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目的:探讨5-Aza-dC及TSA对人胃癌细胞系SGC-7901中抑癌基因Runx3启动子区甲基化、mRNA及蛋白表达水平的影响.方法:单独或联合应用5-Aza-dC及TSA处理体外培养的SGC-7901细胞,提取各组细胞的DNA、RNA及蛋白质,应用甲基化特异性定量PCR法(QMSP)检测Runx3基因启动子区甲基化状态,逆转录PCR法(RT-PCR)检测Runx3mRNA的表达,免疫印迹法(Western blotting)法检测Runx3蛋白表达水平.结果:5-Aza-dC和TSA均能降低Runx3基因启动子区的甲基化水平(5-Aza-dC组及TSA组分别为对照组的0.70倍、0.63倍),提高mRNA表达水平(0.29±0.01、0.28±0.03vs0.14±0.03,P<0.05)及蛋白表达水平(0.35±0.02、0.37±0.02vs0.09±0.01,P<0.05);与单独使用5-Aza-dC和TSA相比,两药联合组Runx3基因启动子区甲基化水平(对照组的0.37倍)及mRNA表达水平(0.45±0.02)和蛋白表达水平(0.50±0.01)均较单药组效果更明显(P<0.05).结论:5-Aza-dC和TSA均能逆转胃癌细胞SGC-7901 Runx3基因的甲基化水平,恢复其mRNA和蛋白表达,且具有协同作用,为5-Aza-dC和TSA应用于胃癌的临床治疗提供了试验依据.
Objective: To investigate the effect of 5-Aza-dC and TSA on the methylation, mRNA and protein expression of Runx3 promoter in human gastric cancer cell line SGC-7901.Methods: 5-Aza-dC and TSA alone or in combination with 5-Aza- TSA treatment SGC-7901 cells in vitro, DNA, RNA and protein were extracted from each group of cells, methylation status of Runx3 promoter region was detected by methylation specific quantitative PCR (QMSP), reverse transcription PCR The expression of Runx3 mRNA was detected by Western blotting.Results: Both 5-Aza-dC and TSA could reduce the methylation level of Runx3 promoter (5-Aza-dC (0.29 ± 0.01,0.28 ± 0.03vs0.14 ± 0.03, P <0.05) and protein expression level (0.35 ± 0.02,0.37 ± 0.02 Compared with 5-Aza-dC and TSA alone, the methylation level of Runx3 promoter in the two groups (0.37-fold of the control group) and mRNA expression level (0.45-fold ± 0.02) and protein expression level (0.50 ± 0.01) were more obvious than single drug group (P <0.05) .Conclusion: Both 5-Aza-dC and TSA can reverse the change of SGC-7901 Runx3 Methylation level, restore its mRNA and protein expression, and having a synergistic effect, which provides experimental evidence for the 5-Aza-dC and TSA for clinical treatment of gastric cancer.