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HBx抑制RASSF1A表达参与肝细胞癌发生的可能机制
作者:宋蕴薇1 2  仇雪梅1 3  陆森4  樊红1 
单位:1. 东南大学医学院 遗传与发育生物学系, 江苏 南京 210009;
2. 江苏省中医药研究院, 江苏 南京 210028;
3. 枣庄市妇幼保健院, 山东 枣庄 277100;
4. 江苏省人民医院, 江苏 南京 210029
关键词:RAS相关结构域家族1A HBx DNA甲基化 肝细胞癌 
分类号:R735.7;Q786
出版年·卷·期(页码):2018·37·第三期(401-406)
摘要:

目的:探讨乙型肝炎病毒关键基因HBx对肝细胞癌相关抑癌基因RASSF1A表达的影响及其可能的调控方式,以期理解HBx参与肝细胞癌发生的分子机制。方法:采用RT-PCR、qPCR法检测肝细胞癌细胞系中RASSF1A的表达水平差异,并评估RASSF1A表达水平与HBx表达的相关性;应用HBx转染的肝细胞系、肝癌细胞系和DNMT1敲低的肝癌细胞系为研究对象,以qPCR、BGS等方法分析HBxRASSF1A表达的影响及其调控方式。结果:与肝细胞系相比,抑癌基因RASSF1A在肝细胞癌细胞系中呈低表达趋势,且RASSF1A的表达水平与HBx表达呈反相关关系。转染HBx基因后,肝癌细胞系中RASSF1A的表达水平下降。应用DNA甲基化酶抑制剂5'-aza处理肝癌细胞后RASSF1A的表达水平升高,siRNA抑制主要DNA甲基转移酶DNMT1后RASSF1A表达水平亦得到回复。BGS方法分析显示抑制DNMT1的表达影响了RASSF1A启动子区CpG位点的甲基化状态。结论:肝癌细胞中HBx能够抑制RASSF1A的表达,并可能通过改变其启动子区DNA甲基化状态调控其表达而参与肝细胞癌的发生。

Objective: To investigate the expression level of RASSF1A induced by HBx and to explore the potential epigenetic regulation mechanisms by HBx in HBV-associated hepatocellular carcinoma(HCC).Methods: The expression level of RASSF1A and HBx was examined by RT-PCR and/or quantitative real-time PCR(qPCR) in HCC cells. The relationship between the expression level of RASSFIA and HBx was evaluated in HCC cell lines.qPCR was employed to detect the RASSF1A expression level induced by HBx. Meanwhile, bisulfite genomic sequencing(BGS) was used to explore the potential epigenetic regulation of RASSF1A by HBx in HCC cells transfected with HBx, and HCC cells knocked-down of DNMT1.Results: The expression level of RASSF1A was relative decreased in 7 HCC cell lines compared with L02 and QSG cells. RASSF1A expression was suppressed in HBx transfected HCC cells. 5'-aza, an inhibitor of DNA methyltransferase, restored RASSF1A expression in HCC cell lines. Knock-down of DNMT1 recovered RASSF1A expression by demethylatedpromoter CpG sites of RASSF1A in SMMC-7721 cells.Conclusion: HBx suppresses the expression of RASSF1A via changing methylation status in its promoter region, which may be involved in HBV-associated hepatocellular carcinogenesis.

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