短发夹状双链RNA抑制丙型肝炎病毒IRES介导的基因表达

Inhibition of HCV IRES controlled reporter gene expression by RNA interference

目的研究载体表达的短发夹状双链RNA(shRNA)对丙型肝炎病毒(HCV)IRES介导的基因表达的特异性抑制作用。方法构建HCV IRES调控的绿色荧光蛋白表达载体(pIRES-GFP)和虫荧光素酶表达载体(p5’ UTR-Luc),以及针对HCV IRES的shRNA表达载体(pshRNA-HCV),共转染HepG2细胞,于转染后24、48,72 h 观察绿色荧光的强弱,用Western blot检测绿色荧光蛋白的表达,半定量逆转录聚合酶链反应法检测GFP的mRNA水平,双荧光素酶系统检测虫荧光素酶活性。结果pshRNA-HCV作用组绿色荧光强度明显弱于未干扰组,GFP蛋白表达量及虫荧光素酶活性降低60％-70％,半定量逆转录聚合酶链反应显示pshRNA-HCV导致了GFP基因mRNA水平的降低。结论针对HCV IRES的shRNA能够显著和特异地抑制该区域调控的蛋白表达水平及mRNA水平,该研究结果为利用RNA干扰技术治疗HCV感染进行了初步探索。

Objective To develop a RNAi approach that specifically targets the HCV IRES sequence by vectorexpressed short hairpin RNA （shRNA） in vitro, and to assess the inhibitory effect of the shRNA on reporter gene expression. Methods Eukaryotic expressing plasmids, pIRES-GFP and p5′ UTR-Luc containing GFP or luciferase gene controlled by HCV IRES were cotransfected into HepG2 cells with either a RNAi plasmid pshRNA-HCV or a control plasmid pTZU6＋ 1. At 24, 48, 72 hours post transfection, the fluorescence in the transfected cells was studied using fluorescence microscopy. The levels of GFP RNA were determined using RT-PCR and those of protein were determined using Western blot. The activities of luciferase were assayed using a dual luciferase assay system. Results The introduction of RNAi plasmid efficiently and specifically downregulated the expression of the reporter gene. RT-PCR showed that the RNAs of GFP gene were distinctly reduced （about 60%） when the pIRES-GFP was cotransfected with pshRNA-HCV, whereas the control vector did not exhibit inhibitory effect on the mRNA level, according to Western blot assay. The luciferase activity also decreased by 60%-70% in comparison to the control plasmid. Conclusion Our results demonstrate that the shRNA targeting HCV IRES shows a strong inhibitive effect on the expression of the reporter gene controlled by this sequence, suggesting that RNAi-based anti-HCV strategy may represent a potential approach in the therapy of HCV infection.