6C), suggesting that a vast abundance of miR-141 could also disrupt the stability of DLC-1 mRNA. Next, we examined whether alterations in DLC-1 protein levels in HCV-infected human primary hepatocytes influence viral RNA level. We assessed the changes in HCV RNA levels within the infected cells, as well as in virions released in culture media of HCV1a-infected hepatocytes. Changes in HCV RNA were quantified by way of nested RT-PCR13 of infected cells’ RNA (Fig. 6A), and the effects of miR-141 modulation on virus released in the culture media of HCV1a-infected hepatocytes were find more analyzed by way

of quantitative RT-PCR (Fig. 6B). The results represent genomic equivalents of HCV RNA normalized with World Health Organization standards learn more for HCV. We depleted intracellular miR-141 through transfection with the miR-141 antagomirs or artificially increased miR-141 inside cells through transfection with miR-141 mimics. The depletion of miR-141 resulted in inhibition of HCV RNA replication in infected hepatocytes, whereas artificially increasing miR-141 resulted in increased viral RNA replication (Fig. 6A, lanes 1-3). Thus, HCV RNA replication in infected hepatocytes appears to be inversely related to the intracellular level of miR-141 and perhaps to its targeted DLC-1 (Figs. 5 and 6). The efficiency of virus released into the culture media of HCV1a-infected primary hepatocytes (Fig. 6B)

appears to be quantitatively more severely affected by the depletion of miR-141. Similarly, the increase in virus released

into the culture medium in response to artificially increased miR-141 (through transfection with miR-141 mimics) was higher than the increase in HCV RNA within the infected cells (compare the percentage inhibition of HCV RNA replication and the released viral RNA in Figs. 6A and 6B). Although the reasons for the quantitative differences in miR-141 modulated see more HCV replication and mature virus particles are not entirely clear, the collective results suggest that HCV replication in infected hepatocytes relies on miR-141–mediated depletion of tumor suppressor DLC-1. The reciprocal relation between the cellular DLC-1 protein level and miR-141 in HCV-infected cells suggests that virus replication modulates the abundance of DLC-1 tumor suppressor protein, which subsequently influences the efficiency of viral RNA replication and the release of mature virus particles from infected hepatocytes. However, these findings do not support a direct role of either miR-141 or DLC-1 protein in the regulation of HCV replication. Functional validation of the role of DLC-1 as a tumor suppressor has been examined based on its effect on cell growth.28 We next asked whether intracellular changes in DLC-1 protein influence the propagation of HCV-infected primary hepatocytes. Cell proliferation was analyzed by way of immunostaining for Ki67 nuclear antigen (Fig. 7).