Specifically, 50 nM of miR-196 mimic decreased the firefly luciferase activity by ≈59% (Supporting Fig. 1B), whereas MMNC had no significant effect on reporter luciferase activity (Supporting Fig. 1C). Next, we cotransfected 9-13 cells with luciferase reporter construct containing a 4-nt mutant Bach1 3′-UTR, which we called pGL3-Bach1-Mut (Fig. 4A), and with pRL-TK, and with miR-196 mimic or miR-155 mimic22 (a negative miR, with no changes of predicted miR-155 binding sites Liproxstatin-1 ic50 in pGL3-Bach1-WT and pGL3-Bach1-Mut), and assayed the luciferase reporter activity. miR-196 mimic transfection significantly decreased luciferase activity in a dose-dependent fashion, whereas miR-196 did not change reporter activity

in cells transfected with the reporter construct containing mutant binding sites for miR-196 (Fig. 4B). miR-155 significantly decreased reporter activity in both pGL3-Bach1-WT

and pGL3-Bach1-Mut (Fig. 4C). These results demonstrate that miR-196 directly RO4929097 supplier regulates Bach1 gene expression and miR-196 mediates down-regulation of Bach1 though the 3′-UTR of Bach1 mRNA. To further establish the direct interaction between miR-196 and Bach1–3′-UTR, we created mutant miR-196 (Fig. 5A) in which seed match sites for the 3′-UTR of Bach1 mRNA were abolished. Cells were cotransfected with pGL3-Bach1-WT, and with pRL-TK, and with increasing concentrations of miRNA mimic negative control, miR-196 mimic, or mutant miR-196 mimic, and luciferase reporter activity was assayed. As anticipated, miR-196 resulted in a significant reduction in luciferase activity in a dose-dependent fashion, which was consistent with our previous observations, whereas miRNA negative controls and mutant miR-196 did medchemexpress not affect luciferase activity (Fig. 5B), further indicating the direct interaction between miR-196 and the 3′-UTR of Bach1 mRNA. We predicted that mutant miR-196 (miR-196-Mut) containing base complementarity with mutant pGL3-Bach1 (pGL3-Bach1-Mut) should restore its

effect on mutant reporter (Bach1-3′-UTR-Mut) activity, because they again match perfectly in their seed regions (Fig. 5C). As we predicted, mutant miR-196 mimic significantly inhibited luciferase activity in cells transfected with pGL3-Bach1-Mut, which was mutated to fit mutant miR-196, whereas no significant effects of miR-196-WT on mutant reporter (pGL3-Bach1-Mut) luciferase activity were observed, further establishing the direct interaction between miR-196 and the 3′-UTR of Bach1 mRNA (Fig. 5D). To determine whether miR-196 represses HCV mRNA and protein expression, HCV replicon cell lines Con1 and 9-13 cells were transfected with miR-196 mimic or miRNA mimic negative control. As shown in Fig. 6A,B, 50 nM of miR-196 mimic resulted in a significant reduction of HCV replication by ≈50% in Con1 cells and NS5A protein levels by ≈52%, compared with the same concentration of miRNA mimic negative control. When Bach1 gene expression was silenced by Bach1-siRNA (Fig.