Objective To assess the expression of OPCML ( opioid - binding protein/cell adhesion molecule like ) in gastric cancer cells, and its correlation with methylation of promoter CpG islands. Methods ( 1) The stomach cancer ceU 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 analysis 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 OPCML 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 rnsuppressor 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.%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.
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