国际肿瘤学杂志

国际肿瘤学杂志 ›› 2017, Vol. 44 ›› Issue (11): 812-818.doi: 10.3760/cma.j.issn.1673-422X.2017.11.003

• 论著 • 上一篇    下一篇

miR-92a调控KLF4表达影响结肠癌细胞增殖的机制研究

黄虞，杜冀晖，龚慧，王秀，王磊，李一凡   

  1. 518052 广东医科大学深圳南山临床医学院中心实验室
  • 收稿日期:2017-04-19 出版日期:2017-11-08 发布日期:2017-11-24
  • 通讯作者: 杜冀晖 E-mail:jihuidu@163.com
  • 基金资助:

    深圳市科技计划（JCYJ20140411092959841）

miR-92a promotes colorectal cancer cell proliferation by regulation of KLF4 gene expression

Huang Yu, Du Jihui, Gong Hui, Wang Xiu, Wang Lei , Li Yifan   

  1. Central Laboratory, Shenzhen Nanshan Clinical Medicine College of Guangdong Medical University, Shenzhen 518052, China
  • Received:2017-04-19 Online:2017-11-08 Published:2017-11-24
  • Contact: Du Jihui E-mail:jihuidu@163.com
  • Supported by:

    Shenzhen Science and Technology Project of China (JCYJ20140411092959841)

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摘要： 目的探讨微小RNA92a（miR92a）靶向调控Krüppel样因子4（KLF4）对结肠癌细胞增殖的影响。方法采用实时荧光定量PCR（qRTPCR）方法检测21例结肠癌和对应癌旁正常组织及4种结肠癌细胞（HT29、SW480、SW620、HCT116）中miR92a的表达水平。选取结肠癌细胞株HCT116、SW620分别瞬时转染miR92a mimic、inhibitor，在构建miR92a高表达和抑制表达的细胞模型后，采用CCK8比色法检测细胞增殖活性，运用流式细胞仪检测细胞周期，采用Western blotting方法检测KLF4的蛋白表达水平，采用双荧光素酶报告基因检测细胞荧光素酶活性。结果结肠癌组织中miR92a的表达水平为（0.648±0.489）fmol/μg总RNA，显著高于对应癌旁正常组织的（0.064±0.062）fmol/μg总RNA，差异具有统计学意义（t=-5.420，P＜0.001）。4种人结肠癌细胞株中HCT116的miR92a表达水平相对最低，SW620的表达水平相对最高。上调miR92a表达，HCT116细胞72 h增殖活性高于阴性对照组（0.919±0.014∶0.765±0.025），差异具有统计学意义（t=-9.309，P=0.001），S期细胞增多［（41.670±0.461）%∶（38.703±0.554）%，t=-7.127，P=0.002］，KLF4蛋白表达水平下调（0.460±0.048∶0.758±0.109，t=22.865，P=0.028）；抑制miR92a表达，SW620细胞72 h增殖活性低于阴性对照组（0.608±0.011∶0.713±0.005），差异具有统计学意义（t=15.920，P＜0.001），S期细胞减少［（31.935±0.365）%∶（34.955±0.465）%，t=8.849，P=0.001］，KLF4蛋白表达水平上调（0.694±0.121∶0.479±0.044，t=-5.246，P=0.034）。miR92a3p mimic与KLF4 3′UTR野生型质粒共转染HCT116细胞后，与阴性对照组比较，上调细胞miR92a表达能使Firefly荧光素酶活性略有降低，但差异无统计学意义（t=0.878，P=0.429）。结肠癌组织中miR92a表达与KLF4蛋白表达之间有一定的负相关趋势，但无统计学意义（r=-0.163，P=0.699）。结论miR92a在结肠癌组织中高表达，可能通过促进细胞周期从G0G1期向S期转化而增强结肠癌细胞增殖能力。结肠癌细胞中高表达miR92a可能通过抑制KLF4蛋白表达发挥调控作用，但KLF4不是miR92a的直接作用靶基因。

关键词: 结肠肿瘤, 细胞增殖, 微小RNA-92a, Krüppel样因子4

Abstract: ObjectiveTo evaluate the effect of microRNA92a (miR92a) on regulating cell proliferation by targeting Krüppellike factor 4 (KLF4) in colon cancer. MethodsThe miR92a expressions in 21 colon cancer tissues and matched normal tumoradjacent tissues and 4 colon cancer cells (HT29, SW480, SW620, HCT116) were detected using quantitative realtime polymerase chain reaction (qRTPCR). Models of overexpression and suppression of miR92a were established by transient transfection of miR92a3p mimic to HCT116 and transient transfection of miR92a3p inhibitor to SW620, respectively. Cell proliferation activity was detected by the CCK8 colorimetry method, cell cycles were detected by flow cytometry, KLF4 protein expression was detected by Western blotting, and cell luciferase activity was detected by the dual luciferase reporter gene experiment. ResultsThe expression level of miR92a in colon cancer tissues was (0.648±0.489) fmol/μg total RNA, significantly higher than that in matched normal tumoradjacent tissues ［(0.064±0.062) fmol/μg total RNA］, with statistically significant difference (t=-5.420, P＜0.001). In 4 colon cancer cell lines, the miR92a expression level in HCT116 cells was the lowest, and highest in SW620 cell. When the expression of miR92a was upregulated, the cell proliferation activity of 72 h in HCT116 cells was higher than that in the negative control group (0.919±0.014 vs. 0.765±0.025), with statistically significant difference (t=-9.309, P=0.001), the proportion of S phase cells was also significantly increased ［(41.670±0.461)% vs. (38.703±0.554)%, t=-7.127, P=0.002), and KLF4 protein expression was decreased (0.460±0.048 vs. 0.758±0.109, t=22.865, P=0.028). When the expression of miR92a was downregulated, the cell proliferation activity of 72 h in SW620 cells was lower than that in the negative control group (0.608±0.011 vs. 0.713±0.005), with statistically significant difference (t=15.920, P＜0.001), while the proportion of S phase cells was decreased ［(31.935±0.365)% vs. (34.955±0.465)%, t=8.849, P=0.001］, and KLF4 protein expression was increased (0.694±0.121 vs. 0.479±0.044, t=-5.246, P=0.034). KLF4 3′UTR wildtype dual luciferase report plasmids were cotransfected with miR92a3p mimic to HCT116 cell, and dual luciferase assay showed that miR92a slightly repressed firefly luciferase actively, but the difference was not statistically significant (t=0.878, P=0.429). There was a negative correlation between the expression of miR92a and the expression of KLF4 protein in colon cancer tissues, but with no statistical significance (r=-0.163, P=0.699). ConclusionmiR92a is highly expressed in colon cancer tissues. It can promote colon cancer cells proliferation via enhancement of the cell cycle transition of G0G1 phase to S phase. Upexpression of miR92a may play a role in downregulating the expression of KLF4 protein in colon cancer cells. However, KLF4 is not a direct target gene of miR92a.

Key words: Colonic neoplasms, Cell proliferation, MicroRNA-92a, Krüppellike factor 4