IRES特异性IRNA对HCV IRES启动蛋白翻译细胞内抑制作用

Inhibitory effect of IRES specific inhibitor RNA on HCV IRES mediated protein translation

目的:研究核糖体内部进入位点(internalribosomeentrysite,IRES)特异性抑制性RNA(inhibitorRNA,IRNA)细胞内对丙型肝炎病毒(HCV)IRES介导蛋白翻译的抑制作用.方法:体外应用脂质体细胞转染法,将IRNA及其突变体mIRNA真核表达载体pcRz-IRNA/pcRz-mIRNA转染人肝癌细胞株(HHCC),经G418筛选4wk后建立IRNA及mIRNA表达株;以相同的方法构建pcHCVcluc转染株;以脂质体介导细胞转染法将pCMVNCRLuc转染IRNA及mIRNA细胞株,于转染后48h检测荧光素酶表达量;将IRNA及mIRNA真核表达体转染pcHCVcLuc表达株,于转染后不同时间检测荧光素酶表达量.结果:HCVIRES介导蛋白翻译在IRNA表达株明显受到抑制,同样IRNA对HCV翻译复制子的蛋白翻译作用有明显抑制性;突变体mIRNA表达株和空载体对照株中未见相似的抑制性.结论:pcHCVcluc在HHCC细胞中获得有效表达;IRES特异性IRNA能有效的抑制HCVIRES介导细胞内蛋白翻译作用.

AIM:To explore the inhibitory effect of internal ribosome entry site (IRES) specific inhibitor RNA (IRNA) on HCV IRES mediated protein translation in vivo. METHODS:Human hepatic carcinoma cell line (HHCC) was transfected with the eukaryotic vectors of IRNA or mIRNA (pcRz-IRNA or pcRz-mIRNA),and then selected with G418 for 4 weeks.HHCC expressing IRNA or mIRNA was cotransfected with pCMVNCRluc containing HCV IRES.HHCC stably expressing pcHCVcluc was transfected with pcRz-IRNA, and pcRz-mIRNA,respectively, the luciferase activity was examined at desired time post-transfection. RESULTS:The pCMVNCRluc was efficiently suppressed in HHCC expressing IRNA rather than the cell line expressing mIRNA.The IRES specific IRNA inhibited expression of HCV IRES mediated luc gene by 20 % to 80 % in pcHCVcluc expressing cell after transfection; However, no inhibitory effect of the mutant IRNA was observed. CONCLUSION:pcHCVcluc could be expressed successfully in HHCC, and IRNA inhibited HCV IRES mediated gene expression in vivo.