国际肿瘤学杂志

国际肿瘤学杂志 ›› 2019, Vol. 46 ›› Issue (2): 72-76.doi: 10.3760/cma.j.issn.1673422X.2019.02.002

• 论著 • 上一篇    下一篇

miR-182-5p靶向叉头蛋白O3a促进A549肺癌细胞增殖及抑制其凋亡

龚倩1, 谌赟1, 廖德华1, 符一岚1, 曹丽芝1, 姚敦武1, 杨小红2   

  1. 1湖南省肿瘤医院药学部，长沙410013；2湖南师范大学医学院实验中心，长沙410013
  • 出版日期:2019-02-08 发布日期:2019-04-03
  • 通讯作者: 杨小红，Email: 34063304@qq.com E-mail:34063304@qq.com
  • 基金资助:

    湖南省卫生厅科研基金课题（B2013098）；湖南省卫生健康委科研课题（C2019065）

miR-182-5p enhances proliferation and inhibits apoptosis of A549 lung cancer cells by targeting forkhead box O3a

 Gong Qian1, Chen Yun1, Liao Dehua1, Fu Yilan1, Cao Lizhi1, Yao Dunwu1, Yang Xiaohong2   

  1. 1Department of Pharmacy, Hunan Cancer Hospital, Changsha 410013, China; 2Central Laboratory, Medical College, Hunan Normol University, Changsha 410013, China
  • Online:2019-02-08 Published:2019-04-03
  • Contact: Yang Xiaohong, Email: 34063304@qq.com E-mail:34063304@qq.com
  • Supported by:

    Scientific Research Project of Health Department of Hunan Province of China (B2013098); Scientific Research Project of Health Commission of Hunan Province of China (C2019065)

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摘要： 目的  探讨微小RNA-182-5p（miR-182-5p）靶向叉头蛋白O3a（FOXO3a）对非小细胞肺癌（NSCLC）A549细胞增殖及凋亡的影响。方法  比较人正常肺上皮细胞BEAS-2B、NSCLC细胞A549中miR-182-5p的表达差异。选取A549细胞进行转染，分为miR-182-5p mimic组（转染miR-182-5p mimic）、miR-182-5p inhibitor组（转染miR-182-5p inhibitor）、NC mimic组（转染negative control mimic）和NC inhibitor组（转染negative control inhibitor）。采用反转录聚合酶链反应（RT-PCR）检测miR-182-5p表达，Western blotting检测FOXO3a蛋白表达，四甲基偶氮唑蓝(MTT)法检测细胞增殖活性，流式细胞术检测细胞凋亡。双荧光素酶实验验证miR-182-5p与FOXO3a的靶向关系。结果  A549细胞和BEAS-2B细胞中miR-182-5p相对表达量分别为3.21±0.24、1.01±0.11，差异具有统计学意义（t=14.209，P＜0.001）。NC mimic组、miR-182-5p mimic组、NC inhibitor组和miR-182-5p inhibitor组中miR-182-5p相对表达量分别为1.09±0.20、12.80±1.10、1.03±0.11、0.47±0.08，组间差异具有统计学意义（F=87.872，P＜0.001）；上述4组的FOXO3a相对表达量分别为118.34±16.71、50.89±11.58、125.33±20.87、289.26±34.51，组间差异具有统计学意义（F=62.125，P＜0.001）；4组72 h细胞增殖活性分别为1.12±0.13、1.70±0.14、1.07±0.13、0.71±0.11，组间差异具有统计学意义（F=31.336，P＜0.001），miR-182-5p mimic组细胞增殖活性较NC mimic组显著升高（P＜0.05），miR-182-5p inhibitor组细胞增殖活性较NC inhibitor组显著降低（P＜0.05）;4组细胞凋亡率分别为(5.51±1.80)%、(1.41±0.50)%、(6.24±1.71)%、(47.93±5.12)%，组间差异具有统计学意义（F=211.081，P＜0.001），miR1825p mimic组细胞凋亡率较NC mimic组显著降低（P＜0.05），miR-182-5p inhibitor组细胞凋亡率较NC inhibitor组显著升高（P＜0.001）。miRNA靶基因预测软件预测miR-182-5p能作用于FOXO3a 3′非翻译区。与转染NC mimic相比较，共转染miR-182-5p mimic与FOXO3a-Wt后A549荧光素酶活性显著降低（1.20±0.14∶0.62±0.10，t=5.839，P=0.004）。结论  miR-182-5p可通过靶向FOXO3a促进A549细胞增殖，并抑制细胞凋亡。  

