沉默ESRP1基因表达对卵巢癌OVCAR5细胞中FGFR2亚型转化的影响  

Silencing ESRP1 gene expression can induce FGFR2 isoform switch in ovarian cancer OVCAR5 cells

作　　者：郭欣[1] 易祎[1] 杨宇[1] 胡锦辉[1] 李娜[1] 张米妮 朱琳[1] 黄石榴 孙贤青[1] 耿佳娇 肖方祥[1] 袁成福[1] 

机构地区：[1]三峡大学医学院生物化学教研室,湖北宜昌443002

出　　处：《肿瘤》2016年第11期1187-1195,共9页Tumor

基　　金：国家自然科学基金资助项目(编号:81550029);三峡大学人才启动基金(编号:KJ2014B064);湖北省自然科学基金(编号:2015CFB198);肿瘤微环境与免疫治疗湖北省重点实验室(三峡大学)开放基金课题(编号:2015KZL02);宜昌市科学技术局项目(编号:A16-301-31)~~

摘　　要：目的 :研究沉默上皮剪接调节蛋白(1epithelial splicing regulatory protein 1,ESRP1)基因表达对卵巢癌OVCAR5细胞中成纤维细胞生长因子受体2(fibroblast growth factor receptor 2,FGFR2)亚型转换的影响。方法:构建靶向ESRP1基因的ESRP1-sh RNA慢病毒表达载体(p LVTHM/ESRP1-sh RNA1和p LVTHM/ESRP1-sh RNA2),同时设置阴性对照(negative control,NC)质粒(p LVTHM/NC-sh RNA);将3种质粒分别转染到293T细胞中制备慢病毒。慢病毒感染卵巢癌OVCAR5细胞后,分别采用实时荧光定量PCR及蛋白质印迹法检测ESRP1 m RNA及蛋白的表达水平;采用限制性内切酶AvaⅠ和Hin CⅡ检测沉默ESRP1基因后对FGFR2亚型转换的影响;MTT法检测沉默ESRP1基因后对OVCAR5细胞增殖的影响;蛋白质印迹法检测上皮标志物E-钙黏蛋白(E-cadherin)、间质标志物N-钙黏蛋白(N-cadherin)及β-链蛋白(β-catenin)表达的变化。结果:成功构建靶向ESRP1基因的sh RNA慢病毒表达载体并包装获得慢病毒;与阴性对照组比较,靶向ESRP1基因的sh RNA能明显抑制OVCAR5细胞中ESRP1 m RNA及蛋白的表达(P值均<0.05);沉默ESRP1基因的表达能诱导OVCAR5细胞中FGFR2由上皮亚型(FGFR2b)向间质亚型(FGFR2c)的转换,并促进OVCAR5细胞的增殖(P<0.05);同时,下调E-cadherin的表达水平而上调N-cadherin的表达水平,并激活β-catenin(P值均<0.05)。结论:沉默ESRP1基因表达能诱导OVCAR5细胞中FGFR2亚型的转换,促进OVCAR5细胞增殖,并诱导OVCAR5细胞发生上皮-间质转化(epithelial-mesenchymal transition,EMT),该过程可能涉及Wnt/β-catenin信号通路。Objective： To investigate the effects of silencing epithelial splicing regulatory protein 1 （ESRP1） gene expression on isoform switch of fibroblast growth factor receptor 2 （FGFR2） in ovarian cancer OVCAR5 cells. Methods： The two lentivirus expression vectors carrying ESRP1- shRNA1/2 targeting ESRP1 gene （named as pLVTHM/ESRP1-shRNA1 and pLVTHM/ESRP1-shRNA2, respectively） were constructed, while the vector pLVTHM/ NC-shRNA was used as the negative control. The three plasmids were separately transfected into 293T cells to prepare the recombinant lentivirus, and the recombinant lentivirus carrying ESRP1- shRNA1/2 and NC-shRNA were infected into OVCAR5 cells, respectively. Then the expression levels of ESRP1 mRNA and protein in OVCAR5 cells were determined by real-time fluorescent quantitative PCR and Western blotting, respectively. The effect of ESRP1 gene silencing on FGFR2 isoform switch was detected by restrictive endonucleases Ava I and HinC II. The effect of ESPR1 gene silencing on the proliferation of OVCAR5 cells was detected using M-IF method. The changes of E-cadherin, N-cadherin and β-catenin expressions were detected by Western blotting. Results： The lentivirus expression vectors containing ESRP1-shRNA1/2 were constructed successfully, and their recombinant lentivirus were obtained. Compared with the negative control group, the expressions of ESRP1 mRNA and protein in OVCAR5 cells were significantly inhibited by ESRP1 shRNA （both P 〈 0.05）. ESPR1 gene silencing could induce the FGFR2 isoform switch from epithelial subtype （FGFR2b） to mesenchymal subtype （FGFR2c） in OVCAR5 cells, and could promote cell proliferation, down-regulate the expression of E-cadherin, up-regulate the expression of N-cadherin, and activate β-catenin in OVCAR5 cells （all P 〈 0.05）. Conclusion： ESPR1 gene silencing could induce the isoform switch of FGFR2 gene switch, promote cell proliferation and induce epithelialmesenchymal transition （EMT） in OVCAR5 cells. The processes may involve t

关 键 词：卵巢肿瘤 上皮剪接调节蛋白1 受体 成纤维细胞生长因子 2型 上皮-间质转化 

分 类 号：R737.31[医药卫生—肿瘤]