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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 effect of epirehelial splicing regulatory protein 1 (ESRP1) gene on the subtypes of fibroblast growth factor receptor 2 (FGFR2) in ovarian cancer OVCAR5 cells. METHODS: ESRP1-sh RNA lentiviral vector targeting ESRP1 gene (p LVTHM / ESRP1-sh RNA1 and p LVTHM / ESRP1-sh RNA2) was constructed and a negative control (NC) plasmid -sh RNA). Three plasmids were transfected into 293T cells respectively to prepare lentivirus. The lentivirus-infected OVCAR5 cells were detected by real-time fluorescence quantitative PCR and Western blotting respectively ESRP1 m RNA and protein expression levels; using restriction endonucleases Ava Ⅰ and Hin C Ⅱ silencing ESRP1 gene after FGFR2 subtype conversion The effect of silencing ESRP1 gene on the proliferation of OVCAR5 cells was detected by MTT assay. The expression of epithelial markers E-cadherin, interstitial markers N-cadherin and β - Changes in the expression of β-catenin. Results: The shRNA targeting sh RNA of ESRP1 gene was successfully constructed and packaged into lentivirus. Compared with the negative control group, sh RNA targeting ESRP1 gene significantly inhibited the expression of ESRP1 mRNA and protein in OVCAR5 cells (P value (P <0.05). The silencing of ESRP1 gene expression induced the translocation of FGFR2 from epithelial subtype (FGFR2b) to stromal subtype (FGFR2c) and the proliferation of OVCAR5 cells in OVCAR5 cells (P <0.05) -cadherin and up-regulated the expression of N-cadherin, and activated |Â-catenin (all P <0.05). CONCLUSIONS: Silencing ESRP1 gene expression can induce the transformation of FGFR2 subtype in OVCAR5 cells, promote the proliferation of OVCAR5 cells and induce the epithelial-mesenchymal transition (EMT) in OVCAR5 cells, which may involve Wnt / β-catenin signal path.