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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 effect of hepatitis B virus X protein (HBx) on promoter methylation of p16 INK4A gene and its mechanism. Methods Hep G2 cells and Hep G2 / GFP-HBx cells stably expressing GFP / HBx fusion protein were used as experimental cells and Hep G2 / GFP and Hep G2 / GFP-HBx cells were inoculated subcutaneously in nude mice to establish subcutaneous xenografts of hepatocellular carcinoma in nude mice. The si RNA (X-si RNA) and control si RNAs targeting HBV X were synthesized, and the cells and nude mice were treated with X-si RNA, 5-Aza-CdR, alone or in combination The methylation of p16 INK4A gene was detected by methylation PCR and the m RNA expression of DNMT1, DNMT3A and DNMT3B was detected by RT-PCR. Results The methylation of p16 INK4A was detected in all Hep G2 / GFP-HBx cells and xenografts in Hep G2 / GFP-HBx group but no methylation of p16 was detected in Hep G2 / GFP group. The methylation of p16 INK4A gene in Aza-Cd R-treated cells and transplanted tumor tissues was decreased. The expression of DNMT1 and DNMT3A in GFP-HBx / Hep G2 group was significantly higher than that in Hep G2 group Statistical significance (P = 0.000 2, P = 0.000 5) was also significantly higher than that in GFP / Hep G2 cells (P = 0.001 1, P = 0.000 5) There was no significant difference between the GFP / Hep G2 and GFP-HBx / Hep G2 groups (P> 0.05). Conclusion HBX may induce the methylation of p16 INK4A promoter by up-regulating the transcription of DNMT1 and DNMT3A m RNA.