5-氮杂-2'-脱氧胞苷对人胃癌SGC-7901细胞株生长及EDNRB基因启动子异常甲基化的影响

Treatment with 5-Aza-2'-deoxycytidine induces promoter demethylation of the EDNRB gene and inhibits cell proliferation in human gastric carcinoma cell line SGC-7901

目的:探讨去甲基化药物5-氮杂-2'-脱氧胞苷(5-Aza-CdR)对人胃癌细胞系SGC-7901细胞株的生长及EDNRB基因启动子异常甲基化的影响.方法:使用1、2、5、10μmol/L5-Aza-CdR干预胃癌SGC-7901细胞,甲基化特异性PCR(MSP)和逆转录聚合酶链反应(RT-PCR)分别检测药物干预前后EDNRB基因的甲基化状态和EDNRB mRNA的表达,MTT法检测细胞增殖活性,流式细胞术分析细胞周期及细胞凋亡的改变.结果:未经5-Aza-CdR处理的SGC-7901细胞中EDNRB基因启动子区域CpG岛高甲基化,且EDNRB mRNA不表达,经1、2、5、10μmol/L5-Aza-CdR处理4d后,EDNRB基因启动子区域高甲基化状态得到逆转,细胞中EDNRB mRNA表达恢复.4种浓度5-Aza-CdR处理的SGC-7901细胞后,细胞增殖受到抑制,且呈时间和剂量依赖性;并抑制SGC-7901细胞生长周期,其细胞周期阻滞于S期,5、10μmol/L5-Aza-CdR实验组细胞凋亡率显著高于对照组,且差异有统计学意义(7.13%±0.87%,13.34%±1.12% vs 3.69%±0.52%,P=0.032,P<0.001).结论:5-Aza-CdR能有效逆转人胃癌SGC-7901细胞EDNRB基因的异常甲基化,从而激活因高甲基化导致EDNRB基因沉默的再转录,诱导该基因的表达,抑制该细胞的生长.

AIM：To investigate the effects of 5-Aza-2＇-deoxycytidine on promoter hypermethylation of the EDNRB gene and cell proliferation in human gastric carcinoma cell line SGC-7901.METHODS：After SGC-7901 cells were treated with different concentrations（1,2,5,10 μmol/L） of 5-Aza-2＇-deoxycytidine,the promoter methylation status of the EDNRB gene in SGC-7901 cells was analyzed using methylation-specific polymerase chain reaction（MSP）;the expression of EDNRB gene in SGC-7901 cells was detected by reverse transcription polymerase chain reaction（RT-PCR）;cell proliferation was measured by MTT assay;and cell cycle distribution and apoptosis were analyzed by flow cytometry.RESULTS：Before treatment with 5-Aza-2＇-deoxycytidine,promoter hypermethylation of the EDNRB gene was detected in SGC-7901 cells.Accordingly,the expression of EDNRB mRNA was not detected in SGC-7901 cells.After treatment of cells with 5-Aza-2＇-deoxycytidine for four days,promoter demethylation of the EDNRB gene was induced and EDNRB mRNA expression was detected.Treatment with 5-Aza2＇-deoxycytidine restrained the proliferation of SGC-7901 cells in a time-and dose-dependent manner.Treatment with 5-Aza-CdR induced cell cycle arrest in S phase.The apoptosis rate was significantly higher in SGC-7901 cells treated with 5 or 10 μmol/L 5-Aza-CdR than in control cells（7.13% ± 0.87%,13.34% ± 1.12% vs 3.69% ± 0.52%,both P〈 0.05）.CONCLUSION：5-Aza-2＇-deoxycytidine can effectively induce promoter demethylation of the EDNRB gene,activate EDNRB gene expression,and inhibit cell growth in human gastric carcinoma cell line SGC-7901.