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目的探讨HBV、miR-499a及CFLAR在肝癌细胞中的关系。方法通过实时定量聚合酶链反应(Real-time PCR)检测miR-499a在有HBV复制的肝癌细胞中的表达;通过Real-time PCR、双荧光素酶报告系统、Westernblot证明CFLAR是否为miR-499a的靶基因。结果 miR-499a在Hep G2.2.15细胞中的表达高于Hep G2细胞,在Ad-HBVHep G2细胞中的表达高于Ad-GFP-Hep G2细胞;过表达miR-499a之后在mRNA和蛋白水平发现CFLAR表达降低,抑制miR-499a表达之后在mRNA和蛋白水平发现CFLAR表达升高;miR-499a通过与CFLAR的3’UTR作用来抑制其表达;HBV在mRNA水平降低CFLAR的表达。结论在肝癌细胞中HBV上调miR-499a的表达,CFLAR是miR-499a的靶基因。
Objective To investigate the relationship between HBV, miR-499a and CFLAR in hepatocellular carcinoma cells. Methods Real-time PCR was used to detect the expression of miR-499a in hepatoma cells with HBV replication. Real-time PCR, dual luciferase reporter system and Western blot were used to confirm whether miR-499a was miR-499a Of target genes. Results The expression of miR-499a in Hep G2.2.15 cells was higher than that in Hep G2 cells, and higher in Ad-HBV Hep G2 cells than in Ad-GFP-Hep G2 cells; miR-499a was found at mRNA and protein levels after overexpression of miR-499a CFLAR expression was reduced and miR-499a expression was inhibited, and CFLAR expression was found at mRNA and protein levels. MiR-499a inhibited its expression through 3’UTR interaction with CFLAR, and HBV reduced CFLAR expression at mRNA level. Conclusion HBV can up-regulate the expression of miR-499a in hepatocellular carcinoma cells. CFLAR is the target gene of miR-499a.