Objective To investigate the inhibitory effects of hepatitis B virus(HBV) replication and expression by transfecting artificial microRNA targeted La gene into HepG2. 2. 15 cells. Methods Three amiRNA-HBV plasmids were constructed and transfected into HepG2. 2. 15 cells via LipofectamineTM 2000 reagent. The level of La mRNA and La protein was measured by semi-quantitative RT-PCR and Western blot respectively. HBV antigen secretion was detected in the cells with transient and stable transfection by time-resolved fluoroimmunoassays(TRFIA). HBV DNA replication was examined by fluorescence quantitative PCR. Results Three amiRNA could significanfly reduce the La mRNA and protein expression, the greatest reduction was amiRNA-La-610 transfected group, the expression of La mRNA and protein levels were down-regulated by 57.3％ and 49. 8％ at 72 h(P ＜0. 01 ). In the virus level,three amiRNA-La plasmids could inhibit the secretion of HBsAg and HBeAg,the greatest reduction group was amiRNA-La-610 transfected group, the expression levels of HBsAg and HBeAg were down-regulation by 23.3％％ and 25.8％ after 72 h(P ＜0. 01 ) ,HBV DNA level was down-regulated by 22. 3％ at 72 h(P ＜0. 01 ).Conclusion In HepG2. 2. 15 cells,HBV replication and expression can be inhibited by artificial microRNA targeted La. La gene can be a promising therapy approach for chronic HBV infection.%目的 设计并构建靶向La的microRNA表达载体,观察其对La基因的抑制作用,探讨其在HBV感染基因治疗中的应用价值.方法 以La为靶基因,设计并构建3个针对La不同位点的microRNA表达载体,通过脂质体方法转染HepG2.2.15细胞,RT-PCR和Western blot检测其对La mRNA和蛋白的抑制作用,乙肝五项定量和HBV DNA检测其对HBV的抑制作用.结果 3个外源性miRNA (amiRNA)均能明显降低La mRNA和蛋白的表达,其中amiRNA-La-1130抑制效应最强,转染后72 h La mRNA和蛋白的水平平均下降了58.6%和50.6%(P＜0.01);但仅有amiRNA-La-1130质粒能显著抑制相应细胞株中HBsAg和HBeAg的分泌,抑制率为23.3%和25.8%(P＜0.01),HBV DNA的平均抑制率为22.3%(P＜0.01).结论 靶向La的amiRNA能显著抑制靶基因的表达,进而抑制HBV的复制和表达.La基因可作为慢性HBV感染联合基因治疗的候选靶点之一.
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