siRNA抑制RRM2表达对子宫内膜癌Ishikawa细胞增殖影响的研究-东南大学学报医学版

siRNA抑制RRM2表达对子宫内膜癌Ishikawa细胞增殖影响的研究

单位：1. 东南大学医学院, 江苏南京 210009;
2. 南京大学医学院附属鼓楼医院妇产科, 江苏南京 210008

分类号：R730.54;R737.33

出版年·卷·期（页码）：2015·34·第六期（890-896）

摘要：

目的:探讨小干扰RNA(siRNA)抑制RRM2基因表达对子宫内膜癌Ishikawa细胞增殖及侵袭能力的影响.方法:针对RRM2基因设计siRNA, 利用阳离子脂质体Lipofectamine2000TM将siRNA片段导入Ishikawa细胞中.用荧光显微镜观察转染情况,流式细胞术检测细胞转染率、凋亡率及细胞周期,用MTT比色法检测siRNA对Ishikawa细胞增殖的影响,半定量q-PCR和Western blot分别检测RRM2在mRNA和蛋白水平上表达的变化, 用transwell小室检测细胞体外侵袭能力.结果:设计的siRNA能有效地抑制子宫内膜癌Ishikawa细胞的增殖,降低侵袭能力,诱导细胞凋亡,并使细胞阻滞于G₁期,减少S和G₂期的细胞,同时RRM2在mRNA及蛋白水平上的表达明显减少,而对照的错义序列组Ishikawa/Neg-ative-siRNA则没有上述效应.结论:体外合成的siRNA可降低子宫内膜癌Ishikawa细胞中RRM2的表达,且抑制细胞增殖,降低细胞侵袭能力,促进细胞凋亡并诱导生长停滞.

Objective: To observe the influence of siRNA on Ishikawa cell proliferation and invasion using small interfering RNA(siRNA) to knockdown RRM2 expression in endometrial cancer cell line Ishikawa. Methods: siRNA targeting RRM2 gene was designed and prepared to form ds-siRNA, then ds-siRNA was transfected into Ishikawa cells with Lipofectamine2000TM. The result of transfection was evaluated by fluorescence microscope. The transfection efficiency,apoptotic rate and cell cycle were measured by flow cytometry. The proliferation ability of Ishikawa cells was detected by MTT colorimetry. The expression of RRM2 on mRNA and protein level was analyzed by q-PCR and Western blotting respectively. The ability of cells invasion was detected with transwell chamber model. Results: The siRNA targeting human RRM2 gene effectively inhibited the proliferation and invasion of Ishikawa cells, induced cell apoptosis, increased cell counts in phase G₁ and decreased in S and G₂ phase, and decreased the expression of RRM2 on both mRNA and protein level. But these effects did not appear in scrambled siRNA (Ishikawa/Negative-siRNA) control group. Conclusion: RRM2 expression is reduced by siRNA targeting human RRM2 gene, which results the inhibition of Ishikawa cells proliferation and invasion,thus apoptosis and growth retardation are induced in vitro.

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