中华皮肤科杂志 ›› 2018, Vol. 51 ›› Issue (4): 305-309.doi: 10.3760/cma.j.issn.0412-4030.2018.04.014

• 研究报道 • 上一篇    下一篇

生存素小分子干扰RNA对皮肤鳞状细胞癌A431细胞生存素基因表达及生物学功能的影响

魏明,梁颖红,陈河涛,刘佳,龚艳杰   

  1. 郑州大学第五附属医院
  • 收稿日期:2017-04-17 修回日期:2017-11-03 发布日期:2018-03-29
  • 通讯作者: 魏明 E-mail:gushiweiming@126.com

Effect of small interfering RNA targeting survivin gene on the of survivin and biological function of a human cutaneous squamous cell carcinoma cell line A431

  • Received:2017-04-17 Revised:2017-11-03 Published:2018-03-29
  • Contact: Ming WEI E-mail:gushiweiming@126.com

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摘要： 目的 探讨生存素小分子干扰RNA（siRNA）对皮肤鳞状细胞癌A431细胞生存素基因表达及细胞增殖、凋亡、迁移和侵袭力的影响。方法 培养的A431细胞分为3组：生存素siRNA组、阴性对照组和空白对照组，分别用50.0 nmol/L脂质体包裹的生存素基因序列特异性siRNA、无关序列siRNA阴性脂质体及预制囊泡转染。实时定量反转录-聚合酶链反应（RT?PCR）、Western印迹法分别检测A431细胞生存素mRNA及蛋白的表达。采用MTT比色法检测细胞增殖，流式细胞仪膜联蛋白Ⅴ/碘化丙锭双染法检测细胞凋亡，Transwell实验检测细胞迁移和侵袭力，流式细胞仪检测细胞周期变化。结果 转染后48 h，生存素siRNA组、阴性对照组、空白对照组A431细胞中生存素mRNA水平分别为0.56 ± 0.15、0.88 ± 0.37、0.90 ± 0.43，蛋白表达水平分别为0.59 ± 0.04、0.86 ± 0.05、0.91 ± 0.07，差异均有统计学意义（F = 276.67、243.61，均P < 0.001），且生存素siRNA组生存素mRNA和蛋白表达水平均显著低于阴性对照组和空白对照组，差异均有统计学意义（P < 0.05），而阴性对照组与空白对照组间差异无统计学意义（均P > 0.05）。重复测量方差分析显示，转染生存素siRNA能抑制A431细胞增殖（F = 13.19，P = 0.004），且生存素siRNA组A431细胞增殖抑制率明显高于阴性对照组和空白对照组（均P < 0.05），而阴性对照组与空白对照组比较，差异无统计学意义（P > 0.05）。转染后24 h， 3组间A431细胞凋亡率差异有统计学意义（F = 83.97，P = 0.002），且生存素siRNA组A431细胞凋亡率显著高于阴性对照组（P < 0.05）和空白对照组（P < 0.05），而阴性对照组与空白对照组间差异无统计学意义（P > 0.05）。转染后48 h，生存素siRNA组G2/M期细胞比例显著高于阴性对照组（P < 0.05）和空白对照组（P < 0.05），而生存素siRNA组A431细胞迁移数及侵袭数均显著低于空白对照组（均P < 0.05）和阴性对照组（均P < 0.05）。结论 生存素序列特异性siRNA能抑制A431细胞生存素基因表达和细胞增殖，促进细胞凋亡，抑制细胞迁移和侵袭，提示生存素可成为皮肤鳞状细胞癌基因治疗的靶点。

关键词: 肿瘤, 鳞状细胞, RNA, 小分子干扰, 细胞增殖, 细胞凋亡, 细胞周期, 细胞运动, 生存素, A431细胞

Abstract: Wei Ming, Shi Guangyong, Liu Jia, Gong Yanjie, Chen Hetao, Liang Yinghong, Tu Ling Clinical Laboratory, The Fifth Affiliated Hospital of Zhengzhou University, Zhengzhou 450052, China Corresponding author: Wei Ming, Email: gushiweiming@126.com 【Abstract】 Objective To evaluate the effect of small interfering RNA（siRNA）targeting survivin gene on the of survivin and proliferation, apoptosis, migration and invasion of a human cutaneous squamous cell carcinoma cell line A431 in vitro. Methods A survivin-specific siRNA was designed and synthesized. Cultured A431 cells were divided into 3 groups to be transfected with 50.0 nmol/L liposome complexes containing survivin-specific siRNA （survivin siRNA group）, 50.0 nmol/L liposome complexes containing unrelated siRNA（negative control group） and 50.0 nmol/L prepared vesicles （blank control group）. Real-time quantitative reverse transcription-PCR （RT-PCR） and Western blot analysis were performed to determine the mRNA and protein of survivin in A431 cells, respectively. Methyl thiazolyl tetrazolium （MTT） assay was conducted to evaluate cellular proliferative activity, flow cytometry using annexin-V/propidium iodide （PI） staining to detect cell apoptosis, Transwell assay to estimate migratory and invasive activities of A431 cells, and flow cytometry to detect cell cycle changes. Results At 48 hours after transfection, the mRNA and protein of survivin both significantly differed among the survivin siRNA group, negative control group and blank control group （mRNA: 0.56 ± 0.15, 0.88 ± 0.37, 0.90 ± 0.43, F = 276.67, P < 0.001; protein: 0.59 ± 0.04, 0.86 ± 0.05, 0.91 ± 0.07, F = 243.61, P < 0.001）, the survivin siRNA group showed significantly lower mRNA and protein of survivin compared with the negative control group and blank control group （all P < 0.05）, and there were no significant differences between the negative control group and blank control group （both P > 0.05）. Repeated measures analysis of variance showed that the transfection with survivin siRNA could significantly inhibit the proliferation of A431 cells（F = 13.19, P = 0.004）, the proliferation inhibition rate was significantly higher in the survivin siRNA group than in the negative control group and blank control group （both P < 0.05）, and no significant difference was observed between the negative control group and blank control group （P > 0.05）. At 24 hours after transfection, the apoptosis rate significantly differed among the 3 groups （F = 83.97, P = 0.002）. The survivin siRNA group showed a significantly higher apoptosis rate compared with the negative control group and blank control group （both P < 0.05）, and there was no significant difference between the negative control group and blank control group （P > 0.05）. At 48 hours after transfection, the survivin siRNA group showed a significantly higher proportion of cells at G2/M phase, but lower number of migratory cells and invasive cells compared with the negative control group and blank control group （all P < 0.05）. Conclusion Survivin-specific siRNA can inhibit the of survivin gene and the proliferation of A431 cells, promote cell apoptosis, and suppress cell migration and invasion, indicating that survivin may serve as a genetic target for the treatment of cutaneous squamous cell carcinoma.

Key words: Neoplasms, squamous cell, RNA, small interfering, Cell proliferation, Apoptosis, Cell cycle, Cell movement, Survivin, A431 cells

引用本文

魏明 梁颖红 陈河涛 刘佳 龚艳杰. 生存素小分子干扰RNA对皮肤鳞状细胞癌A431细胞生存素基因表达及生物学功能的影响[J]. 中华皮肤科杂志, 2018,51(4):305-309. doi:10.3760/cma.j.issn.0412-4030.2018.04.014