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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。
Objective To investigate the effect of recombinant trichosanthin (rTCS) on the methylation status and gene expression of p27 gene in cervical cancer HeLa cells. Methods After treated with different concentrations of rTCS (20, 40, 80 μg / mL), the methylation status of p27 gene was detected by MSP method before and after treatment with the method of Real-Ti me PCR The changes of p27 and DNMT1 mRNA in cells were detected by Western-blotting. Results The p27 gene was lowly expressed in HeLa cells and the methylation status of CpG island in gene promoter region. 40μg / mL rTCS treatment resulted in demethylation of CpG island in p27 gene promoter region; After 48 h of 20, 40 and 80 μg / mL rTCS treatment, the relative expression levels of p27 mRNA increased by 2.22, 4.00 and 6.03 times respectively. DNMT1 mRNA was highly expressed in cervical cancer HeLa cells, and DNMT1 mRNA expression was reduced by 78% at 40 μg / mL rTCS for 48 h. Conclusion rTCS can inhibit the expression of p27 gene in cervical cancer HeLa cells by inhibiting the DNMT1 expression and reversing the methylation status of CpG island in promoter region of p27 gene. rTCS can reverse the methylation status of p27 gene in cervical cancer HeLa cells and regulate the expression of p27 and DNMT1.