the complete mutantbinding sites of K-Ras mRNA, thereby disrupting the interactionbetween miR-622 and K-Ras (Figure 4F). These data indicate thatK-Ras is targeted directly by miR-622."}
{"The reporter plasmids were cotransfectedwith miR-622 mimic or miR-622 inhibitor. The wild-typereporter wt-K-Ras plasmids cotransfected with miR-622 mimic exhibitedinhibited luciferase activity compared with the vector controlgroup or the NC mimic group, whereas plasmids cotransfected withmiR-622 inhibitor exhibited increased luciferase activity comparedwith the vector control group or NC inhibitor group. In contrast, theluciferase activity was not affected by mutant type mt-K-Ras plasmidcotransfection with miR-622 mimic and miR-622 inhibitor, potentiallybecause miR-622 was unable to recognize the complete mutantbinding sites of K-Ras mRNA, thereby disrupting the interactionbetween miR-622 and K-Ras (Figure 4F). These data indicate thatK-Ras is targeted directly by miR-622."}
{"Twenty-four and 48 hours later,luciferase activity in BEAS-2B cells cotransfected with pGL3‚Äì4E-BP1 vector and miR-125b was decreased by 80% comparedwith control miRNA (Figure 3C; see Figure E2). When themiR-125b binding site was mutated in the 4E-BP1 39-UTR,luciferase activity was no longer inhibited by miR-125b mimic(Figure 3C; see Figure E2)."}
{"The vectorwas cotransfected with miR-210 or control intoHEK293T cells, and the relative luciferase activity wasdetermined. The results showed that when comparedto the control, the relative luciferase activity was significantlydecreased by miR-210, whereas the luciferaseactivity was not altered by the vector containing themutant 30-UTR (Fig. 4C)."}
{MiR-469 (Fig. 4) significantly decreased luciferase activity in the plasmid containing full-length coding region (103/423 nt) along with 172 bp 3' non-coding region (423/594 nt) of Prm2 (103/594 nt) (psiCHECK-2/Prm2). Similar inhibition was noted in the construct of 3' deletion from nt 386 (103/386 nt) (psiCHECK-2/Prm2-a) when compared to the negative control group with scrambled RNA. However, miR-469 did not have any effect on the plasmid containing isolated fragment from 365-594 nt (psiCHECK-2/Prm2-b).}
{MiR-469 (Fig. 4) significantly decreased luciferase activity in the plasmid containing full-length coding region (103/423 nt) along with 172 bp 3' non-coding region (423/594 nt) of Prm2 (103/594 nt) (psiCHECK-2/Prm2). Similar inhibition was noted in the construct of 3' deletion from nt 386 (103/386 nt) (psiCHECK-2/Prm2-a) when compared to the negative control group with scrambled RNA. However, miR-469 did not have any effect on the plasmid containing isolated fragment from 365-594 nt (psiCHECK-2/Prm2-b).}
{MRE was then proposed to be localized in the coding region rather at 3‚Äô-UTR. mRNA-fold program confirmed the formation of duplex structure of TP2 mRNA (nt 352-383) with perfect match at nt 376-383 to the 5‚Äô seed region of miR-469 (Fig 6B). Significant loss of luciferase activity was observed in the construct containing the MRE sequence alone (psiCHECK-2/TP2-MRE). Mutation of the proposed MRE site in the psiCHECK-2 vector containing either coding region of TP2 (psiCHECK-2/TP2-CD-X) or the isolated MRE element (psiCHECK-2/ TP2-MRE-X) completely recovered the luciferase activity in the presence of miR-469.}