Cx43基因启动子去甲基化与乳腺癌MDA-MB231细胞上皮间质转换的相关性研究

Relationship between demethylation of connexin 43 gene promoter and epithelial-mesenchymal transition in breast cancer MDA-MB231 cells

目的:探讨乳腺癌细胞中连接蛋白43(Cx43)基因启动子甲基化与乳腺癌上皮间质转化(EMT)的关系。方法:甲基化PCR检测Cx43启动子甲基化的频度;5-氮杂脱氧胞苷(5-Aza-dC)培养乳腺癌MD-MBA-231细胞后,反转录PCR(RT-PCR)乳腺癌细胞中Cx43 mRNA,免疫荧光、蛋白质印迹法检测E-cadherin、波蛋白(vimen-tin)及Cx43蛋白表达,甲基化PCR检测启动子甲基化的变化。结果:在应用5-Aza-dC处理前,MD-MBA-231 Cx43基因呈甲基化状态,将4.0μmol/L的5-Aza-dC作用48 h后,乳腺癌细胞Cx43基因甲基化逆转;Cx43mRNA表达水平增加,为对照组的6.5倍。蛋白质印迹检测结果显示,Cx43蛋白相对灰度值为4.3±0.2,明显高于对照组。处理组细胞E-cadherin表达增加,vimentin表达下调。结论:5-Aza-dC能逆转乳腺癌细胞MD-MBA-231的Cx43基因异常甲基化,促进Cx43基因的再表达,在一定程度上逆转乳腺癌MDA-MB231细胞EMT。

OBJECTIVE： To explore the relationship between Cx43 methylation of connexin 43（Cx43） gene promoter and breast cancer epithelial-mesenchymal transition（EMT）.METHODS： Cx43 promoter methylation frequency was detected by methylation PCR detection.MD-MBA-231 cells after being treated by 5-aza-deoxycytidine（5-Aza-dC）,Reverse Transcription PCR（RT-PCR） was used to detect the expression of Cx43 mRNA,Western blot and immunofluorescence for the detection of Cx43,E-cadherin and vimentin protein expression,Cx43 promoter methylation frequency was detected by methylation PCR detection.RESULTS： Before the treatment of 5-Aza-dC,Cx43 gene of MD-MBA-231 cells was presented methylation status.Cx43 gene methylation status of MD-MBA-231 cells was modulated by 4.0 μmol/L of 5-Aza-dC with upregulation expression of Cx43 mRNA to 6.5 fold of control group;Western blot results showed that relative gray value of Cx43 protein was 4.3±0.2,which was significantly higher than that of the control group.E-cadherin was upregulated and vimentin was downregulated.CONCLUSION： These results indicate 5-Aza-dC can modulate the abnormal methylation,promote the expression of Cx43,and reverse the EMT of breast cancer MDA-MB231 cells to some extent.