5-aza-dC和丁酸钠对人胃癌细胞株MKN28细胞周期、凋亡以及抑癌基因表达的影响

Effect of 5-aza-dC and Sodium Butyrate on Cell Cycle,Apoptosis and Expression of Anti-oncogene in Human Gastric Cancer Cell Line MKN28

背景:表遗传修饰的主要方式DNA甲基化和组蛋白乙酰化是胃癌发生机制研究中的热点内容。目的:探讨表遗传学调控对人胃癌细胞株MKN28细胞周期、凋亡以及抑癌基因Runx3、p21^(WAF1)表达的影响。方法:培养人胃癌细胞株MKN28,并分为5-氮-2'-脱氧胞苷(5-aza-dC)组、丁酸钠组、5-aza-dC+丁酸钠组和对照组。以Annexin V-FITC/PI双染法检测细胞周期和凋亡,以逆转录聚合酶链反应(RT-PCR)检测Runx3、p21^(WAF1)mRNA表达,以甲基化特异性PCR(MSP)检测Runx3基因启动子区甲基化状态。结果:与对照组相比,5-aza-dC对细胞周期无明显影响,丁酸钠可使细胞周期阻滞于G0/G1期(P<0.05);5-aza-dC组和丁酸钠组细胞凋亡率均显著增高(8.3%±1.3%、20.8%±2.4%对2.0%±0.5%,P<0.05)。5-aza-dC可诱导Runx3 mRNA重新表达(P<0.05),但对p21^(WAF1)mRNA表达无影响。丁酸钠可增强p21^(WAF1)mRNA表达(P<0.05),但不能诱导Runx3 mRNA表达。联合5-aza-dC和丁酸钠可显著诱导细胞凋亡和增强抑癌基因Runx3、p21^(WAF1)mRNA表达(P<0.05)。5-aza-dC干预后,Runx3基因启动子区呈去甲基化状态。结论:去甲基化制剂5-aza-dC或组蛋白去乙酰化酶抑制剂丁酸钠通过重新表达Runx3或增强p21^(WAF1)表达而诱导胃癌MKN28细胞凋亡,从而发挥抗肿瘤的作用。

Background： Epigenetics including DNA methylation and histone acetylation has been a hot spot in the study of gastric cancer. Aims： To appraise the effect of epigenetic modulation on cell cycle, apoptosis, and expressions of antioncogene Runx3, p21^WAF1 in human gastric cancer cell line MKN28. Methods： Human gastric cell line MKN28 was cultured and divided into 5-aza-2＇- deoxycytidine （5-aza-dC） group, sodium butyrate group, 5-aza-dC＋ sodium butyrate group and control group. Cell cycle and apoptosis were analyzed by Annexin V-FITC/PI double staining. The mRNA expressions of Runx3 and p21^WAF1 were determined by reverse transcriptase polymerase chain reaction （RT-PCR）. The promoter methylation status of Runx3 gene was measured by methylation-specific PCR （MSP）. Results： Compared with control group, cell cycle was not arrested by 5-aza-dC, whereas the ratio of G0/G1 phase was significantly increased by sodium butyrate （P〈0.05）. Cell apoptosis rate induced by 5-aza-dC and sodium butyrate were significantly increased than that in control group （8.3%±1.3%, 20.8%±2.4% vs. 2.0%±0.5%, P〈0.05）. 5-aza-dC induced the reexpression of Runx3 mRNA （P〈0.05）, while it had no effect on expression of p21^WAF 1 mRNA. Expression of p21^WAF1 mRNA was significantly increased （P〈0.05）, whereas the Runx3 mRNA was not reexpressed in sodium butyrate group. Cell apoptosis and mRNA expressions of Runx3 and p21^WAF 1 were signifieantly increased in 5-aza-dC＋sodium butyrate group （P〈0.05）. After the intervention of 5-aza-dC, the promoter of Runx3 gene was demethylated. Conclusions： 5-aza-dC and sodium butyrate induce the apoptosis of MKN28 cells via the reexpression of Runx3 mRNA or up-regulation of p21^WAF 1 mRNA expression, thereby exert their anti-tumor effect.