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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 investigate the effect of liver endogenous microRNA on hepatitis B virus (HBV) replication and gene expression. Methods The miRNA target analysis software was used to find the liver endogenous microRNAs associated with HBV sequences. The corresponding microRNA molecules were chemically synthesized in vitro. The synthetic oligonucleotides and controls were compared with the 1.3-fold HBV genome-wide eukaryotic expression plasmid pUC18- HepG2 cells were co-transfected with Lipofectamine 2000 in HBV1.3, and the supernatants were collected 48h after transfection. The expression of HBsAg and HBeAg was detected by ELISA. The expression of HBcAg was detected by Western blotting. The level of HBV mRNA was detected by fluorescence quantitative PCR. The genomic DNA was extracted and the HBV replication intermediates were detected by Southern blotting. The above detection of HBV protein expression, transcription and replication level evaluation of the corresponding microRNA effect. Results Bioinformatics methods suggested that miR-16 and miR-122 may have potential binding sites for HBV genomic function. Preliminary experiments confirmed that miR-16 can down-regulate the expression of HBV protein and HBVDNA level; miR-122 can down-regulate the expression of HBsAg and HBeAg and up-regulate the level of HBV mRNA. Conclusion Liver endogenous microRNA can regulate HBV replication and gene expression.