摘要
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、1．0、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（MTF）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岛仅呈非甲基化状态。经不同浓度（分别为0．1、1．0、10．0μmob／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。
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-2,3AO, 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 Hypermethylation status of CpG islands of E-cad promoter region was observed in ES-2 and SKOV3 cell fines, but not in 3AO cell lines. After treated with 5-Aza-CdR （0. 1,1.0,10. 0 ±mol/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.
出处
《中华妇产科杂志》
CAS
CSCD
北大核心
2009年第7期538-541,共4页
Chinese Journal of Obstetrics and Gynecology
