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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 metalloproteinase,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Target Scan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 investigate the expression of microRNA-17 (miR-17) and matriv metalloproteinase-2 (MMP-2) in human glioma U87 cells and to elucidate the role of miR- To regulate the role and invasiveness of U87 cells. Methods U87 cells were cultured and the expression of MMP-2 gene was detected by immunofluorescence staining. The matching of miR-17 and MMP-2 genes was predicted by bioinformatics method and identified by dual luciferase reporter gene system. Real-time PCR was used to detect the expression of miR-17 and MMP-2 genes after miR-17 mimics. The expression of MMP-2 protein was detected by Western blotting. Transwell chamber was used to detect the invasiveness of cancer cells in vitro. Results The inverted phase contrast microscope showed that the distribution of U87 cells was uniform and the diversity was obvious. The immunofluorescence showed that the expression of MMP-2 protein in cytoplasm showed green fluorescence. Target gene prediction software miRanda and Target Scan showed good matching of miR-17 and MMP-2 genes. Systematic identification of dual luciferase reporter gene revealed that miR-17 can bind 3’UTR of MMP-2 mRNA and effectively inhibit its expression. Both real-time PCR and Western blotting showed that overexpression of miR-17 downregulated MMP-2 gene expression. Transwell experiments show that miR-17 overexpression can inhibit the invasion of U87 cells. Conclusion miR-17 negatively regulates the expression of MMP-2 in human glioma U87 cells by targeting 3’UTR of MMP-2 mRNA and inhibits its invasion.