5-氮杂-2′-脱氧胞苷对肺癌SPC-A-1细胞p16、MGMT基因甲基化及其表达的影响

Effects of 5-Aza-CdR on DNA methylation and expression of p16 and MGMT gene in lung cancer cell line SPC-A-1

目的观察5-氮杂-2′-脱氧胞苷(5-Aza-CdR)对体外培养的肺癌SPC-A-1细胞p16、MGMT基因启动子区DNA甲基化状态及其表达的影响,探讨肺癌细胞p16、MGMT基因失活的机制及去甲基化制剂对p16、MGMT基因表达的调控。方法 5-Aza-CdR处理体外培养的肺癌SPC-A-1细胞,甲基化特异性PCR(MSP)法检测用药前后细胞p16、MGMT基因的甲基化状态,RT-PCR法检测用药前后细胞p16、MGMT mRNA。结果加入5-Aza-CdR前,SPC-A-1细胞p16、MGMTmRNA表达缺失,其启动子区域表现为DNA甲基化。加入5-Aza-CdR后,SPC-A-1细胞中p16、MGMT基因呈现DNA去甲基化,而且表达缺失的p16、MGMT mRNA重新表达。结论启动子区高甲基化是肺癌细胞p16、MGMT基因失活的主要原因之一,去甲基化制剂5-Aza-CdR能逆转p16、MGMT基因甲基化状态,从而调控p16、MGMT基因表达。

Objective To investigate the effects of 5-Aza-2′-deoxycytidine（5-Aza-CdR） on DNA methylation and expression of p16 and MGMT gene in the human lung cancer cell line SPC-A-1.Methods SPC-A-1 cells were cultured with RPMI 1640 medium and were treated with 5 μmol/L DNA methyltransferase inhibitor 5-Aza-CdR.Methylation-specific polymerase chain reaction（MSP） was used to detect the promoter methylation state of the p16 and MGMT gene.RT-PCR was used to detect the mRNA expression of p16 and MGMT before and after treatment with 5-Aza-CdR,respectively.Results Before treatment with 5-Aza-CdR,p16 and MGMT expressions were absent,and promoter hypermethylation of the p16 and MGMT gene were detected in SPC-A-1 cells,After treatment with 5-Aza-CdR,the promoter region of the p16 and MGMT gene exhibited a demethylation state,and their mRNA expressions were increased.Conclusion Promoter hypermethylation is a major mechanism of p16 and MGMT gene silencing in human lung cancer cells,and can be reversed by the demethylating agent 5-Aza-CdR,which can regulate the expressions of the p16 and MGMT gene.