摘要
目的:观察去甲基化制剂——5-Aza-CdR对体外培养的胃癌细胞SGC7901p16基因启动子区甲基化状态及表达的影响,探讨胃癌细胞p16基因失活的机制及去甲基化制剂对p16基因表达的调控.方法:应用不同浓度的5-Aza-CdR(1×10-7,5×10-7,1×10-6,5×10-6mol/L)处理体外培养的胃癌细胞SGC7901后,MSP法检测用药前后细胞中p16基因的甲基化状态,RT-PCR及Western-blot法检测用药前后细胞中p16基因mRNA及蛋白表达的变化.结果:p16基因在胃癌细胞系SGC7901中启动子区呈异常甲基化状态,在mRNA及蛋白水平低表达.经过1×10-7,5×10-7,1×10-6,5×10-6mol/L5-Aza-CdR处理后,p16基因启动子区呈去甲基化状态,各组mRNA及蛋白表达相应的比值分别与处理前的比例为2.21±0.36,2.01±0.31;2.82±0.39,2.22±0.33;2.98±0.42,3.15±0.43及3.35±0.55,3.75±0.61.结论:5-Aza-CdR能逆转胃癌细胞p16基因甲基化状态,调控p16基因表达.
AIM To investigate the effects of 5-aza-2＇- deoxycytidine （5-Aza-CdR） on methylation and expression of p16 gene in the human gastric cancer cell line SGC7901, and to discuss the mechanism of p16 gene silencing in human gastric cancer cells, as well as the regulating effect of the demethylating agent on p16 gene expression.
METHODS： SGC7901 cells were cultured in RPMI 1640 medium and were treated with different concentrations （1 × 10^-7 mol/L, 5 ×10^-6mol/L, 1× 10^-6 mol/L, 5×10^-6 mol/L） of DNA methyltransferase inhibitor 5-Aza-CdR. Methylation-specific polymerase chain reaction （MSP） was used to detect the promoter methylation state of the p16 gene. RT-PCR and Western blotting were used to detect expression of p16 mRNA and protein before and after treatment with 5-Aza- CdR, respectively.
RESULTS： Promoter hypermethylation of the p16 gene was detected in SGC7901 cells, and p16 was expressed at a low level before treatment. After treatment with 5-Aza-CdR, the promoter region of the p16 gene exhibited a demethylation state, and its expression was increased at the mRNA and protein levels.
CONCLUSION： Promoter hypermethylation is a major mechanism of p16 gene silencing in hu- man gastric cancer cells, and can be reversed by the demethylating agent 5-Aza-CdR. Demethyl- ating agents can regulate the expression of the p16 gene. The corresponding ratio of the group mRNA and protein expression prior to treatment when the concentration of 5-Aza-CdR is 1× 10^-7 5 ×10^7, 1×10^-7, 5×10^-6mol/L are 2.21± 0.36, 2.01± 0.31; 2.82± 0.39, 2.22 ± 0.33; 2.98 ± 0.42, 3.15 ± 0.43, 3.35 ± 0.55, and 3.75 ± 0.61 respectively.
出处
《世界华人消化杂志》
CAS
北大核心
2007年第19期2082-2086,共5页
World Chinese Journal of Digestology
基金
国家自然科学基金资助项目
No.30572162
No.30271477
教育部高校博士点基金资助项目
No.20050159001
