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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 investigate the relationship between promoter methylation of connexin 43 (Cx43) gene and breast epithelial mesenchymal transition (EMT) in breast cancer cells. Methods: The frequency of methylation of Cx43 promoter was detected by methylation PCR. After MD-MBA-231 cells were cultured with 5-azacytidine (5-Aza-dC) ) Cx43 mRNA, immunofluorescence and Western blotting were used to detect the expression of E-cadherin, vimen-tin and Cx43 in breast cancer cells. The methylation of promoter was detected by methylation PCR. Results: MD-MBA-231 Cx43 gene was methylated before 5-Aza-dC treatment. Methylation of Cx43 gene in breast cancer cells was reversed after 4.0 μmol / L 5-Aza-dC treatment for 48 h ; Cx43 mRNA expression increased, 6.5 times that of the control group. Western blot results showed that the relative gray value of Cx43 protein was 4.3 ± 0.2, which was significantly higher than that of the control group. E-cadherin expression increased and vimentin expression decreased in treatment group. CONCLUSION: 5-Aza-dC can reverse the abnormal methylation of Cx43 gene in MD-MBA-231 breast cancer cells, promote the re-expression of Cx43 gene and reverse the EMT of breast cancer MDA-MB231 cells to a certain extent.