hMLH1基因启动子甲基化与非小细胞肺癌细胞顺铂耐药方面的研究

hMLH1 promoter hypermethylation correlates with platinum-resistance in non-small cell lung cancer cells

背景与目的:hMLH1启动子甲基化是肺癌发生、发展的因素之一,但与肺癌细胞耐药方面的研究尚未见报道,本研究旨在探讨是否能通过去甲基化作用恢复hMLH1基因表达从而逆转A549/DDP细胞对顺铂的耐药。方法:RT-PCR检测A549及A549/DDP细胞的hMLH1表达情况,及5-氮杂-2'-脱氧胞苷(5-Aza-2'-eoxycytidine,5-Aza-CdR)干预A549/DDP细胞后hMLH1表达情况;MSP检测A549、A549/DDP细胞及5-Aza-CdR干预A549/DDP细胞后hMLH1甲基化状态。MTT试验、Hoechst33258染色、流式细胞仪(FCM)分别观察5-Aza-CdR干预前后顺铂对A549/DDP细胞抑制率、凋亡的形态学变化和凋亡指数。结果:hMLH1mRNA在A549中的表达高于A549/DDP细胞;A549细胞hMLH1为非甲基化状态,A549/DDP细胞为部分甲基化状态,经5-Aza-CdR去甲基作用后hMLH1呈非甲基化状态;A549组、A549/DDP组、经10μmol/L5-Aza-CdR干预后的A549/DDP组经顺铂作用后的IC50值分别为(4.7±0.7)、(30.1±1.8)和(6.9±0.6)μmol/L;5-Aza-CdR干预后的A549/DDP细胞经顺铂作用后,比单用顺铂抑制A549/DDP细胞的凋亡形态学改变明显,凋亡小体以及核固缩现象增多。结论:hMLH1甲基化可能参与了A549/DDP细胞的顺铂耐药过程;5-Aza-CdR能抑制hMLH1甲基化,恢复hMLH1表达,并能增强顺铂对A549/DDP细胞增殖抑制和凋亡。

Background and purpose：hMLH1 promoter methylation is one of the factors in the development of lung cancer.However,little is known about hMLH1 promoter methylation in association with chemotherapy resistance in lung cancer cells.This study aimed to investigate whether resistance to DDP can be overcame by restoring the expression of hMLH1 in A549/DDP cells after treatment with a demethylating agent,5-Aza-2＇-deoxycytidine （5-Aza-CdR）.Methods：hMLH1 mRNA was determined by RT-PCR in A549,A549/DDP and A549/DDP treated with 5-Aza-CdR;MSP was carried out to determine the methylation status of hMLH1 in A549 and A549/DDP,A549/DDP treated with 5-Aza-CdR.MTT test,Hoechst 33258 staining and flow cytometry were used to determine the change of cell proliferation,apoptotic morphology and the cell apoptosis index,respectively.Results：hMLH1 mRNA was expressed higher in A549 than in A549/DDP.The unmethylation status of hMLH1 was observed in A549 cells whereas partial methylation status of hMLH1 in A549/DDP cells.After treatment with 5-Aza-CdR,the A549/DDP cells displayed an unmethylation of the hMLH1 gene.After the treatment with cisplatin,the IC50 of A549,A549/DDP and A549/DDP treated by 5-Aza-CdR were （4.7±0.7）,（30.1±1.8） and （6.9±0.6）μmol/L,respectively.Apoptotic bodies were less in A549/DDP cells than in A549/DDP cells treated with the 5-Aza-CdR.The rate of apoptosis of A549/DDP treated by 5-Aza-CdR was higher than that of A549/DDP.Conclusion：The methylation of hMLH1 may be relevant to the resistance to platinum-based chemotherapy in the A549/DDP cell line.5-Aza-CdR can inhibit hMLH1 methylation,restore the expression of hMLH1,and enhance cell apoptosis as well as the inhibition of cell proliferation of A549/DDP cells after the treatment of cisplatin.