肝脏内源性microRNA调控乙型肝炎病毒基因的表达与复制

Endogenous Liver MicroRNA Regulates HBV Gene Expression and Replication

目的探讨肝脏内源性microRNA对乙型肝炎病毒(HBV)复制与基因表达的影响。方法通过miRNA靶点分析软件寻找与HBV序列之间相关联的肝脏内源性microRNA,体外化学合成相应的microRNA分子,将合成寡核苷酸及对照与1.3倍HBV全基因组真核表达质粒pUC18-HBV1.3采用Lipofectamine2000共转染HepG2细胞,转染48h后收集细胞培养上清;通过ELISA检测HBsAg、HBeAg的表达水平;Western印迹检测HBcAg的表达水平;Trizol抽提转染细胞RNA,逆转录后用荧光定量PCR检测HBVmRNA的水平;提取细胞基因组DNA,Southern印迹检测HBV的复制中间体。经以上检测从HBV蛋白表达、转录和复制水平评价相应的microRNA作用效应。结果生物信息学方法提示miR-16和miR-122存在与HBV基因组作用的可能结合位点。经试验初步证实miR-16可下调HBV蛋白的表达及HBVDNA水平;miR-122可下调HBsAg、HBeAg的表达,上调HBVmRNA的水平。结论肝脏内源性microRNA可以调节HBV的复制与基因表达。

Objective To study HBV gene expression and replication regulated by endogenous liver mieroRNA. Methods Liver endogenous mieroRNAs associated with HBV genome was detected by using miRNA target analysis software. The corresponding microRNAs were synthesized in vitro, and then were co-transfected, with synthetic oligonucleotides and 1.3 copy of HBV genome eukaryotie expression vector pUC18-HBV1.3, to HepG2 cell lines by using Lipofeetamine 2000. Fortyeight hours after the transfection, cell culture supematants were collected, HbsAg and HBeAg expressions were detected by ELISA. Western blotting were used for the detection of HBcAg. RNA was extracted from the transfected cells by Trizol. Fluorescence quantitative reverse transcription PCR was used for the detection of HBV mRNA. DNA was extracted from the transfected cells and was detected by Southern blotting for HBV replication intermediates. Results Through bioinformatic methods miR-16 and miR-122 possible binding sites with HBV genome were predicted. The results showed that miR-16 down-regulated HBV protein expression and HBV DNA. MiR-122 appeared to down-regulate the expression of HBsAg, HBeAg and up-regulate HBV mRNA level. Conclusion Endogenous liver microRNA could regulate HBV gene expression and replicationand this provides a new clue for the further study of HBV regulation in liver cells.