5-氮杂-2′脱氧胞苷对肺癌 A549／DDP 细胞hMLHl，MGMT 基因甲基化及其表达的影响

摘要

Objective To investigate the effects of 5-Aza-2′-deoxycytidine (5-Aza-Cde)on DNA methylation and expression of hMLH1 and MGMT gene in the human lung cancer cell line A549/DDP.Methods A549/DDP cells were cultured with RPMI 1 640 medium and were treated with 5 μmol/L DNA methyhransferase inhibitor 5-Aza-Cde.Methylation-specific pol-ymerase chain reaetioll (MSP)was used to detect the promoter methylation state of the hMLH1 and MGMT gene.RT-PCR was used to detect the mRNA expression of hMLH1 and MGMT before and after treatment with 5-Aza-Cde,respectively. Results Before treatment with 5-Aza-Cde,hMLH1 and MGMT expressions were absent,and promoter hypermethylation of the hMLH1 and MGMT gene were detected in A549 cells.After treatment with 5-Aza-Cde,the promoter region of the hM-LH1 and MGMT gene exhibited a demethylation state,and their mRNA expressions were increased.Conclusion Promoter hypermethyhtion is amajor mechanism of hMLH1 and MGMT gene silencing in human lung cancer cells,and can be reversed by the demethylating agent 5-Aza-Cde,which can regulate the expressions of the hMLH1 and MGMT gene.%目的：观察5-氮杂-2′脱氧胞苷（5-Aza-Cde）对体外培养的顺铂（DDP）耐药株肺癌 A549／DDP 细胞 hMLH1，MG-MT 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 hMLH1和 MGMT 基因失活的机制及去甲基化制剂对 hMLH1和 MGMT 基因表达的调控。方法5-Aza-Cde 处理体外1640培养的肺癌 A549／DDP 细胞，甲基化特异性PCR（MSP）法检测用药前后细胞 hMLH1和 MGMT 基因的甲基化状态，RT-PCR 法检测用药前后细胞 hMLH1和 MG-MT mRNA 的表达。结果在对照组 A549细胞当中 hMLH1基因是非甲基化状态和高表达，而 MGMT 显示为低甲基化（部分甲基化）状态和高表达；而在顺铂耐药株 A549-DDP 中，hMLH1和 MGMT 基因均显示高甲基化状态，mRNA 表达下调。结论hMLH1和 MGMT 基因甲基化修饰程度与 mRNA 的表达呈相关性改变，证明 hMLH1，MGMT 基因甲基化可能是 A549对顺铂耐药的因素之一。