天津医药

天津医药 ›› 2016, Vol. 44 ›› Issue (2): 162-165.doi: 10.11958/56744

• 细胞与分子生物学 • 上一篇    下一篇

miR-200a靶向调控AP-2γ表达抑制神经母细胞瘤SK-N-AS细胞增殖

高顺利， 王立忠， 刘海英， 刘丹莉   

  1. 南华大学附属第一医院儿科 （邮编421001）
  • 收稿日期:2014-07-28 修回日期:2015-11-02 出版日期:2016-02-15 发布日期:2016-02-15
  • 基金资助:
    湖南省教育厅课题 （13C802）

miR-200a inhibits cell proliferation by targeting AP-2γ expression in neuroblastoma cells SK-N-AS

GAO Shunli, WANG Lizhong, LIU Haiying, LIU Danli   

  1. Department of Pediatrics, the First Affiliated Hospital, University of South China, Hengyang, Hunan 421001, China
  • Received:2014-07-28 Revised:2015-11-02 Published:2016-02-15 Online:2016-02-15

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摘要： 目的 明确 miR-200a 具有靶向调控转录因子活化蛋白 （AP） -2γ表达而抑制神经母细胞瘤细胞增殖的能力， 进一步揭示 miR-200a 抗瘤的分子机制。方法 通过双荧光素酶报告基因分析检测 miR-200a 对 AP-2γ 3′UTR- 荧光素酶活性的影响； 将 miR-200a 模拟物转染神经母细胞瘤细胞， 利用逆转录多聚酶链反应 （RT-PCR） 和蛋白印迹（Western blot） 方法检测 AP-2γ表达水平的改变； 将 AP-2γ shRNA 转染 SK-N-AS 细胞， MTS 细胞增殖活性检测分析干扰 AP-2γ表达对神经母细胞瘤细胞增殖能力的影响； 将 AP-2γ重组质粒与 miR-200a 模拟物共转染神经母细胞瘤细胞， 通过 MTS 检测细胞增殖能力。结果 miR-200a 处理组细胞活性(66.33±5.13)与对照组的(100±0)相比， 细胞增殖受到明显抑制（F=114， P＜0.05）。与未转入的相对荧光素酶活性（0.982±0.119）相比， miR-200a 转染明显抑制 AP-2γ的 3′UTR 驱动的荧光素酶活性 （0.624±0.051）。且 miR-200a 模拟物明显降低神经母细胞瘤细胞中 AP-2γ的 mRNA 和蛋白表达水平； 此外， 与对照组相比， shRNA 干扰 AP-2γ表达能明显抑制 SK-N-AS 细胞的增殖(62.5±2.4)，它能部分模拟 miR-200a 的抑制增殖能力。最后， 发现过表达 AP-2γ能部分逆转 miR-200a 对 SK-N-AS 细胞的增殖抑制作用 （92.4±1.4）。结论 miR-200a 通过靶向下调 AP-2γ表达而抑制神经母细胞瘤细胞的增殖。

关键词: 神经母细胞瘤, 微 RNAs, 细胞增殖, AP-2γ, miR-200a

Abstract: Objective To investigate whether miR-200a suppresses cell proliferation by targeting AP-2γ expression, and reveal molecular mechanism that miR-200a functions as a tumor-suppressor in neuroblastoma cells. Methods Dualluciferase reporter gene assay was employed to examine the effect of miR-200a on AP-2γ promotor luciferase activity. Neu⁃ roblastoma cells were transfected with miR-200a mimics, and the expressions of AP-2γ mRNA and protein were detected by RT-PCR and Western blot assay. The effects of AP-2γ down-regulation on cell proliferation were observed after AP-2γ shRNA was transfected into neuroblastoma cells. Neuroblastoma cell proliferation was detected by MTS assay after being cotransfected with miR-200a mimics and AP-2γ plasmid. Results Results showed that miR-200a could inhibit proliferation of neuroblastoma cells at cell viability (66.33±5.13) compared with that of control group (100±0), and also miR-200a can bind to the 3′untranslated region of AP -2γ promotor and inhibit its luciferase activity with an inhibit ratio at (0.624±0.051). AP-2γ mRNA and protein expressions were significantly down-regulated when miR-200a was over-expressed in neuroblas⁃ toma cells. Furthermore, results showed that shRNA-mediated down-regulation of AP-2γ that suppressed the cell prolifera⁃ tion of neuroblastoma at (62.5±2.4) by comparing with the control group (100±0). Moreover, restoring AP-2γ expression re⁃ versed the effect of miR-200a with a cell viability suppression at (92.4±1.4). Conclusion miR-200a suppresses cell prolif⁃ eration by targeting AP-2γ expression in neuroblastoma cells.

Key words: neuroblastoma, microRNAs, cell proliferation, AP-2γ, miR-200a