p21^(Waf1/Cip1)甲基化调控细胞衰老

Cellular senescence is regulated by p21^(Waf1/Cip1) methylation

目的探讨p21Waf1/Cip1启动子甲基化改变对细胞衰老进程的影响。方法采用克隆、测序的方法定量检测p21Waf1/Cip1启动子在人胚肺二倍体成纤维细胞(2BS)衰老过程中的甲基化改变;RT-PCR和Western blot检测p21Waf1/Cip1的表达;采用5-氮杂-2-脱氧胞苷去甲基化处理2BS细胞,通过MTT和β-半乳糖苷酶染色检测细胞衰老。结果 p21Waf1/Cip1启动子在年轻2BS中,CpG甲基化的发生率为1.25%;在中年细胞中为27.27%;在衰老2BS细胞中为0.64%;p21Waf1/Cip1的表达在细胞衰老过程中呈波动性变化,先增加,后降低,到细胞开始衰老时,表达又显著增加;中年2BS细胞经5-氮杂-2-脱氧胞苷处理后,p21Waf1/Cip1表达增加,细胞增殖速率较正常中年细胞显著降低,同时细胞β-半乳糖苷酶染色阳性率增加。结论 p21Waf1/Cip1去甲基化加速细胞衰老。

Objective To explore the effect of p21Waf1/Cip1 methylation changes on the process of cellular senescence .Methods Bisulfite sequencing was used to analyze the methylation changes of p21Waf1/Cip1 in the process of cellular senescence;p21Waf1/Cip1 ex‐pression was detected by RT‐PCR and Western‐blot ;Middle‐aged 2BS cells was treated by 5‐aza‐CdR and cellular senescence was detected by MTT and SA‐β‐Gal staining .Results Bisulfite sequencing analysis of p21Waf1/Cip1 promoter showed that CpGs were methylated by 1 .25% in the young 2BS cells ,by 27 .27% in the middle‐aged 2BS cells ,while only by 0 .64% in the senescent cells . The expression of p21Waf1/Cip1 was low in the young（28 PD） 2BS cells ,it increased first（35 PD） but decreased later in the middle‐aged（42 PD） cells .In the senescent 2BS cells ,p21Waf1/Cip1 expression was further increased .5‐aza‐CdR treatment resulted in de‐creased growth rate but increasedβ‐Gal staining of middle‐aged 2BS cells .Conclusion The process of cellular senescence is regula‐ted by the status of p21Waf1/Cip1 methylation ,and p21Waf1/Cip1 demethylation accelerates cellular senescence .