国际肿瘤学杂志

国际肿瘤学杂志 ›› 2018, Vol. 45 ›› Issue (3): 134-138.doi: 10.3760/cma.j.issn.1673422X.2018.03.002

• 论著 • 上一篇    下一篇

miR-1280通过激活p21基因的表达对膀胱癌细胞周期及增殖的影响

叶志华，黄耿，付金伦，桂定文   

  1. 435000 鄂东医疗集团黄石市中心医院（湖北理工学院附属医院）泌尿外科 肾脏疾病发生与干预湖北省重点实验室
  • 收稿日期:2017-09-11 出版日期:2018-03-08 发布日期:2018-05-16
  • 通讯作者: 黄耿 E-mail:huanggsir@163.com

Effects of miR-1280 expression on the cell cycle and proliferation of bladder cancer by activating p21 gene expression

Ye Zhihua, Huang Geng, Fu Jinlun, Gui Dingwen   

  1. Department of Urological Surgery, Huangshi Central Hospital of Edong Healthcare Group (Affiliated Hospital of Hubei Polytechnic University); Hubei Key Laboratory of Kidney Disease Pathogenesis and Intervention, Huangshi 435000, China
  • Received:2017-09-11 Online:2018-03-08 Published:2018-05-16
  • Contact: Huang Geng E-mail:huanggsir@163.com

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摘要： 目的探讨微小RNA-1280（miR-1280）对膀胱癌细胞株BIU87细胞p21基因表达的激活作用及对膀胱癌细胞周期及增殖的影响。方法实时定量荧光聚合酶链反应（qRT-PCR）检测膀胱癌细胞株T24、5637、J82、BIU87和正常膀胱上皮细胞SV-HUC-1中miR-1280表达水平。选取表达量最低的膀胱癌细胞，分别转染miR1280模拟物（实验组）和miRNC（对照组），qRTPCR检测两组细胞中miR1280和p21 mRNA表达水平。染色质免疫共沉淀（ChIP）实验验证miR-1280与p21基因启动子的靶向作用。Western blotting分别检测两组细胞中p21、细胞周期依赖性激酶1（CDK1）、细胞周期蛋白A2（Cyclin A2）mRNA和蛋白的表达。流式细胞术检测细胞周期。四甲基偶氮唑蓝（MTT）法检测细胞增殖能力。结果qRTPCR结果提示，膀胱癌细胞株T24、5637、J82、BIU87和正常膀胱上皮细胞SVHUC1中miR1280表达分别为0.503±0.094、0.611±0.054、0.567±0.077、0.257±0.032和1.014±0.090，差异有统计学意义（F=1.880，P<0.001）。与膀胱癌细胞株T24、5637、J82相比，BIU87细胞株中的miR1280表达量最低（P=0.026，P=0.003，P=0.008）。与对照组相比，实验组BIU87细胞中miR1280表达显著升高（1041.000±157.500∶1.023±0.118，t=6.606，P<0.001），p21 mRNA表达亦显著升高（5.280±0.660∶1.007±0.070，t=6.440，P<0.001）。Western blotting结果显示P21蛋白表达上调，CDK1和Cyclin A2蛋白表达下调。ChIP实验结果显示，相比miRNC转染组，生物素修饰的miR1280在p21基因启动子区域的富集倍数显著增加（1.246±0.171∶0.519±0.087，t=3.787，P=0.009）。实验组BIU87细胞G0G1期细胞比例较对照组明显升高（68.360%±3.064%∶46.970%±3.971%，t=4.263，P=0.005）。MTT结果显示，与对照组相比，转染miR1280后的BIU87细胞从第3天（0.826±0.099∶1.224±0.057，t=3.505，P=0.013）开始增殖能力明显降低。结论miR1280可通过结合p21基因启动子区域，激活膀胱癌细胞BIU87中p21基因的表达，从而阻滞细胞周期的进展，抑制细胞的增殖，为膀胱癌的靶向治疗提供了一个新靶点。

关键词: 微RNAs, 膀胱肿瘤, 原癌基因蛋白质p21（ras）, RNA激活

Abstract: Objective To investigate the activation effect of microRNA1280 (miR1280) on the expression of p21 gene in bladder cancer cell line BIU87 and its effect on cell cycle and proliferation of bladder cancer cell line. MethodsRealtime fluorescence quantitative polymerase chain reaction (qRTPCR) was used to detect the expressions of miR1280 in bladder cancer cell lines T24, 5637, J82, BIU87 and normal bladder epithelial cells SVHUC1. miR1280 mimics (experimental group) and miRNC (control group) were transfected into the bladder cancer cells with the lowest expression of miR1280. The expressions of miR1280 and p21 mRNA were detected by qRTPCR. Chromatin immunoprecipitation (ChIP) was used to verify the targeting effect of miR1280 and p21 gene promoter. Western blotting was used to detect the expressions of p21, cell cycledependent kinase 1 (CDK1), Cyclin A2 mRNA and protein in the two groups. Cell cycle was detected by flow cytometry, and cell proliferation was detected by methyl thiazolyl tetrazclium (MTT) assay. ResultsThe results of qRTPCR indicated that the expression levels of miR1280 in bladder cancer cell lines T24, 5637, J82 and BIU87 and normal urothelium cell line SVHUC1 were 0.503±0.094, 0.611±0.054, 0.567±0.077, 0.257±0.032 and 1.014±0.090 respectively, with a significant difference (F=1.880, P<0.001). Compared with bladder cancer cell lines T24, 5637 and J82 cells, the expression of miR1280 in BIU87 cell was the lowest (P=0.026, P=0.003, P=0.008). Compared with the control group, the expression of miR1280 in BIU87 cell was significantly increased (1041.000±157.500 vs. 1.023±0.118, t=6.606, P<0.001), and the expression of p21 mRNA was also significantly increased (5.280±0.660 vs. 1.007±0.070, t=6.440, P<0.001). Western blotting showed that p21 protein expression was upregulated, CDK1 and Cyclin A2 protein expressions were downregulated. ChIP experiments showed that compared with the miRNC transfection group, the concentration of biotin modified miR1280 in the p21 gene promoter region was significantly increased (1.246±0.171 vs. 0.519±0.087, t=3.787, P=0.009). The proportion of G0G1 cells in the experimental group BIU87 cells was significantly higher than that in the control group (68.360%±3.064% vs. 46.970%±3.971%, t=4.263, P=0.005). The results of MTT showed that compared with the control group, the cell proliferation ability of BIU87 cells after being transfected miR1280 was significantly decreased starting from day 3 (0.826±0.099 vs. 1.224±0.057, t=3.505, P=0.013). ConclusionmiR1280 can activate the expression of p21 gene in bladder cancer cell line BIU87 by binding the promoter region of p21 gene, blocking the progression of cell cycle and inhibiting cell proliferation, which provides a new direction for bladder cancer targeted therapy theory.

Key words: MicroRNAs, Urinary bladder neoplasms, Protooncogene proteins p21 (ras), RNA activation