miR-7沉默EGFR/PI3K通路逆转脑胶质瘤U251细胞的恶性表型  

MicroRNA-7 silencing EGFR/PI3K pathway reverses the malignant phenotype of U251 glioma cells

作　　者：刘晓智[1] 姜忠敏[2] 陈镭[1] 刘洪良[1] 史万超[1] 张合亮[1] 刘振林[1] 

机构地区：[1]天津市第五中心医院神经外科,天津300450 [2]天津市第五中心医院病理科,天津300450

出　　处：《肿瘤》2011年第11期987-992,共6页Tumor

基　　金：天津市自然科学基金面上资助项目(编号:09JCYBJC09500);国家自然科学基金(青年科学基金资助项目)(编号:81000901)

摘　　要：目的:探讨利用微RNA(microRNA-7,miR-7)靶向沉默表皮生长因子受体(epidermal growth factor receptor,EGFR)/磷脂酰肌醇3激酶(phosphatidylinositol kinase-3,PI3K)通路途径逆转脑胶质瘤的恶性表型。方法:利用脂质体转染法将带有miR-7基因序列的表达质粒转入人脑胶质瘤细胞株U251中;采用实时荧光定量-PCR(real-time fluorogenic quantitative-PCR,RFQ-PCR)方法检测转染细胞中miR-7的表达水平;免疫细胞化学法和蛋白质印迹法检测EGFR、PI3K和AKT2蛋白的表达情况;绘制细胞生长曲线,FCM法检测细胞周期变化,Transwell小室法检测瘤细胞迁移能力,软琼脂克隆形成实验检测肿瘤细胞致瘤性。结果:RFQ-PCR结果显示,miR-7基因转染组细胞中miR-7水平明显高于对照组和无义序列组;蛋白质印迹法检测结果显示,转染组U251细胞中EGFR、PI3K和AKT2蛋白的水平较对照组均有明显降低(P<0.05);U251细胞增殖速度减慢,处于S期的细胞所占比例减少,细胞迁移及软琼脂克隆形成能力均有明显降低(P<0.05)。结论:转染miR-7可有效沉默胶质瘤U251细胞中EGFR/PI3K通路主要成员蛋白的表达,进而逆转其恶性表型,有望成为一种新的胶质瘤基因治疗方法。Objective：To investigate the malignant phenotype of human glioma cells by microRNA-7（miR-7） silencing epidermal growth factor receptor（EGFR）/phosphatidylinositol kinase-3（PI3K） pathway.Methods：The human U251 glioma cells were transfected with pri-miR-7.The expression of miR-7 was analyzed by real-time fluorogenic quantitative-PCR（RFQ-PCR）.The expressions of epidermal growth factor receptor（EGFR）,PI3K and AKT2 proteins were detected by immunocytochemistry and Western blotting.The cell growth curves were drawn,and the cell cycle distribution induced by miR-7 was determined by flow cytometry（FCM）.The cell migration ability and tumorigenicity were detected by Transwell chamber assay and soft agar colony assay,respectively.Results：The result of RFQ-PCR showed that the expression level of miR-7 was up-regulated in human glioma cells transfected with pri-miR-7.The expression levels of EGFR,PI3K and AKT2 proteins were down-regulated in glioma U251 cells transfected with pri-miR-7（P0.05）.The cell proliferation rate was slowed down,and the proportion of S phase cells was reduced.The abilities of cell migration and soft agar colony formation of U251 cells transfected with pri-miR-7 were significantly reduced（P0.05）.Conclusion：MiR-7 transfection can effectively silence the expressions of key members of EGFR/PI3K pathway and reverse the malignant phenotype of U251 cells,and which is expected to become a new choice of glioma gene therapy.

关 键 词：神经胶质瘤 转染 受体 表皮生长因子 细胞增殖 侵袭 U251细胞 

分 类 号：R737.9[医药卫生—肿瘤]