雷公藤甲素对乳腺癌细胞P53、P73基因甲基化的影响以及对细胞增殖的抑制作用  

Influence of triptolide on P53/P73 gene methylation and inhibition effect against proliferation of breast carcinoma MCF-7 cells

作　　者：梅怡[1] 史永照[2] 冯雯[1] 殷庆章[2] 李方明[2] 

机构地区：[1]上海中医药大学附属普陀医院普外科,上海200062 [2]复旦大学附属上海市第五人民医院普外科,上海200240

出　　处：《第二军医大学学报》2012年第4期380-384,共5页Academic Journal of Second Military Medical University

基　　金：国家自然科学基金(81072956)~~

摘　　要：目的研究雷公藤甲素(triptolide,TP)对人乳腺癌MCF-7细胞系增殖的影响及其与P53、P73基因表达和甲基化状态的关系。方法用不同浓度(10、20、40 ng/ml)TP处理MCF-7细胞,采用MTT法检测TP对MCF-7细胞增殖的作用;RT-PCR检测MCF-7细胞甲基转移酶1(DNMT1)、DNMT3a、DNMT3b mRNA的表达;甲基特异性PCR检测TP对MCF-7细胞P53、P73基因甲基化的影响;蛋白质印迹分析检测MCF-7细胞中P53、P73蛋白的表达。结果 TP剂量依赖性地抑制MCF-7细胞的增殖(P<0.05,P<0.01),其半数抑制浓度(IC50)约为20 ng/ml,高浓度(40 ng/ml)时抑制率达(70.1±3.52)%。TP可明显抑制MCF-7细胞中DNMT1、DNMT3a、DNMT3b mRNA的表达(P<0.05,P<0.01)。TP处理MCF-7细胞后P53启动子区的甲基化降低,TP(20 ng/ml)处理后P53 mRNA的表达升高,而在高浓度(40 ng/ml)TP作用下表达明显上调(P<0.01);TP处理MCF-7细胞后P73基因启动子区的甲基化则明显降低,且TP(10 ng/ml)处理后P73 mRNA的表达亦明显增强(P<0.05),并呈剂量依赖性。蛋白质印迹分析检测TP(20 ng/ml)处理MCF-7细胞后,P53、P73蛋白的表达均增强。结论TP可通过抑制甲基转移酶活性和抑制P53特别是P73基因启动子区甲基化促进P53、P73的表达,并且能够抑制MCF-7细胞的增殖。Objective To study the effect of triptolide（TP） on the proliferation of breast carcinoma cell line MCF-7 and its association with P53/P73 gene expression and methylation.Methods MCF-7 cells were treated with different concentrations of TP（10 ng/ml,20 ng/ml,and 40 ng/ml）,and the proliferation of MCF-7 cells was measured by MTT method.The expressions of methyltransferase DNMT1,DNMT3a and DNMT3b mRNA were measured by RT-PCR in MCF-7 cells,P53/P73 gene methylation was analyzed by methylation specific PCR,and the protein expression of p53/P73 in MCF-7 cells was examined by Western blotting assay.Results TP inhibited the proliferation of MCF-7 cells in a dose-dependent manner（P〈0.05,P〈0.01）,with the inhibitory rate being（70.1±3.52）% at 40 ng/ml TP,and the IC50 of TP was 20 ng/ml.TP significantly inhibited DNMT1,DNMT3a,and DNMT3b mRNA expression in MCF-7 cells（P〈0.05,P〈0.01）,and it also significantly inhibited methylation of P53 promoter region.TP increased P53 gene expression at 20 ng/ml and the increase was significant at 40 ng/ml（P〈0.01）.TP reversed the hypermethylation of P73 gene in MCF-7 cells;it also significantly increased P73 mRNA expression at 10 ng/ml（P〈0.05）,and the increase was in a dose-dependent manner.Western blotting analysis showed that TP（20 ng/ml） increased the protein expression of P53 and P73 in MCF-7 cells.Conclusion TP can promote the expression of P53 and P73 genes through inhibiting methyltransferase-dependent gene methylation,and further inhibit the proliferation of MCF-7 cells.

关 键 词：雷公藤内酯醇 甲基化 甲基转移酶类 P53基因 P73基因 乳腺肿瘤 

分 类 号：R737.9[医药卫生—肿瘤]