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Inhibition of Hepatitis C Virus Replication by Small Interfering RNA in Cells Infected by HCV

Author: XingXiaoKang
Tutor: ChenZhi
School: Zhejiang University
Course: Internal Medicine
Keywords: siRNA HCV Huh-7.5.1cell Hepatitis C HCV infected cell
CLC: R363
Type: Master's thesis
Year: 2012
Downloads: 32
Quote: 0
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Abstract


Objective:To observe the inhibition effect-of siRNA on virus replication in HCV infected cells as well as joint effects of the siRNA in different sections of targeting HCV genome on HCV.Methods:HCV genome plasmid was transcribed into RNA in vitro, and Huh-7.5.1cells were transfected by lipofectin. The culture supernatant containing virus particles of transfected cells was collected to infect normal Huh-7.5.1cells to construct HCV infection cell model, and the expression of HCV RNA and HCV core protein in the infected cells were detected using PCR agarose gel electrophoresis and Western blot respectively. Then infected cells were treated with different concentrations of IFNa-2b, and the relative amount of intracellular HCV RNA was detected by fluorescence quantitative PCR. siRNA sequences specific to four gene segments of HCV (Core, NS3, NS4B, NS5B) were designed and chemically synthesized, and then were used singly and in pair respectively to transfect HCV infected cells with different concentration (0.1nM,10nM,100nM). Relative amounts of intracellular HCV in each group were detected with PCR agarose gel electrophoresis and fluorescence quantitative PCR. PKR mRNA of siRNA transfected cells was detected by fluorescence quantitative PCR and the activity of transfected cells was measured with CCK-8test to further analyze nonspecific effects of siRNA.Results:There were expressions of HCV RNA and HCV core protein detected in HCV infected cells, and HCV could be effectively inhibited by IFNa-2b in dose dependent manner. Compared with the control group, HCV RNA in infected cells decreased significantly when4kinds of siRNA (siCore, siNS3, siNS4B, siNS5B) were transfected in the concentrations of10nM and100nM (P<0.01). In the concentrations of0.1nM and10nM, HCV RNA in cells transfected with siCore, siNS3, siNS5B in pair were significantly decreased in the comparison of the groups transfected singly (P<0.01). There were no significant changes in intracellular PKR mRNA levels in the transfected cells by4siRNA, and there were no obvious effects on cell activity.Conclusion:siRNA of targeting HCV genome can effectively inhibit virus replication in HCV infected cells. At low concentrations, the use of siCore, siNS3, siNS5B in pair has obvious combined antiviral effect.

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