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目的探讨人前列腺癌PC3细胞中miR-873和生存蛋白(Survivin)基因的表达,阐明miR-873对生存蛋白基因的靶向调控作用以及对PC3细胞侵袭性的影响。方法培养PC3细胞并应用免疫荧光化学技术检测生存蛋白基因的表达;采用生物信息学方法对miR-873和生存蛋白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-873模拟物后,行实时定量PCR(qRT-PCR)检测miR-873和生存蛋白基因的表达,Western印迹检测生存蛋白的表达,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细胞均匀分布,形态规则均一;免疫荧光化学显示,胞质内有生存蛋白的表达;靶基因预测软件miRanda和Target Scan显示,miR-873和生存蛋白基因匹配良好,双荧光素酶报告基因系统鉴定发现,miR-873能够结合生存蛋白mRNA 3’UTR并有效抑制其表达。qRT-PCR和Western印迹检测结果均表明,miR-873的过表达能够下调生存蛋白基因表达。Transwell实验显示,miR-873的过表达能够抑制PC3细胞的侵袭。结论 miR-873通过靶向作用于生存蛋白mRNA 3’UTR负性调控人前列腺癌PC3细胞中生存蛋白的表达,并能够抑制其侵袭。
Objective To investigate the expression of miR-873 and Survivin in human prostate cancer PC3 cells and to elucidate the role of miR-873 in the regulation of survivin genes and their impact on the invasiveness of PC3 cells. Methods PC3 cells were cultured and the expression of survivin gene was detected by immunofluorescence staining. The matching relationship between miR-873 and survivin gene was predicted by bioinformatics method and identified by dual luciferase reporter gene system. -873 mimics. Real-time quantitative PCR (qRT-PCR) was used to detect the expression of miR-873 and survivin genes. Western blotting was used to detect the expression of survivin. Transwell chamber was used to detect the invasiveness of cancer cells in vitro. Results Immunofluorescent staining showed that there were survivin in the cytoplasm. The target gene prediction software miRanda and Target Scan showed that the genes of miR-873 and survivin matched well, double-fluorescence Systematic identification of the enzyme reporter gene revealed that miR-873 binds to the 3’UTR of the survivin mRNA and effectively inhibits its expression. The results of qRT-PCR and Western blot showed that overexpression of miR-873 could down-regulate survivin gene expression. Transwell experiments showed that miR-873 overexpression can inhibit PC3 cell invasion. Conclusion miR-873 negatively regulates the expression of survivin in human prostate cancer PC3 cells by targeting 3’UTR of survivin mRNA and can inhibit its invasion.