HBV X蛋白对抑癌基因p16的表达和启动子甲基化的影响

康艳红; 李威; 占伟丽; 尚佳; 杨林

doi:10.3969/j.issn.1001-5256.2016.03.017

HBV X蛋白对抑癌基因p16的表达和启动子甲基化的影响

DOI: 10.3969/j.issn.1001-5256.2016.03.017

1. 河南省人民医院感染科
2. 中山大学附属第三医院感染科

基金项目:

国家自然科学基金(81071409)；

详细信息

中图分类号: R735.7
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出版历程
- 出版日期: 2016-03-20

Effects of HBV X protein on expression and promoter methylation of p16 tumor suppressor gene

Department of Infectious Diseases, Henan Provincial People′s Hospital

Research funding:

摘要

摘要: 目的探讨HBV X蛋白（HBx）对抑癌基因p16的表达、p16启动子甲基化的影响,从表观遗传学的角度探讨HBx参与HBV相关性HCC发生发展的相关机制。方法以人肝母细胞瘤细胞Hep G2、表达绿色荧光蛋白（GFP）的Hep G2/GFP、稳定表达HBx蛋白的Hep G2/GFP-HBx细胞为实验系统;采用Western Blot法检测Hep G2、Hep G2/GFP、Hep G2/GFP-HBx细胞中p16蛋白的表达水平。以DNA甲基转移酶（DNMT）抑制剂5-氮杂-2’脱氧胞苷（5-Aza-2’-DC）处理Hep G2/GFP-HBx细胞,采用甲基化特异性PCR（MSP）法检测Hep G2、Hep G2/GFP细胞及药物处理和未处理的Hep G2/GFP-HBx细胞中抑癌基因p16启动子区域的甲基化情况。计量资料多组间比较采用单因素方差分析。结果 Western Blot分析示Hep G2/GFP-HBx细胞中p16蛋白相对灰度值（23.68±3.93）显著低于Hep G2（91.23±6.87）、Hep G2/GFP（94.55±8.40）细胞,差异均有统计学意义（P值分别为0....
- 细胞系,肿瘤 /
- 基因,p16 /
- DNA甲基化
Abstract: Objective To explore the effects of hepatitis B virus X protein( HBx) on the expression and promoter methylation of the p16 tumor suppressor gene,and to investigate the epigenetic role of HBx in the development and progression of hepatitis B virus( HBV)- associated hepatocellular carcinomas( HCC). Methods Experiments were performed in the human hepatoblastoma cell line Hep G2,Hep G2 cells expressing green fluorescent protein( Hep G2 / GFP),and Hep G2 cells stably expressing GFP- HBx fusion protein( Hep G2 / GFP- HBx).Western blot was used to determine the expression levels of the p16 protein in Hep G2 cells,Hep G2 / GFP cells,and Hep G2 / GFP- HBx cells. Hep G2 / GFP- HBx cells were treated with a universal inhibitor of DNA methyltransferase( DNMT),5- aza- 2'- deoxycytidine( 5-aza- 2'- d C). Methylation- specific polymerase chain reaction( MSP) was used to determine the promoter methylation of the p16 tumor suppressor gene in Hep G2 cells,Hep G2 / GFP cells,and Hep G2 / GFP- HBx cells treated with or without 5- aza- 2'- d C. Multiple- group comparison was made by analysis of variance. Results According to the results of Western blot,Hep G2 / GFP- HBx cells had a significantly lower expression level of the p16 protein than Hep G2 cells and Hep G2 / GFP cells( P = 0. 0007; P = 0. 0014); there was no significant difference in the expression level of the p16 protein between Hep G2 / GFP and Hep G2 cells( P > 0. 05). The MSP assay revealed partial Cp G methylation in the p16 promoter region in Hep G2 / GFP- HBx cells. No promoter methylation was detected in Hep G2 cells or Hep G2 / GFP cells. Non- methylation in the p16 promoter region was restored in Hep G2 / GFP- HBx cells treated with 5- aza- 2'- d C. Conclusion In the hepatoblastoma cell line,HBx down- regulates the expression of the p16 tumor suppressor gene by inducing methylation in its promoter region. The DNMT inhibitor,5- aza- 2'- d C,restores non- methylation in the p16 promoter region. The reversible modification provides new insights for the treatment and prevention of HBV- associated HCC.
- cell line,tumor /
- genes,p16 /
- DNA methylation

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