{"The mRNA expression level of NDRG3 was detected by RT-PCR and qPCR 48 h post-transfection. The result (Fig. 3B and D) showed that the mRNA expression levels of NDRG3 in cells transfected with pS-miR122 were significantly reduced as compared to the mock and untreated cells. The NDRG3 protein level was determined by Western blot analysis 72 h post-transfection (Fig. 3C), demonstrating that NDRG3 protein was significantly reduced in cells transfected with pS-miR122, as compared to the mock and untreated cells. These data suggest that NDRG3 is one of the direct downstream targets regulated by miR-122.Figure 2. NDRG3 is a candidate target of miR-122. (A) By querying MiRanda and TargetScan, NDRG3 was identified in both programs as a potential target gene of miR-122. The prediction results and score are listed. (B) The TargetScan prediction shows a common binding sequence on the 3'-UTR of NDRG3 conserved across vertebrate species, including the human, chimpanzee, Rhesus and mouse. (C) The duplex of miR-122 and the 3'-UTR of NDRG3 are shown. The free energy of miR-122 binding to the targeting site of NDRG3 was calculated using RNAhybrid (mfe, -25.8 kcal/mol).Figure 3. The expression level of NDRG3 is down-regulated by transfection with pS-miR122. (A) pEGFP-N1 was co-transfected with pS-miR122 into HepG2.2.15 cells. Compared to the mock and untreated cells, the expression level of miR-122 48 h after pS-miR122 transfection was elevated in the HepG2.2.15 cells (P<0.001). (B) RT-PCR was performed 48 h post-transfection. mRNA of NDRG3 in cells transfected with pS-miR122 was significantly reduced compared to the mock and untreated cells (P<0.01). (C) The results of Western blot analysis show that the protein expression level of NDRG3 was down-regulated 72 h after transfection with pS-miR122, as compared to the mock and untreated cells (P<0.01). (D) The level of NDRG3 mRNA was detected by real-time qPCR 48 h post-transfection. The transcription of NDRG3 in cells transfected with pS-miR122 was significantly decreased, as compared to the mock and untreated cells (P<0.01)."}