关键词: 癌, 非小细胞肺, 微小RNA-182-5p, 叉头蛋白O3a

Abstract: Objective  To evaluate the effect of microRNA-182-5p (miR-182-5p) on proliferation and apoptosis of non-small cell lung cancer (NSCLC) A549 cells by targeting forkhead box O3a (FOXO3a). Methods  The difference of miR-182-5p expression between human normal lung epithelial cells BEAS-2B and NSCLC cells A549 was compared. The A549 cells were chosen, and miR-182-5p mimic (miR-182-5p mimic group), miR-182-5p inhibitor (miR-182-5p inhibitor group), negative control mimic (NC mimic group) and negative control inhibitor (NC inhibitor group) were transfected respectively. The expression of miR-182-5p was detected by reverse transcriptionpolymerase chain reaction (RT-PCR). The protein expression of FOXO3a was detected by Western blotting. The cell proliferation activity was detected by methyl thiazolyl tetrazolium (MTT) method. The cell apoptosis was detected by flow cytometry. The targeted relationship between miR-182-5p and FOXO3a was detected by dualluciferase experiment. Results  The miR-182-5p expression of A549 cells and BEAS-2B cells respectively was 3.21±0.24 and 1.01±0.11, and the difference was statistically significant (t=14.209, P＜0.001). The miR182-5p expression of NC mimic group, miR-182-5p mimic group, NC inhibitor group and miR-182-5p inhibitor group respectively was 1.09±0.20, 12.80±1.10, 1.03±0.11 and 0.47±0.08, and the difference was statistically significant (F=87.872, P＜0.001). The FOXO3a expression of the above four groups respectively was 118.34±16.71, 50.89±11.58, 125.33±20.87 and 289.26±34.51, and the difference was statistically significant (F=62.125, P＜0.001). The 72 h proliferation activity of the four groups respectively was 1.12±0.13, 1.70±0.14, 1.07±0.13 and 0.71±0.11, and the difference was statistically significant (F=31.336, P＜0.001). The proliferation activity of miR-182-5p mimic group was significantly higher than that of NC mimic group (P＜0.05), and the proliferation activity of miR-182-5p inhibitor group was significantly lower than that of NC inhibitor group (P＜0.05). The apoptosis rate of the four groups respectively was (5.51±1.80)%, (1.41±0.50)%, (6.24±1.71)% and (47.93±5.12)%, and the difference was statistically significant (F=211.081, P＜0.001). The apoptosis rate of miR1825p mimic group was significantly lower than that of NC mimic group (P＜0.05), and the apoptosis rate of miR-182-5p inhibitor group was significantly higher than that of NC inhibitor group (P＜0.001). The miRNA target genes prediction software test results showed that miR-182-5p could act on FOXO3a 3′-untranslated region (UTR). Compared with transfection NC mimic, co-transfection miR-182-5p mimic and FOXO3aWt could make luciferase activity of A549 significantly decreased (1.20±0.14 vs. 0.62±0.10; t=5.839, P=0.004）. Conclusion  miR-182-5p can enhance proliferation and inhibit apoptosis of A549 cell by targeting FOXO3a.

Key words: Carcinoma, non-small-cell lung, MicroRNA-182-5p, Forkhead box O3a