HBx对HBV相关性肝癌增殖和迁移能力的影响及其机制

Effect of HBx on proliferation and migration of HBV-associated hepatocellular carcinoma and its mechanism Tang

目的探讨乙型肝炎病毒X蛋白(HBx)对HBV相关性肝细胞癌(肝癌)增殖和迁移能力的影响及其作用机制。方法构建稳定过表达HBx的肝癌细胞系(SMMC7721、Huh7),采用CCK-8法、细胞平板克隆实验和Transwell迁移实验检测HBx在肝癌细胞增殖和迁移能力中的作用。microRNA(miRNA,miR)芯片数据分析HBV相关性肝癌差异表达的miRNA。荧光定量PCR检测lncRNA PTENpg1、miR-21的表达情况,Western blot法检测PTEN的表达情况。构建过表达miR-21的肝癌细胞系(SMMC7721、Huh7),采用CCK-8法、细胞平板克隆实验和Transwell迁移实验检测miR-21对肝癌细胞增殖和迁移能力的影响。结果细胞平板克隆形成实验、CCK-8法检测和Transwell迁移实验显示,过表达HBx促进肝癌细胞克隆形成、生长和迁移。miRNA芯片数据分析显示miR-21为HBV相关性肝癌的差异表达基因。荧光定量PCR检测显示,HBx过表达后,肝癌细胞lncRNA PTENpg1表达明显下调,miR-21表达明显上调。Western blot检测显示,HBx和miR-21过表达后PTEN蛋白水平下调。细胞平板克隆形成实验、CCK-8法检测和Transwell迁移实验显示,上调miR-21促进肝癌细胞的增殖、迁移能力。结论HBx可能通过调控lncRNA PTENpg1-miR-21-PTEN分子信号通路,促进HBV相关性肝癌发生、发展。

Objective To evaluate the effect of hepatitis B virus X protein(HBx)on the proliferation and migration of HBV-associated hepatocellular carcinoma(HCC)and the mechanism.Methods The HCC cell lines(SMMC7721 and Huh7)stably overexpressing HBx were constructed.The effect of HBx upon the proliferation and migration of HCC cells was assessed by CCK-8 assay,plate cloning experiment and Transwell chamber test.The differentially-expressed microRNAs(miRNAs,miR)in HBV-associated HCC were identified by miRNA chip data.The expression levels of lncRNA PTENpg1 and miR-21 were measured by fluorescent quantitative PCR.The expression level of PTEN was detected by Western blot.The HCC cell lines(SMMC7721 and Huh7)overexpressing miR-21 were constructed.The effect of miR-21 on the proliferation and migration of HCC cells was evaluated by CCK-8 assay,plate cloning experiment and Transwell chamber test.Results Plate cloning experiment,CCK-8 assay and Transwell chamber test showed that overexpression of HBx promoted the colony formation,proliferation and migration of HCC cells.Analysis of siRNA chip data demonstrated that miR-21 was a differentially-expressed gene of HBV-associated HCC.Fluorescent quantitative PCR revealed that the expression level of lncRNA PTENpg1 was significantly down-regulated,whereas that of miR-21 was significantly up-regulated after HBx overexpression.Western blot showed that the expression level of PTEN protein was down-regulated while the HBx and miR-21 were over expressed.Plate cloning experiment,CCK-8 assay and Transwell chamber test indicated that up-regulation of miR-21 promoted the proliferation and migration of HCC cells.Conclusions HBx can promote the incidence and progression of HBV-associated HCC by regulating the lncRNA PTENpg1-miR-21-PTEN molecular signaling pathway.