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目的观察病毒白细胞介素-6(viral interleukin-6,vIL-6)对抑癌基因p16启动子区甲基化及蛋白表达的影响。方法以vIL-6真核表达质粒转染293T细胞(转染组)为实验,空载体转染的293T细胞(空载体组)和未转染的293T细胞(未转染组)为对照,3组均采用Western blot法检测DNA甲基转移酶1的表达情况,甲基化特异性PCR法检测p16启动子区甲基化状态,荧光定量RT-PCR和Western blot法检测p16基因的表达。结果转染组DNA甲基转移酶1蛋白表达较未转染组和空载体组增加;转染组p16基因启动子区出现甲基化改变;转染组p16mRNA表达量明显低于未转染组和空载体组,差异有统计学意义(P<0.05);转染组P16蛋白表达较未转染组和空载体组降低。结论 vIL-6可上调DNA甲基转移酶1表达,导致抑癌基因p16启动子区甲基化,抑制p16基因表达;vIL-6所致的DNA甲基化改变,可能是其致瘤机制之一。
Objective To investigate the effect of virus interleukin-6 (vIL-6) on the methylation and protein expression of the oncogene p16 promoter. Methods 293T cells transfected with eukaryotic expression vector vIL-6 (transfected group) were used as control. 293T cells transfected with empty vector (empty vector group) and untransfected 293T cells (untransfected group) The expression of DNA methyltransferase 1 was detected by Western blot. The methylation status of p16 promoter was detected by methylation-specific PCR. The expression of p16 gene was detected by quantitative RT-PCR and Western blot. Results The expression of DNA methyltransferase 1 in transfection group was increased compared with untransfected group and empty vector group. The methylation of p16 gene promoter in transfection group was changed. The expression of p16 mRNA in transfected group was significantly lower than that in untransfected group And empty vector group, the difference was statistically significant (P <0.05); P16 protein expression in transfected group than the non-transfected group and empty vector group decreased. Conclusion vIL-6 can up-regulate the expression of DNA methyltransferase 1, leading to the methylation of the p16 promoter region of tumor suppressor gene and inhibiting the expression of p16 gene. The DNA methylation induced by vIL-6 may be the mechanism of its tumorigenicity one.