EZH2基因在人卵巢癌顺铂耐药株中的表达及其对细胞耐药性的影响  

Expression of EZH2 gene in cisplatin-resistant ovarian cell line A2780/DDP and its effect on cisplatin resistance

作　　者：胡沙[1] 王静[1] 李智敏[1] 郭剑锋[1] 于利利[1] 杨纯[1] 王泽华[1] 

机构地区：[1]华中科技大学同济医学院附属协和医院妇产科,武汉430022

出　　处：《肿瘤》2010年第9期748-752,共5页Tumor

基　　金：国家自然科学基金资助项目(编号:30901585)

摘　　要：目的:检测果蝇zeste基因增强子同源物2(enhancer ofzestehomolog 2,EZH2)基因在人卵巢癌顺铂(cisplatin,DDP)耐药细胞株A2780/DDP及其亲本细胞株A2780中的表达差异,探讨以EZH2基因为靶向的RNA干扰能否有效逆转A2780/DDP细胞对DDP的耐药性。方法:采用实时荧光定量PCR(real-time fluorogenic quantitative PCR,RFQ-PCR)和Western印迹法检测EZH2 mRNA和蛋白在A2780/DDP及A2780细胞中的表达差异。人工构建4个靶向沉默EZH2基因的短发夹状RNA(short hairpin RNA,shRNA)质粒表达载体,脂质体法瞬时转染至A2780/DDP细胞,RFQ-PCR检测EZH2基因的沉默水平并挑选沉默效果最佳的质粒载体,G418加压筛选得到转染稳定的细胞株。RFQ-PCR和Western印迹法检测稳定转染后A2780/DDP细胞中EZH2基因的表达,MTT法检测转染细胞对DDP的耐药性。结果:A2780/DDP细胞中EZH2 mRNA和蛋白的表达量分别是A2780细胞表达量的(3.50±1.06)和(2.10±0.29)倍,差异有统计学意义(P<0.01)。稳定转染EZH2 shRNA后的A2780/DDP细胞,EZH2的mRNA及蛋白水平的表达量分别下降(83.66±5.65)%和(77.57±3.90)%(P<0.001),其对DDP的半数抑制浓度(half inhibitory concentration,IC50)值下降(61.33±11.40)%,差异有统计学意义(P<0.05)。结论:EZH2基因在A2780/DDP细胞中的表达明显升高,沉默EZH2基因能有效逆转A2780/DDP细胞对DDP的耐药性。Objective:To examine the enhancer of zeste homolog 2(EZH2) gene expression in cisplatin-resistant ovarian cancer cell line A2780/DDP and its parental cell line A2780,and to investigate whether silencing EZH2 by RNA interference could reverse cisplatin resistance of A2780/DPP cells.Methods:Expressions of EZH2 mRNA and protein in cells were detected by real-time fluorogenic quantitative PCR(RFQ-PCR) and Western blotting,respectively.The difference of EZH2 expression was compared between A2780/DDP and A2780 cells.Four EZH2 shRNA plasmid vectors were constructed and transfected into A2780/DDP cells and the best effective vector was identified by RFQ-PCR.The stable cell line was screened by G418.RFQ-PCR and Western blotting were used to validate whether the EZH2 gene expression was silenced.The effect of cisplatin on cell growth was evaluated using MTT assays.Results:The EZH2 mRNA and protein expression levels in A2780/DDP cells were(3.50±1.06) and(2.10±0.29) folds of those in A2780 cells.The difference was significant(P<0.01).After the EZH2 shRNA plasmid was stably expressed in A2780/DDP cells,the mRNA and protein expressions of EZH2 were decreased by(83.66±5.65)% and(77.57±3.90)%,respectively(P<0.001) and the IC50 of cisplatin was decreased by(61.33±11.40)%(P<0.05).The difference was significant.Conclusion:EZH2 is over-expressed in A2780/DDP cell line compared with parental cell line A2780.Silencing EZH2 by RNA interference effectively reverse the resistance of A2780/DDP cells to cisplatin.

关 键 词：EZH2基因 人卵巢癌 顺铂 耐药株 表达量 细胞耐药性 cisplatin cell line DDP WESTERN印迹法 inhibitory concentration 稳定转染 统计学意义 检测 耐药细胞株 A2780细胞 表达差异 PCR 质粒表达载体 实时荧光定量 

分 类 号：R73[医药卫生—肿瘤]