卵巢上皮性癌细胞中E钙黏素基因的甲基化状态及去甲基化的意义

目的 研究卵巢上皮性癌(卵巢癌)细胞中E钙黏素(E-cad)基因启动子区5'二核苷酸胞嘧啶(5'CpG)岛的甲基化状态,观察DNA甲基转移酶抑制剂--5-杂氮脱氧胞苷(5-Aza-CdR)去甲基化后对卵巢癌细胞的生长、侵袭以及E-cad蛋白表达的影响.方法 采用甲基化特异性PCR(MSP)技术检测卵巢癌细胞系ES-2、3AO及SKOV3细胞中E-cad基因启动子区5'CpG岛的甲基化状态.以不同浓度(分别为0.1、110、10.0μmoL/L)的5-Aza-CdR处理卵巢癌细胞后,电镜下观察细胞形态的变化,四甲基偶氮唑蓝(MTT)比色法检测细胞生长情况,蛋白印迹法检测细胞中E-cad蛋白表达的变化,体外侵袭实验检测细胞侵袭能力的变化.结果 MSP技术检测显示,ES-2及SKOV3细胞中E-cad基因启动子区5'CpG岛呈高度甲基化状态和非甲基化状态,3AO细胞中E-cad基因启动子区5'CpG岛仪呈非甲基化状态.经不同浓度(分别为0.1、1.0、10.0 μmol/L)的5-Aza-CdR处理后,ES-2及SKOV3细胞的体积变小,皱缩,核/质比例缩小,核分裂象减少,随着药物浓度的增高,此种趋势逐渐明显;ES-2和SKOV3细胞中E-cad蛋白相对表达水平分别为0.274、0.320、0.398和0.415、0.507、0.638,均明显高于对照细胞(分别为0.131和0.342,P＜0.01);ES-2和SKOV3细胞穿膜细胞数分别为(88.8±2.5)、(60.7±2.4)、(36.1±3.0)个和(88.2±2.1)、(60.5±2.2)、(36.2±3.0)个,均明显低于对照细胞[分别为(121.9±2.3)、(97.6±2.7)个,P＜0.01].结论 启动子区5'CpG岛的高度甲基化是卵巢癌细胞中E-cad基因异常表达的重要机制之一,5-Aza-CdR能通过降低E-cad基因启动子区5'cpG岛的甲基化而恢复其在卵巢癌细胞中的表达,抑制卵巢癌细胞的增殖和侵袭.

E-cadherin promoter methylation and demethylation in epithelial ovarian carcinoma cells

Objective To investigate the cytidylyl phosphate guanosine(CpG) islands methylation status of E-cadherin (E-cad) promoter region in human ovarian carcinoma cell lines (ES-2,3 AO, SKOV3 ), and the effect of 5-azacytidine-2 '-deoxycytidines (5-Aza-CdR ) on the cell proliferative ability, invasion and the expression of E-cad protein. Methods Methylation specific PCR(MSP) was used to detect CpG islands methylation status of E-cad promoter region in ES-2,3AO and SKOV3 cell lines. After treated with different concentrations of 5-Aza-CdR, morphological changes of cell lines were observed under microscope. The proliferative ability was evaluated by methyl thiazolyl tetrazolium(MTT) assay. E-cad protein expression was detected by western-blot and cellular invasion was investigated by 24-well matrigel invasion chambers. Results Hypermethylatian status of CpG islands of E-cad promoter region was observed in ES-2 and SKOV3 cell lines, but not in 3AO cell lines. After treated with 5-Aza-CdR (0.1,1.0,10.0 μmol/L), ES-2 and SKOV3 cell lines displayed morphological evidence of differentiation. 5-Aza-CdR was found to decrease proliferation as evidenced by cell growth curve , to increase the level of E-cad protein expression (P ＜ 0.01 ), and effectively inhibit the ability of cell invasion(P ＜0.01 ). Conclusions CpG hypermethylation is an important mechanism of E-cad gene inactivation in ES-2 and SKOV3 cell lines. 5-Aza-CdR be found to inhibit proliferation and invasion, and increase the expression of E-cad probably by the inhibition of hypermethylation.