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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。
Objective To investigate the effect of hepatitis B virus X protein on the expression of DNA methyltransferase 1 and its regulatory mechanism. Methods Hep G2, Hep G2 / EGFP and Hep G2 / EGFP-HBx cells were used as experimental cells. Real-time PCR and Western blot were used to detect the expression of m RNA and protein in DNMT1 cells. Hep G2 / EGFP-HBx cells were treated with different doses of MEK1 / 2 specific inhibitor U0126, and the total ERK1 / 2, phosphorylated ERK1 / 2 and DNMT1 protein levels were detected by Western blot. Results The average DNMT1 mRNA expression in Hep G2 / EGFP-HBx cells was 9.464 ± 0.22, significantly higher than that in Hep G2 cells (1.00 ± 0.0) (t = 60.64, P = 0.0002) and Hep G2 / EGFP cells ± 0.29), the difference was statistically significant (t = 32.78, P = 0.0011). Compared with Hep G2 and Hep G2 / EGFP cells, the phosphorylation levels of ERK1 / 2 and DNMT1 in Hep G2 / EGFP-HBx cells were significantly increased. However, the phosphorylation of ERK1 was induced by MEK1 / 2 specific inhibitor U0126 / 2 phosphorylation and DNMT1 protein expression was significantly lower. Conclusion HBx can up-regulate the expression of DNMT1 through activation of ERK1 / 2 signaling pathway, which may be one of the mechanisms by which HBx regulates cell methylation and participates in the development of HBV-related hepatocellular carcinoma.