5-氮杂-2′-脱氧胞苷诱导卵巢癌细胞系p16基因去甲基化及其表达

目的观察去甲基化制剂5-氮杂-2′-脱氧胞苷(5-Aza-CdR)对体外培养的卵巢癌细胞CAOV3p16基因启动子区甲基化状态及表达的影响,探讨卵巢癌细胞p16基因失活的机制及去甲基化制剂对p16基因表达的调控。方法应用不同浓度的5-Aza-CdR(1×10-7mol/L,5×10-7mol/L,1×10-6mol/L,5×10-6mol/L)处理体外培养的卵巢癌细胞CAOV3后,甲基化特异性聚合酶链反应(PCR)(MSP)法检测用药前后细胞中p16基因的甲基化状态,RT-PCR法及Western-blot法检测用药前后细胞中p16 mRNA及蛋白表达的变化。结果p16基因在卵巢癌细胞系CAOV3中启动子区呈异常甲基化状态,在 mRNA及蛋白水平低表达。经过5-Aza-CdR处理后,p16基因启动子区呈去甲基化状态,其 mRNA及蛋白表达显著增强(P<0.01)。结论启动子区异常甲基化是卵巢癌细胞p16基因失活的主要原因之一,去甲基化制剂-5-Aza-CdR能逆转p16基因甲基化状态,从而调控p16基因表达。

5-Aza-CdR induces demethylation of p16 gene in human ovarian cancer cell line and up-regulates its expression

Objective To investigate the effect of 5-AzaCdR on methylation state and expression of p16 gene in human ovarian cancer cell line CAOV3,and to discuss the mechanism of p16 gene silencing in human ovarian cancer cell as well as the regulating effect of demethylation agent on its expression.Methods CAOV3 cells were cultured in RPMI1640 and were treated with different concentrations(1×10-7mol/L,5×10-7mol/L,1×10-6mol/L,5×10-6mol/L) of DNA methyltransferase inhibitor 5-aza2′-deoxycytidine(5-Aza-CdR).Methylation-specific PCP(MSP) was used to detect promoter methylation state of p16 gene.RT-PCR and western blot were used to detect expression of mRNA and protein of p16 gene before and after treatment with 5-Aza-CdR respectively.Results Promoter hypermethylation of p16 gene was detected in ovarian cancer cell CAOV3,and p16 gene was expressed in CAOV3 cells at a low level before treatment.After treated with 5-Aza-CdR,the promoter region of p16 gene exhibits demethylation state,and its expression was increased significantly at mRNA and protein level(P<0.01).Conclusion Promoter hypermethylation is a main mechanism of p16 gene silencing in human ovarian cancer cell line,and can be reversed by demethylation agent 5-Aza-CdR.Demethylation agent regulates the expression of p16 gene.