重组天花粉蛋白体外诱导宫颈癌HeLa细胞p27基因去甲基化的研究

目的研究重组天花粉蛋白(recombinant trichosanthin,rTCS)对宫颈癌HeLa细胞中p27基因甲基化状态和基因表达的影响。方法应用不同质量浓度的rTCS(20、40、80μg/mL)处理体外培养的宫颈癌HeLa细胞后,MSP法检测用药前后细胞中p27基因的甲基化状态,Real-Ti me PCR法检测用药前后细胞中p27和DNA甲基转移酶-1(DNMT1)mRNA水平的变化,Western-blotting法检测用药前后细胞中p27蛋白表达的变化。结果HeLa细胞中p27基因为低表达,基因启动子区CpG岛呈部分甲基化状态。40μg/mLrTCS处理导致p27基因启动子区CpG岛去甲基化;在20、40、80μg/mL的rTCS作用48 h后,p27基因mRNA相对表达水平分别升高2.22、4.00、6.03倍,p27蛋白水平表达也逐渐增加;宫颈癌HeLa细胞中DNMT1 mRNA高表达,40μg/mLrTCS处理48 h可至DNMT1 mRNA表达水平降低78%。结论rTCS通过抑制DNMT1,逆转p27基因启动子区CpG岛的甲基化状态,使宫颈癌HeLa细胞中p27基因表达活化。rTCS能逆转宫颈癌HeLa细胞p27基因甲基化状态,调控p27基因和DNMT1的表达。

In vitro introduction of recombinant trichosanthin on demethylation of p27 in HeLa cells

Objective To investigate the effects of recombinant trichosanthin (rTCS) on methylation status and expression level of p27 gene in HeLa cells. Methods HeLa cells was treated by different con-centration (20 μg/mL, 40 μg/mL, and 80 μg/mL) of rTCS for 48 h and then methylation-specific poly- merase chain reaction (MSP) was used to detect the promoter methylation status of the p27 gene, real-time PCR was used to detect levels of p27 and DNMT1 mRNA, and Western blotting assay was used to detect expression level of p27 protein before and after treatment with rTCS. Results Low expression level and promoter methylation status of the p27 gene were detected in HeLa cells. Treatment with 40 μg/mL rTCS totally demethylated p27 promoter. Treatment with 20 μg/mL, 40 μg/mL or 80 μg/mL rTCS resulted in a 2.22-, 4.00- or 6.03-folds increase in p27 mRNA level, respectively, and also a great increase in p27 pro- tein level. A high DNMT1 expression level was observed in HeLa cells and treatment with 40μg/mL rTCS resulted in a 78% decrease at the DNMT1 mRNA expression. Conclusion rTCS could reverse promoter hypermethylation and re-activate the expression of p27 gene by inhibiting DNMT1 expression in HeLa cells, which indicates its potential use in cancer therapy.