DNA甲基化抑制剂对涎腺腺样囊性癌中PTEN基因表达的影响

Effects of DNA methylation inhibitor on the expression of PTEN in adenoid cystic carcinoma cells

目的:研究5-氮杂-2′-脱氧胞苷(5-aza-2,-deoxycytidine,5-Aza-dc)对涎腺腺样囊性癌(adenoid cystic carcinoma,ACC)细胞中抑癌基因第10号染色体同源丢失性磷酸酶张力蛋白基因(phosphatase and tensin homology deleted on chromosome ten,PTEN)的影响及可能的机制。方法:利用RT-PCR检测正常涎腺细胞和涎腺腺样囊性癌细胞中PTEN基因mRNA的表达水平,后运用"Methprimer"软件对PTEN基因启动子区进行分析,预测CpG岛,通过甲基化特异性PCR(methylation-specific PCR,MSP)检测PTEN启动子区CpG岛的甲基化状态;利用RT-PCR检测涎腺腺样囊性癌细胞经甲基转移酶抑制剂5-Aza-dc作用后,PTEN基因mRNA的表达水平;western blot检测5-Aza-dc干预对PTEN蛋白表达的影响。结果:涎腺腺样囊性癌细胞中PTEN基因的表达明显低于正常涎腺细胞中的表达,存在统计学意义(p<0.05),通过"Methprimer"软件表明:涎腺腺样囊性癌细胞PTEN基因启动子区存在CpG岛,同时,通过甲基化特异性PCR(methylation-specific PCR,MSP)检测发现,涎腺腺样囊性癌细胞中PTEN基因启动子甲基化水平呈高表达;而在一定时间内,经5-Aza-dc作用后,涎腺腺样囊性癌细胞中PTEN基因mRNA及蛋白表达水平逐渐增加,且存在统计学意义(p<0.05),PTEN mRNA表达水平改变与PTEN蛋白的表达基本一致。结论:涎腺腺样囊性癌细胞系中PTEN的低表达可能与PTEN基因启动子区高水平的甲基化状态相关。

Objective：To detect the effects and mechanism of 5-aza-2＇-deoxycytidine on the expression of PTEN in adenoid cystic carcinoma.Methods：Using the expression of mRNA of PTEN in the normal salivary gland epithelial cells and ACC-2 cells were analyzed by RT-PCR,after using ＂Methprimer＂software,prediction of CpG island in promoter of＇ PTEN gene,the status of methylation of CpG island in promoter of PTEN gene was analyed methylation-specific PCR（MSP）.Using The expression of mRNA and protein of PTEN were analyzed by RT-PCR,western blot after treatment with methyltransferase inhibitor 5-aza-2＇-deoxycytidine（5-Aza-dc）.Results：RT-PCR analysis showed that the expression of PTEN in ACC-2 cells was significantly lower than that in the normal salivary gland epithelial cells（p0.05）.Using ＂Methprimer＂software predicted,the CpG islands were found in the promoter region of PETN.The existence of CpG island methylation in PETN promoter region in ACC-2 cells was super-expression by Methylation-Specific PCR（MSP） analysis.After the treatment with methyltransferase inhibitor 5-aza-2＇-deoxycytidine（5-Aza-dc）,the levels of mRNA and protein of PTEN in adenoid cystic carcinoma cells were increased dramatically（p0.05）,the levels of PTEN mRNA and protein is consistent.Conclusion：The low expression of PTEN in adenoid cystic carcinoma cells may be related to The hypermethylation of CpG island in promoter of PTEN.