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、q PCR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q PCR、BGS等方法分析HBx对RASSF1A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Cp G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 RASSF1 A induced by HBx and to explore the potential epigenetic regulation mechanisms by HBx in HBV-associated hepatocellular carcinoma(HCC). Methods: The expression level of RASSF1 A and HBx was examined by RT-PCR and/or quantitative real-time PCR(q PCR) in HCC cells. The relationship between the expression level of RASSFIA and HBx was evaluated in HCC cell lines. q PCR was employed to detect the RASSF1 A expression level induced by HBx. Meanwhile,bisulfite genomic sequencing(BGS) was used to explore the potential epigenetic regulation of RASSF1 A by HBx in HCC cells transfected with HBx,and HCC cells knocked-down of DNMT1. Results: The expression level of RASSF1 A was relative decreased in 7 HCC cell lines compared with L02 and QSG cells. RASSF1 A expression was suppressed in HBx transfected HCC cells. 5’-aza,an inhibitor of DNA methyltransferase,restored RASSF1 A expression in HCC cell lines. Knockdown of DNMT1 recovered RASSF1 A expression by demethylatedpromoter Cp G sites of RASSF1 A in SMMC-7721 cells. Conclusion: HBx suppresses the expression of RASSF1 A via changing methylation status in its promoter region,which may be involved in HBV-associated hepatocellular carcinogenesis.
作者
宋蕴薇
仇雪梅
陆森
樊红
SONG Yun-wei1,2, QIU Xue-mei1,3 , LU Sen4, FAN Hong1(1. Department of Medical Genetics and Developmental Biology, School of Medicine, Southeast University, Southeast University, Nanjing 210009, China; 2.Jiangsu Provinee Academy of Traditional Chinese Medicine, Nanjing 210028, China; 3. Zaozhuang Maternal and Child Health Care Hospital, Zaozhuang 277100, China ; 4. Jiangsu Provinee Hospital, Nanjing 210029, Chin)
出处
《东南大学学报(医学版)》
CAS
2018年第3期401-406,共6页
Journal of Southeast University(Medical Science Edition)
基金
国家自然科学基金资助项目(91229107)