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

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、40ng/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20ng/ml（P<0.01），高浓度（40ng/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（P<0.05，P<0.01）的表达。TP处理MCF-7细胞后P53启动子区的甲基化降低，TP(20ng/ml)处理后P53 mRNA的表达升高,而在高浓度(40ng/ml) TP作用下表达明显上调(P<0.0501)；TP处理MCF-7细胞后P73基因启动子区的甲基化则明显降低，并呈剂量依赖性，且TP(20ng10ng/ml)处理后P73 mRNA的表达亦明显增强(P<0.0105) ，并呈剂量依赖性。蛋白质印迹分析检测TP(20ng/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、抑制P53特别是P73基因启动子区甲基化，促进P53/P73的表达，从而抑制MCF-7细胞的增殖。

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

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 anner. 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.