miR-17靶向调控 MMP-2基因对人脑胶质瘤 U87细胞侵袭性的影响

摘要

目的：探讨人脑胶质瘤 U87细胞中微小 RNA-17（microRNA-17，miR-17）和基质金属蛋白酶（matriv metal-loproteinase，MMP）-2基因的表达，阐明 miR-17对 MMP-2基因的靶向调控作用以及对 U87细胞侵袭性的影响。方法培养 U87细胞并应用免疫荧光化学技术检测 MMP-2基因的表达；采用生物信息学方法对 miR-17和 MMP-2基因的匹配关系进行预测并应用双荧光素酶报告基因系统鉴定；转染 miR-17模拟物后，行实时 PCR 检测 miR-17和MMP-2基因的表达，Western 印迹检测 MMP-2蛋白的表达，Transwell 小室检测癌细胞体外的侵袭性。结果倒置相差显微镜下可见 U87细胞分布均匀，多样性明显；细胞免疫荧光表明胞质内有 MMP-2蛋白的表达，显示绿色荧光。靶基因预测软件 miRanda 和 TargetScan 显示 miR-17和 MMP-2基因匹配良好，双荧光素酶报告基因系统鉴定发现 miR-17可结合 MMP-2 mRNA 3′UTR 并有效抑制其表达。实时 PCR 和 Western 印迹检测结果均表明 miR-17的过表达可下调 MMP-2基因表达。Transwell 实验表明 miR-17的过表达可抑制 U87细胞的侵袭。结论miR-17通过靶向作用于 MMP-2 mRNA 3′UTR 负性调控人脑胶质瘤 U87细胞中 MMP-2的表达并可抑制其侵袭作用。%Objective To identify the role of microRNA-17(miR-17)in human glioma U87 cells invasion which may regulate expression of matriv metalloproteinase(MMP)-2.Methods U87 cells were cultured in vitro,while changes in cellular morphology were observed by phase contrast microscope.The miR-17 which might regulate the expression of MMP-2 was predicted by bioinformatics and identified using dual luciferase report system.Expressions of miR-17 and MMP-2 were determined using real-time PCR and Western blot after transfection of miR-17 mimics.The invasion of U87 cells was detected in vitro by Transwell chamber.Results Expression of MMP-2 was positive by immunofluorescence cytochemistry. Using dual luciferase reporter system,miR-17 could inhibit the expression of MMP-2 by binding to its mRNA 3′UTR. Results of real-time PCR and Western blot showed that over-expression of miR-17 down-regulated expression of MMP-2. The invasion of U87 cells was suppressed by over-expression of miR-17.Conclusion MiR-17 may negatively regulate expression of MMP-2 in human glioma U87 cells and inhibit cell invasion.