论文部分内容阅读
目的探讨miR-485-5p对人结直肠癌HCT116细胞中Survivin基因的靶向调控作用以及对细胞侵袭性的影响。方法培养HCT116细胞,应用免疫荧光检测Survivin基因在细胞中的表达。采用生物信息学方法对mi R-485-5p和Survivin基因的靶向匹配关系进行预测,并应用双荧光素酶报告基因系统鉴定。脂质体2000转染mi R-485-5p模拟物后,Real-time PCR检测mi R-485-5p和Survivin基因的表达,Western blotting检测Survivin蛋白的表达,Transwell小室检测HCT116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;细胞免疫荧光显示胞质内有Survivin蛋白的表达。靶基因预测软件mi Randa和Target Scan显示mi R-485-5p和Survivin基因匹配良好,双荧光素酶报告基因系统鉴定发现mi R-485-5p能够结合Survivin m RNA 3’UTR,并有效抑制其表达。Real-time PCR和Western blotting检测结果显示,过表达mi R-485-5p能够导致Survivin基因表达的下降。Transwell实验结果显示,mi R-485-5p的过表达能够抑制HCT116细胞的侵袭。结论 mi R-485-5p通过靶向作用于Survivin mRNA 3’UTR能够负性调控Survivin基因的表达,并能够抑制人结直肠癌HCT116细胞的侵袭。
Objective To investigate the regulatory effect of miR-485-5p on Survivin gene in human colorectal cancer HCT116 cells and its effect on cell invasiveness. Methods HCT116 cells were cultured and the expression of Survivin gene was detected by immunofluorescence. Bioinformatics methods were used to predict the target-matching relationship between mi R-485-5p and Survivin genes and identified by dual luciferase reporter assay. Real-time PCR was used to detect the expression of mi R-485-5p and Survivin gene after transfection with mi R-485-5p mimics. The expression of Survivin protein was detected by Western blotting. The invasiveness of HCT116 cells in vitro was detected by Transwell chamber . Results The cells were evenly distributed under inverted phase contrast microscope and the morphology was uniform. The expression of Survivin protein in cytoplasm was detected by immunofluorescence. The target gene prediction software mi Randa and Target Scan showed good matching of mi R-485-5p and Survivin genes. The system identification of dual luciferase reporter gene revealed that mi R-485-5p can bind to Survivin m RNA 3’UTR and effectively inhibit its expression expression. Real-time PCR and Western blotting results showed that overexpression of mi R-485-5p could result in the decrease of Survivin gene expression. Transwell experimental results show that overexpression of mi R-485-5p can inhibit the invasion of HCT116 cells. Conclusion mi R-485-5p can negatively regulate the expression of Survivin gene by targeting 3’UTR of Survivin mRNA and inhibit the invasion of human colorectal cancer HCT116 cells.