Establishment Of Hcv-infected Huh7 Cell Model And Inhibition Of MiRNA On HCV RNA

Objective: To establish a HCV-infected Huh7 cell model in vitro, and investigate the inhibitive effects of mi RNA on HCV RNA replication in cells infected by HCV in vitro.Methods: 1.Huh7 was incubated with serum from a HCV patient(HCV 1b) for 72 hours, and the levels of HCV RNA were determined by real time quantitative PCR(Q-PCR). Cells and supernatant were harvested at various time intervals. Both the plus and minus strands of HCV RNA in supernatant were examined by RT-PCR.The expression of HCV Core and NS4 B proteins was determined by Western blot. 2.The recombination vectors of HCV 1b type Core and NS4B(pc DNA6.2-GW/Em GFP-mi R-Core, pc DNA6.2-GW/Em GFP-mi RNS4B)were transfected respectively and co-transfected into infected Huh7 cells by lipofectamine, simultaneously the infected Huh7 cells transfected with pc DNA6.2-GW/Em GFP-mi R-negative and untransfected infected Huh7 cells were used as negative control( respectively transfecte the recombination vectors of 3μg/ml and 6μg/ml), the infected Huh7 cells incubated with interferon as the positive control(the interferon of 3000IU/ml), the levels of HCV RNA were determined by Q-PCR after 48 hours.Results: 1.The high levels HCV RNA of infected Huh7 cells could be detected by Q-PCR. The plus stand of HCV RNA was detected persistently in supernatant and cells after infected 3, 6, 9, 12, 16, 20, 25, 30 days, and the minus stand was detected occasionally in cells after infected for 3, 6, 9, 12, 20 days, HCV Core and NS4 B proteins were detected in the Huh7 cells transfected with HCV RNA. 2.In the infected Huh7 cells of 3μg/ml recombination vectors concentration, the relative levels of 5’NTR m RNA were less in the cells of Groups mi R-Core, mi R-NS4 B, mi R-Core+mi R-NS4 B than those in the cells of Group mi R-negative and Group blank control(p<0.01), and these were higher than positive control(p<0.01), and there were no significant difference between the other groups(P>0.05). In the infected Huh7 cells of 6μg/ml recombination vectors concentration, the relative levels of 5’NTR m RNA were less in the cells of Groups mi R-Core, mi R-NS4 B, mi R-Core+mi R-NS4 B than those in the cells of Group mi R-negative and Group blank control(p<0.01), the Group mi R-Core, mi R-NS4 B were higher than positive control(p<0.01), the Group mi R-Core+mi R-NS4 B were less than positive control(p<0.01), the Group mi R-Core+mi R-NS4 B were less than the Group mi R-Core, mi R-NS4B(p<0.01), and there were no significant difference between the other groups(P>0.05).Conclusions: 1.The HCV-infected Huh7 cell model was successfully established. 2. mi RNA against HCV Core and NS4 B region can effectively inhibit HCV replication in HCV infected cells,and they have synergies.