miR-138靶向调控PDK1基因抑制人结肠癌SW620细胞侵袭

发布时间：2018-05-04 17:12

本文选题：微小RNA + 丙酮酸脱氢酶激酶-　；参考：《肿瘤防治研究》2016年05期

【摘要】：目的探讨人结肠癌SW620细胞中miR-138和PDK1基因的表达,阐明miR-138对PDK1基因的靶向调控作用以及对SW620细胞侵袭性的影响。方法培养SW620细胞并应用免疫荧光检测PDK1基因在细胞中的表达。采用生物信息学方法对miR-138和PDK1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miR-138模拟物后,qRT-PCR检测miR-138和PDK1基因的表达,Western blot检测PDK1蛋白的表达,Transwell小室检测SW620细胞体外的侵袭性。结果倒置相差显微镜下可见人结肠癌SW620细胞均匀分布,形态规则均一;细胞免疫荧光显示细胞内有PDK1蛋白的表达,显示红色荧光。靶基因预测软件miRanda和Target Scan显示miR-138和PDK1基因匹配良好,双荧光素酶报告基因系统鉴定发现miR-138能够结合PDK1 m RNA 3’UTR并有效抑制其表达。qRTPCR和Western blot检测结果表明过表达miR-138能够导致PDK1基因表达的下降。Transwell实验表明miR-138的过表达能够抑制SW620细胞的侵袭。结论 miR-138通过靶向作用于PDK1 m RNA 3’UTR能够负性调控PDK1基因的表达,并能够抑制人结肠癌SW620细胞的侵袭。
[Abstract]:Objective to investigate the expression of miR-138 and PDK1 genes in human colon cancer SW620 cells and to elucidate the targeted regulation of PDK1 gene by miR-138 and its effect on the invasion of SW620 cells. Methods SW620 cells were cultured and the expression of PDK1 gene was detected by immunofluorescence. The target matching relationship between miR-138 and PDK1 genes was predicted by bioinformatics and identified by double luciferase reporter gene system. After transfection of liposome 2000 into miR-138 mimics, the expression of miR-138 and PDK1 genes was detected by qRT-PCR and the expression of PDK1 protein was detected by Western blot. The invasiveness of SW620 cells in vitro was detected by transwell chamber. Results under inverted phase contrast microscope, the SW620 cells of human colon cancer were uniformly distributed, and the expression of PDK1 protein was detected by cell immunofluorescence, which showed red fluorescence. Target gene prediction software miRanda and Target Scan showed that miR-138 and PDK1 genes matched well. Identification of double luciferase reporter gene system found that miR-138 could bind to PDK1 m RNA 3'UTR and effectively inhibit its expression. QRT PCR and Western blot detection results showed that overexpression of miR-138 could lead to the decrease of PDK1 gene expression. Transwell experiment showed that miR-138 overexpression was over expressed. It can inhibit the invasion of SW620 cells. Conclusion miR-138 can negatively regulate the expression of PDK1 gene and inhibit the invasion of human colon cancer SW620 cells by targeting PDK1 m RNA 3'UTR.
【作者单位】：辽宁医学院附属第三医院病理科;
【分类号】：R735.35

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