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miR-138靶向调控PDK1基因抑制人结肠癌SW620细胞侵袭

Role of miR-138 in Invasion of Human Colon Cancer Cells SW620 by Regulating PDK1 Expression

  • 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TargetScan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 mRNA 3’UTR并有效抑制其表达。qRT-PCR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 mRNA 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 determine the expressions of miR-138 and PDK1 in human colon cancer cells SW620 and identify the role of miR-138 in the invasion of SW620 cells by regulating the expression of PDK1.
    Methods SW620 cells were cultured in vitro, and cellular morphology was observed by phase contrast microscope. The miR-138 which may regulate the expression of PDK1 was predicted by bioinformatics and identified using dual luciferase report system. The expressions of miR-138 and PDK1 were determined using qRT-PCR and Western blot after the transfection of miR-138 mimics by Lipofectamine 2000. The invasion of SW620 cells was detected in vitro by Transwell chamber.
    Results SW620 cells showed short spindle shaped with uniform distribution by a phase contrast microscope. The expression of PDK1 detected by immunofluorescence was positive. miRanda and TargetScan showed miR-138 was well complementary with PDK1 gene. Using dual luciferase reporter system, miR-138 was proved to inhibit PDK1 expression by binding to 3′UTR of PDK1 mRNA. Results of qRT-PCR and Western blot showed that the over-expression of miR-138 down-regulated the expression of PDK1. The invasion of SW620 cells was suppressed by the over-expression of miR-138.
    Conclusion miR-138 may negatively regulate the expression of PDK1 by binding to 3′UTR of PDK1 mRNA and inhibit the invasion of human colon cancer cells SW620.

     

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