| 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。 |
| 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 |
| DOI：10.3724/SP.J.1008.2012.00380 |
| 投稿时间：2012-02-02修订日期：2012-03-15 |
| 基金项目:国家自然科学基金(81072956). |
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| Influence of triptolide on P53/P73 gene methylation and inhibition effect against proliferation of breast carcinoma MCF-7 cells |
| MEI Yi1,SHI Yong-zhao2*,FENG Wen1,YIN Qing-zhang2,LI Fang-ming2 |
(1. Department of General Surgery, Putuo Hospital Affiliated to Shanghai University of Traditional Chinese Medicine, Shanghai200062, China
2. Department of General Surgery, Shanghai Fifth People’s Hospital Affiliated to Fudan University, Shanghai200240, China
*Corresponding author.) |
| Abstract: |
| 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. |
| Key words: triptolide methylation methyltransferases P53 gene P73 gene breast neoplasms |
