靶向ASGPR1的外源性microRNA对HBV表达和复制的抑制作用

Inhibition of hepatitis B virus gene expression and replication by artificial microRNA targeted ASGPR1

目的:设计并构建ASGPR1靶向的microRNA表达载体,观察其对ASGPR1基因的抑制作用及其在HBV感染基因治疗中的应用价值.方法:以ASGPR1为靶基因,设计并构建3个针对ASGPR1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ASGPR1mRNA和蛋白的抑制作用,乙肝五项定量和HBVDNA检测其对HBV的抑制作用.结果:ASGPR1mRNA和蛋白的平均水平分别下降了57.3%和49.8%(P<0.01);在病毒水平3种amiRNA均能明显抑制相应细胞株中HBsAg和HBeAg的分泌,其中以a miRNA-ASGPR1-610抑制作用最强,对培养72h的细胞上清中的HBsAg和HBeAg抑制率分别为31.3%和33.6%(P<0.01),HBV DNA的抑制率为29.7%(P<0.01).结论:靶向ASGPR1的外源性microRNA能显著抑制靶基因的表达,进而抑制HBV的复制和表达.ASGPR1可以作为慢性HBV感染基因治疗的候选靶点.

AIM： To investigate the inhibitory effects on hepatitis B virus （HBV） replication and expression by transfecting artificial microRNA targeted ASGPR1 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 ASGPR1 mRNA was measured by semi-quantitative RT-PCR. The level of ASGPR1 protein was measured by western blot. 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 significantly reduced ASGPR1 mRNA and protein expression, and the greatest reduction was seen in amiRNAASGPR1-610 transfected group. Expressions of ASGPR1 mRNA and protein were downregulated by 57.3% and 49.8% at 72 h（P 〈 0.01）. At the virus level, three amiRNA-ASGPR1 plasmids obviously inhibited the secretion of HBsAg and HBeAg with the greatest reduction seen in amiRNA-ASGPR1-610 transfected group. Expression levels of HBsAg and HBeAg were down-regulated by 31.3% and 33.6% after 72 h （P 〈 0.01） and HBV DNA level was downregulated by 29.7% at 72 h （P 〈 0.01）.CONCLUSION： In HepG2.2.15 cells, HBV replication and expression could be inhibited by artificial microRNA targeted ASGPR1. Artificial microRNA targeted ASGPR1 could be a promising therapeutic approach for chronic HBV infection.