5-氮-2′-脱氧胞苷对肺癌A549/DDP细胞凋亡及hMLH1表达的影响

Effects of 5-Aza-CdR on cell apoptosis in lung cancer A549/DDP cell line and expression of tumor suppressor gene hMLH1

目的 探讨5-氮-2′脱氧胞苷（5-Aza-CdR）对耐顺铂（DDP）的人肺癌A549/DDP细胞凋亡及抑癌基因hMLH1表达的影响.方法 以浓度为0.5、5、50μmol/L的5-Aza-CdR处理A549/DDP细胞,常规培养,采用四甲基偶氮唑蓝（MTT）比色法观察细胞的生长活性,甲基化特异性聚合酶链反应（MSP）检测hMLH1基因甲基化状态,以实时荧光定量PCR法检测hMLH1 mRNA的表达,应用流式细胞术检测细胞凋亡率.结果 5-Aza-CdR能明显抑制肿瘤细胞的生长,随5-Aza-CdR浓度增加、培养时间延长,细胞生长抑制率升高（P＜0.05）.细胞凋亡率与5-Aza-CdR剂量呈正相关（P＜ 0.001）.5-Aza-CdR处理后hMLH1mRNA表达升高（P＜0.05）.A549/DDP细胞hMLH1启动子甲基化阳性,其mRNA为阴性,应用5-Aza-CdR干预培养后,mRNA为阳性.结论 5-Aza-CdR能使hMLH1基因去甲基化,促进细胞凋亡,增强抑癌功能.

Objective To investigate the effects of 5-Aza-2′-deoxycytidine （5-Aza-CdR） on the apoptosis of A549/DDP cells and the expression of hMLH1 gene.Methods A549/DDP cells were treated with 5-Aza-CdR at 0.5,5,50 μmol/L.The growth curve of A549/DDP cells was investigated by MTT assay.The methylation status of hMLH1 gene was detected by methylation specific PCR （MSP）.The expression of hMLH1 mRNA was evaluated by FQ-PCR.The apoptosis rate of A549/DDP cells was analyzed by flow cytometry.Results A549/DDP cells treated with 5-Aza-CdR showed a slow growth in comparison with the control cells,and the growth rates were decreased with the increasing of 5-Aza-CdR concentration.The apoptosis rate after treatment was higher than that before treatment in A549/DDP cells （P 〈 0.05）,and had a positive correlation with 5-Aza-CdR dose （P 〈 0.001）.hMLH1 mRNA expression level was increased in a 5-Aza-CdR concentration dependent manner （P 〈 0.05）.hMLH1 promoter in A549/DDP cells was methylated and hMLH1 mRNA was negatively expressed before treatment,but the mRNA was positively expressed after treatment with 5-Aza-CdR.Conclusions 5-Aza-2′-CdR can induce apoptosis of A549/DDP cells by inducing demethylation of hMLH1 promoter and thereby enhancing hMLH1 gene expression and its tumor suppressor function.