【摘要】 目的探讨乙型肝炎病毒X蛋白对DNA甲基转移酶1表达的影响及调控机制。方法以Hep G2、Hep G2/EGFP及Hep G2/EGFP-HBx细胞作为实验细胞,采用Real-time PCR法及Western blot法检测细胞DNMT1的m RNA及蛋白表达水平。用不同剂量的MEK1/2特异性抑制剂U0126处理Hep G2/EGFP-HBx细胞,采用Western blot法检测总ERK1/2、磷酸化ERK1/2及DNMT1蛋白表达水平。结果 Hep G2/EGFP-HBx细胞的DNMT1 m RNA相对表达量平均值为9.464±0.22,明显高于Hep G2细胞(1.00±0.0)(t=60.64,P=0.0002)及Hep G2/EGFP细胞(0.95±0.29),差异有统计学意义(t=32.78,P=0.0011)。与Hep G2、Hep G2/EGFP细胞相比,Hep G2/EGFP-HBx细胞磷酸化ERK1/2及DNMT1蛋白表达水平有明显升高;而MEK1/2特异性抑制剂U0126处理细胞后,磷酸化ERK1/2磷酸化水平及DNMT1蛋白表达水平明显降低。结论 HBx可通过活化ERK1/2信号通路上调DNMT1的表达,这可能是HBx调控细胞基因甲基化及参与HBV相关肝细胞癌发生的机制之一。更多还原

【Abstract】 Objective To explore the effect of hepatitis B virus X protein(HBx) on the DNA methyltransferase 1(DNMT1) and its regulatory mechanism. Methods DNMT1 m RNA and protein level in Hep G2, Hep G2 / EGFP and Hep G2 / EGFP-HBx cells expressing HBx were detected by Real-time PCR and Western blot, respectively. Hep G2 / EGFPHBx cells were treated with different doses of MEK1 / 2 specific inhibitor U0126, and then total(t)-ERK1 / 2, phospho(p)-ERK1 / 2 and DNMT1 protein level were detected by Western blot. Result The relative m RNA expression level of DNMT1 in Hep G2 / EGFP-HBx cells was 9.464±0.22,which was significantly higher than that in Hep G2 cells(1.00±0.0)(t =60.64,P =0.0002)and Hep G2 / EGFP cells(0.95 ±0.29)(t =32.78,P =0.0011). Compared with Hep G2 cells and Hep G2 / EGFP cells, Hep G2 / EGFP-HBx cells exhibited higher levels of phosphorylated ERK1 / 2 and DNMT1 protein,but such effects were obviously suppressed by using MEK1 / 2 specific inhibitor U0126. Conclusion The HBx induced DNMT1 up-regulation via activation of ERK1 / 2 pathway. This may be an important mechanism by which HBx modulate DNA methylation, and involves in HBV related hepatocellular carcinoma(HCC).更多还原