SiRNA对丙型肝炎病毒5′非翻译区的干扰作用实验研究

Effect of short interfering RNAs on hepatitis C virus 5' untranslated region expression

目的研究多种siRNA对丙型肝炎病毒(HCV)5′非翻译区(5′-UTR)的干扰作用。方法以绿色荧光蛋白基因(GFP)为报告基因,构建HCV5′UTR与GFP核酸序列连接的表达载体:pcDNA-HCV-5′UTR-GFP。设计针对HCV5′UTR区3段小干扰RNA(siRNA),siRNA与表达质pcDNA-HCV-5′UTR-GFP共转染HepG2细胞中,采用荧光显微镜观测细胞内荧光强度改变,并且用流式细胞术量化分析细胞荧光强度变化,评价多种siRNA对HCV基因表达的作用。结果成功构建含HCV5′UTR区的绿色荧光蛋白基因,siRNA能特异性地抑制绿色荧光蛋白基因的表达,siRNAA、siRNAB和siRNAC的抑制率分别为68.4%、72.6%、75.6%;siRNA+siRNAB、siRNB+siRNAC和siRNA+siRNAC的抑制率分别为91.8%、87.2%、92.4%;siRNA+siRNAB+siRNAC的抑制率最高,为95.7%。结论多种siRNA对HCV5′UTR区具有干扰作用,组合使用效果更好。

Objective To evaluate interfering effect of several short interfering RNAs （siRNA） on HCV 5＇ untranslated region（5＇ UTR）.Methods The green fluorescent protein （GFP） was used as reporter gene. A fused gene of HCV-5＇UTR and GFP was constructed. It was cloned into the plasmid pCDNA3.1 named as pcDNA- HCV-5＇UTR-GFP. Three siRNAs were designed and transfected into HepG2 cells with pcDNA-HCV-5＇UTR-GFP. The change of the fluorescence intensity of HepG2 cells was shown by fluorescence microscopy and numerically detected under 488 nm wave length by flow cytometry. Results The fused gene of HCV-5＇ UTR and GFP was successfully constructed. The seven groups displayed inhibitory effects on the gene expression of GFP. The inhibition rates of siRNA A, B and C were 68.4% , 72.6% and 75.6% , respectively. The inhibitory rates of siRN A ＋ B, siRN B ＋ C and siRN A ＋ C were 91.8%, 87.2% and 92.4%, respectively. The inhibitory rates of siRN A ＋ B ＋ C was the highest, up to 95.7%. Conclusion These siRNAs could inhibit expression of HCV 5＇ UTR gene, the inhibitory effect of combined siRNA was better than that of single siRNAs.