启动子甲基化致OPCML基因失活在胃癌发病中的作用

Promoter methylation-induced OPCML expression silence in progression of gastric cancer

目的探讨OPCML基因在胃癌细胞株中的失表达及其与基因启动子CpG岛甲基化的关系,并分析OPCML对胃癌细胞增殖的影响。方法 (1)实验组为胃癌细胞株(SGC7901、AGS、MKN28、MKN45、N87、KATOⅢ和SNU1),对照组为正常胃组织,分别采用RT-PCR检测两组细胞OPCML mRNA的表达。(2)用亚硫酸盐修饰正常胃组织的DNA和胃癌细胞MKN45的DNA,分别采用甲基化特异性PCR法检测OPCML基因启动子区甲基化情况。(3)实验组为药物处理组,即5-氮胞苷(5-AZA)去甲基化处理胃癌细胞MKN45,对照组为未经药物处理的MKN45,分别采用RT-PCR检测OPCML mRNA的表达变化。(4)RT-PCR检测转染OPCML表达载体及pcDNA3.1空载体后mRNA的表达情况。(5)实验组为OPCML表达载体转染胃癌细胞AGS,对照组为空载体转染胃癌细胞AGS,使用浓度为0.5 mg/mL的新霉素(G418)对转染细胞进行筛选,观察OPCML对胃癌细胞AGS集落形成的影响。结果 (1)在胃癌细胞株中OPCML mRNA的表达率显著低于正常胃组织。(2)胃癌细胞AGS和MKN45的甲基化程度明显高于正常胃组织。(3)5-AZA处理MKN45细胞后,OPCML表达上调。(4)OPCML表达载体转染HEK293A、AGS、MKN45细胞后,RT-PCR检测显示OPCML高表达。(5)外源性表达OPCML可抑制胃癌细胞AGS的集落形成率。结论 OPCML基因作为肿瘤抑制基因,在胃癌细胞中的表达下调。OPCML表达下调与该基因启动子区甲基化密切相关。药物性去甲基化处理能够恢复OPCML的表达。在表达沉默的AGS细胞中外源性表达OPCML,抑制集落形成率。

Objective To assess the expression of OPCML （ opioid - binding protein/ceU adhesion molecule like） in gastric cancer cells, and its correlation with methylation of promoter CpG islands. Methods （1） The stomach cancer cell lines, which included SGC7901, AGS, MKN28, MKN45, N87, KATO III and SNU1, were cultured as experimental group whereas the normal stomach tissue as control. OPCML mRNA expression was assesses by RT - PCR. （2） The DNA extracted form normal stomach tissue and stomach cancer cells MKN45 was modified by sulfites for further analy- sis of OPCML gene promoter methylation with MSP （Methylation specific PCR）. （3） MKN45 cells were demethylated by 5 - AZA （5＇ - aza -2＇ - deoxycytidine） for comparison with those without demethylation on OPCML mRNA expression by RT -PCR. （4） The OPCML mRNA expression was assessed by RT- PCR in stomach cancer cells transfected with OPC- ML expression vector or empty carrier. （5） AGS cells transfected with OPCML expression vector and empty carrier were screened with neomycin （G418, 0. 5 mg/mL）. Results （ 1 ） OPCML expression in gastric cancer cells was aberrantly reduced in contrast to normal gastric tissue. （2） Significant up - regulation of methylation of OPCML gene promoter was observed in gastric cancer AGS and MKN45 cell lines comparing with that of normal gastric tissues. （3） OPCML expression was up -regulated as 5 -AZA -indudced demethylation. （4） OPCML expression was up -regulated by transfection of OPCML expression vector in HEK293A, AGS and MKN45 cells. （5） Ectopic expression of OPCML in gastric cancer cells with endogenous silencing led to strong inhibition of cell colony formation. Conclusion The OPCML is a tumor sup-pressor gene and down - regulated in gastric cancer cells, which is induced by methylation of promoter CpG island. This process is restored with demethylation by 5 - AZA. Ectopic expression of OPCML in gastric cancer cells with endogenous silencing leads to strong inhibition of cell colony formation.