Zebularine调控卵巢癌细胞hMLH1基因再表达

Zebularine Induces Demethylation and Re-expression of hMLH1 Gene in Human Ovarian Cancer Cell Line

目的探讨去甲基化药物Zebularin(eZeb)对体外培养的顺铂耐药的卵巢癌细胞A2780/cp70hMLH1基因甲基化状态的影响及表达调控作用。方法应用不同浓度的Zeb(50μmol/L,100μmol/L,200μmol/L)处理体外培养的卵巢癌细胞A2780/cp70后,甲基化特异性PCR(Methylation-specific PCR,MSP)法检测用药前后细胞中hMLH1基因的甲基化状态,RT-PCR法及Western-blot法检测用药前后细胞中hMLH1基因mRNA及蛋白表达的变化。结果 hMLH1基因在卵巢癌细胞A2780/cp70中呈异常甲基化状态,在mRNA及蛋白水平表达阴性。经过Zeb处理后,hMLH1基因呈去甲基化状态,其mRNA及蛋白重新表达。结论异常甲基化是卵巢癌细胞A2780/cp70hMLH1基因失活的原因,去甲基化药物Zeb能使hMLH1基因去甲基化从而调控其重新表达。

Objective To investigate the effect of demethylation agent zebularine（Zeb） on methylation state and expression of hMLH1 gene in human cisplatin-resistant ovarian cancer cell line A2780/cp70.Methods A2780/cp70 cells were cultured in RPMI 1640 and were treated with different concentrations （50 μmol/L,100 μmol/L,200 μmol/L） of DNA methyl-transferase inhibitor Zeb.Methylation-specific PCR （MSP） was used to detect methylation state of hMLH1 gene.RT-PCR and Western blot were used to detect expression of mRNA and protein of hMLH1 gene before and after treatment with Zeb respectively.Results Promoter hypermethylation of hMLH1 gene was detected in ovarian cancer cell A2780/cp70,and no expression was detected before treatment.After treated with Zeb,hMLH1 gene exhibited demethylation and its expression occured at mRNA and protein level.Conclusion Zeb can induce demethylation of hMLH1 gene and regulate its re-expression.