DLC-1 has been shown to be inactivated PF-562271 in HCC7, 27 and to serve as a tumor suppressor gene,28, 29 and thus represents an important choice for further analysis. Validation of

miRNAs (miR-141 and miR-200a) target recognition was based on luciferase reporter vectors containing the 3′-UTR of DLC-1 mRNA. We observed that the introduction of miR-141 and miR-200a mimics inhibited luciferase, whereas the transfection of miRNA antagomirs (2′-O-methyl–modified antisense oligonucleotides) restored luciferase expression (Supporting Information Fig. 2). The results suggest that the DLC-1 3′-UTR indeed harbors target sequences for miR-141 and miR-200a, and that alterations in the miRNA levels could regulate intracellular DLC-1 expression. miR-141 and miR-200a share identical 5′-seed sequences (Supporting Information Fig. 3); these 3-deazaneplanocin A cost studies have focused on the biological validation of miR-141–targeted DLC-1 expression and its effect on HCV replication. To validate whether the increase in intracellular miR-141 during HCV infection targets DLC-1 expression, we introduced luciferase DLC-1 3′-UTR reporter in HCV1a-infected

cells, with or without miR-141 antagomirs (Fig. 3). Expression of DLC-1 3′-UTR luciferase was down-regulated in HCV1a-infected cells. The expression of DLC-1 in HCV-infected cells was restored when miR-141 antagomirs were introduced by way of cotransfection (Fig. 3). The results suggest that DLC-1 expression in HCV1a-infected cells

MCE is regulated by intracellular miR-141. We next examined whether increased miR-141 in HCV-infected cells reduced DLC-1 protein in host cells. Western blot analysis of HCV-infected hepatocytes (infected either with HCV genotypes 1a, 2a, or the JFH1 strain) showed reduced DLC-1 protein levels (between 50% and 60% within 72 hours postinfection) compared with uninfected cells (Fig. 4). Next, we validated the effects of miR-141 on DLC-1 expression (in uninfected and HCV-infected hepatocytes) by either depleting miR-141 with antagomirs or artificially increasing the miR-141 levels by transfection with miR-141 mimic oligonucleotides (Fig. 5). Increasing miR-141 inhibited DLC-1 protein in uninfected cells (Fig. 5, lanes 2 and 5); whereas depleting miR-141 with miR-141 antagomirs derepressed DLC-1 expression (Fig. 5, lanes 3 and 6). There was no further inhibition of DLC-1 in HCV-infected cells upon addition of the miR-141 mimic (Fig. 5, lane 5), presumably because the miR-141 target sites within DLC-1 3′-UTR are saturated with the increased levels of miR-141. These findings suggest that miR-141 regulates DLC-1 protein expression inside cells. The inhibition of DLC-1 protein was not accompanied by a parallel decrease in DLC-1 mRNA, suggesting that miR-141 primarily targets translational inhibition of DLC-1.