人胚肺成纤维细胞衰老过程中P66Shc启动子区CpG岛甲基化状态

The methylation status of CpG in the promoter of P66Shc during cellular senescence of human embryonic lung fibroblasts

目的探讨人胚肺成纤维细胞复制性衰老及过氧化氢诱导的早衰过程中P66Shc的表达改变及其启动子区域的甲基化水平变化。方法荧光定量PCR方法检测细胞衰老过程中的mRNA表达改变,甲基化特异性PCR(MSP)定性检测甲基化的变化情况,亚硫酸氢盐修饰基因组结合克隆测序定量检测启动子区域CpG岛甲基化水平。结果人胚肺成纤维细胞复制性衰老及过氧化氢诱导的早衰过程中,P66Shc的mRNA表达水平逐渐升高,中年细胞组及复制性衰老细胞组mRNA表达分别升高至1.78和1.73倍,而早衰组却显著增高,至7.16倍;年轻细胞组、复制性衰老细胞组及早衰组P66Shc启动子区域CpG岛呈高甲基化水平,分别为94%、90%和90%。结论人胚肺成纤维细胞复制性衰老过程中P66Shc的mRNA表达水平逐渐升高,其启动子区CpG岛呈高的甲基化水平,不参与该基因表达调控。

Objective To explore the expression and the methylation level in its promoter of P66Shc during cellular replicative senescence and premature senescence induced by hydrogen peroxide of human embryonic lung fibroblasts（HEFs）.Methods The mRNA level of P66Shc was detected by Q-PCR.The methylation status in the promoter region was observed by methylation-specific PCR.The CpGs island methylation level was detected by bisulfite sequencing.Results The mRNA level of P66Shc increased 1.78-fold in mid-aged and 1.73-fold in replicative senescent cells during cellular replicative senescence of HEFs.However,a 7.16-fold increase was observed in premature senescent cells.The average rate of methylation was 94% in the young cells,90% in the replicative senescent cells and 90% in the premature sensescent cells.Conclusion The elevated levels of P66Shc is associated with replicative and premature senescence of HEFs,not regulated by the high methylation level in the CpG island in its promoter.