摘要
目的探讨果蝇zeste基因增强子同源物2(enhancer of zeste homolog 2,EZH2)与E-钙粘蛋白(E-cadherin)在卵巢癌中的关系。方法采用短发夹状RNA的真核表达载体稳定转染人卵巢癌细胞C13*,建立稳定沉默EZH2基因的人卵巢癌细胞系shEZH2-C13*,采用Real-time PCR、Western blot和细胞免疫荧光方法检测EZH2下调后E-钙粘蛋白在mRNA水平和蛋白水平表达的变化,了解EZH2对E-钙粘蛋白表达的影响。结果 shEZH2质粒表达载体转染C13*获得的稳定转染细胞株shEZH2-C13*中,相对于未转染组,EZH2的mRNA和蛋白水平分别下降了(67.33±6.43)%和(71.21±5.77)%,差异有统计学意义(均P<0.05)。在稳定沉默EZH2的细胞中,E-钙粘蛋白的mRNA水平和蛋白水平相对于未转染组分别升高(52.33±19.03)%和(60.45±3.28)%,差异有统计学意义(均P<0.05)。细胞免疫荧光检测结果显示,EZH2沉默的细胞中E-钙粘蛋白表达明显高于未转染组,而空载体转染组没有明显变化。结论成功构建EZH2shRNA表达载体,获得EZH2基因稳定沉默的人卵巢癌细胞系shEZH2-C13*,同时发现在卵巢癌细胞中EZH2下调能升高E-钙粘蛋白的表达水平,推测EZH2可能通过催化H3K27三甲基化,介导DNA甲基化或组蛋白甲基化,抑制E-钙粘蛋白转录,进一步触发其下游事件,影响卵巢癌的侵袭和转移。
Objective To explore the association of enhancer of zeste homolog 2（EZH2）and E-cadherin（CDH1）in epithelial ovarian carcinomas.Methods We constructed short hair RNA（shRNA）eukaryotic expression vector targeting EZH2 and established the stable EZH2-knockdown ovarian cancer cell line C13＊（shEZH2-C13＊）by gene engineering technology.The mRNA and protein expression levels of EZH2and E-cadherin were determined by real-time PCR,Western blot and cellular immunofluorescence,respectively.Results The expression of EZH2mRNA and protein was decreased by（67.33±6.43）% and（71.21±5.77）%,respectively,in shEZH2-transfected cells as compared with untransfected cells.Moreover,the levels of E-cadherin mRNA and protein in shEZH2-transfected cells were increased by（52.33±19.03）%and（60.45±3.28）%,respectively,as compared with untransfected C13＊cells（P0.05）.The cellular immunofluorescence demonstrated that the level of E-cadherin protein expression was restored on the cell membrane after EZH2depletion in shEZH2-C13＊cells.Conclusion The expression of E-cadherin was increased significantly in shEZH2-transfected cells,suggesting that EZH2down-regulation may induce the expression of E-cadherin in ovarian cancer cells and a functional link exists between dysregulation of EZH2and repression of E-cadherin.EZH2 may suppress E-cadherin expression and regulate ovarian cancer progression through catalyzing H3K27methylation as well as mediating DNA methylation or histone methylation.
出处
《华中科技大学学报（医学版）》
CAS
CSCD
北大核心
2013年第4期382-385,390,共5页
Acta Medicinae Universitatis Scientiae et Technologiae Huazhong
基金
国家自然科学基金资助项目(No.30901585)
山西省卫生厅科研基金资助项目(No.201202013)
