乙型肝炎病毒X蛋白诱导p16^(INK4A)基因启动子甲基化及其机制研究  

The mechanism by Hepatitis B virus X protein induce p16^(INK4A) promoter methylation

作　　者：邝建玉[1] 杨林[2] 瞿志军[1] 

机构地区：[1]深圳市龙岗中心医院感染科,广东深圳518116 [2]中山大学附属第三医院感染科,广东广州510000

出　　处：《热带医学杂志》2016年第8期976-979,共4页Journal of Tropical Medicine

基　　金：国家自然科学基金(81071409)

摘　　要：目的探讨乙型肝炎病毒X蛋白(HBx)对p16^(INK4A)基因启动子甲基化的影响及机制。方法以Hep G2细胞和稳定表达GFP/HBx融合蛋白的Hep G2/GFP-HBx为实验细胞,Hep G2/GFP、Hep G2/GFP-HBx细胞接种于裸鼠皮下建立裸鼠肝癌皮下移植瘤;设计合成靶向HBV X的si RNA(X-si RNA)和对照si RNA,用X-si RNA、5-N-2-脱氧胞苷(5-Aza-Cd R)单独或联合处理细胞及裸鼠;甲基化PCR检测细胞及移植瘤组织p16^(INK4A)基因甲基化;RT-PCR法测定DNMT1、DNMT3A和DNMT3B的m RNA表达。结果甲基化PCR检测显示Hep G2/GFP-HBx组细胞及移植瘤组织均存在p16^(INK4A)甲基化而Hep G2/GFP组均未检出p16甲基化;X-si RNA、5-Aza-Cd R处理的细胞及移植瘤组织中p16^(INK4A)基因甲基化均减低;RT-PCR检测显示GFP-HBx/Hep G2组DNMT1、DNMT3A较Hep G2组显著升高,差异有统计学意义(P=0.000 2、P=0.000 5),较GFP/Hep G2细胞组也显著升高,差异有统计学意义(P=0.001 1、P=0.000 5),而DNMT3B在Hep G2、GFP/Hep G2、GFP-HBx/Hep G2细胞组组间检测值差异无统计学意义(P>0.05)。结论 HBX蛋白可能通过上调DNMT1、DNMT3A m RNA的转录表达而诱导p16^(INK4A)基因启动子的甲基化。Objective To investigate the related mechanisms of hepatitis B virus X（HBx） protein on inducing p16^（INK4A） promoter methylation. Methods Human hepatocyte HpG2 cell line stably expressing a green fluorescent protein（GFP）-tagged HBx（Hep G2 / GFP-HBx cells） was used for the experiment,and HepG2 parental and HepG2 / GFP cells was used as the controls.Nude mice were inoculated with Hep G2 / GFP and HepG2 / GFP-HBx cells subcutaneous respectively to establish implant models of hepatocellular carcinoma.The small interfering RNA targeting to HBV X gene（X-si RNA） and control si RNA were synthesized. The cells and nude mice were treated with X-si RNA,5-Aza-Cd R alone or in combination. The methylation of p16 gene promoter was detected by methylation specific polymerase chain reaction（MSP）. The levels of DNMT m RNA were detected by RT-PCR. Results MSP analysis showed that p16 gene methylation was observed in HepG2 / GFP-HBx-caused palpable tumors, while no methylation was detected in HepG2 /GFP group.However,after treatment with X-si RNA or 5-Aza-Cd R,p16 gene methylation reduced both in in vitro and in vivo.RT-PCR showed that the DNMT1, DNMT3 A of GFP-HBx / HepG2 group were significantly elevated compared with Hep G2 group with significant differences（P=0.0002, P=0.0005）, and compared with GFP / HepG2 cell group, they also increased significantly and with statistical difference（P =0.0011, P =0.0005）, but the detection value of DNMT3 B in three groups of cells（HepG2, GFP / HepG2, GFP-HBx / HepG2）, and there were no statistically significant difference between the two groups（P 〈0.05）. Conclusion HBx induce p16^（INK4A）promoter methylation may be via the up-regulation the expression of DNMT1 and DNMT3 A mRNA.

关 键 词：乙型肝炎病毒X蛋白 P16 甲基化 DNA甲基化转移酶 

分 类 号：R373.2[医药卫生—病原生物学]