上海交通大学学报（医学版）

上海交通大学学报（医学版） ›› 2017, Vol. 37 ›› Issue (8): 1051-.doi: 10.3969/j.issn.1674-8115.2017.08.001

• 论著（基础研究） • 上一篇    下一篇

Hsa-miRNA124-3p 调控肺癌细胞株NCI-H460 增殖和迁移的作用机制

王烨，谢旺，张洁，盛洁莹，郭忠良   

  1. 同济大学附属东方医院呼吸内科，上海 200120
  • 出版日期:2017-08-28 发布日期:2017-09-28
  • 通讯作者: 郭忠良，电子信箱：drguozhl@163.com。
  • 作者简介:?王烨（1978—），女，主治医师，硕士生；电子信箱：lianlinxiaoyun@hotmail.com
  • 基金资助:
    国家自然科学基金（81370174）

Mechanism of hsa-miRNA124-3p regulating the proliferation and migration of human lung cancer cell line NCI-H460#br#

WANG Ye, XIE Wang, ZHANG Jie, SHENG Jie-ying, GUO Zhong-liang   

  1. Department of Respiration Medicine, Shanghai East Hospital, Tongji University, Shanghai 200120, China
  • Online:2017-08-28 Published:2017-09-28
  • Supported by:
     National Natural Science Foundation of China，81370174）

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摘要： 目的 · 研究 hsa-miRNA124-3p 调控肺癌细胞 NCI-H460 增殖和迁移的作用及机制。方法 · 取临床肺癌及其癌旁组织标本 4 对， 分别检测组织中hsa-miRNA124-3p 及 Krüppel 样因子4（KLF4）蛋白含量。生物信息学预测hsa-miRNA124-3p 与 KLF4 基因3’-UTR 有理论结合位点，并通过荧光素酶报告基因实验进行验证。转染pshRNA-Sponge-miRNA124 或 pshRNA-KLF4 到 NCI-H460 细胞，转 染后48 h 确定细胞转染效率，MTT 检测 NCI-H460 细胞对数期增殖活性，划线法检测细胞迁移变化。结果 · 在检测的4 对标本中， hsa-miRNA124-3p 在肿瘤组织中的含量明显高于癌旁组织（P<0.01），而肺癌组织中KLF4 蛋白表达明显低于癌旁组织（P<0.01）。 生物信息学分析结果显示，hsa-miRNA124-3p 在 KLF4 基因3’-UTR 有 8 个碱基的理论结合位点“5’-UGCCUUAA-3’”。荧光素酶活 性检测数据显示，hsa-miRNA124-3p 可以结合于KLF4 基因3’-UTR 并对蛋白表达进行负调控。细胞转染后72 h，pshRNA-SpongemiRNA124 转染组细胞增殖活性明显受到抑制（P<0.01）， pshRNA-KLF4 转染组细胞增殖活性较对照组增强（P<0.05）， pshRNASponge-miRNA124 与 pshRNA-KLF4 共转染组细胞增殖活性与对照组比较，无显著差异（P>0.05）。细胞迁移检测数据表明，不同处 理组细胞转染后72 h 细胞迁移能力变化与增殖活性的变化趋势完全一致。结论 · Hsa-miRNA124-3p 通过抑制KLF4 基因表达，增强 NCI-H460 细胞的增殖和迁移；敲减细胞内 hsa-miRNA124-3p，可以通过上调 KLF4 蛋白表达抑制 NCI-H460 细胞增殖和迁移活性。

关键词: &ensp, 肺癌, Krü, ppel 样因子 4, NCI-H460, hsa-miRNA124-3p, 增殖, 迁移

Abstract:

Objective · To study the regulation of hsa-miRNA124-3p on the proliferation and migration of human lung cancer NCI-H460 cells and its mechanism.  Methods · Four pairs of lung cancer and para-carcinoma tissues were harvested in clinical and measured for hsa-miRNA124-3p and Krüppellike factor 4 (KLF4) levels. The theoretical binding site of hsa-miRNA124-3p in 3’-UTR of KLF4 was predicted by bioinformatics, and validated by luciferase report assay. NCI-H460 cells were transfected with pshRNA-Sponge-miRNA124 or pshRNA-KLF4, and 48 hours later, the proliferation of NCI-H460 cells after genetic intervention was assayed by the MTT method, and cell migration ability was observed by streak method.  Results · For all four pairs of samples tested, hsa-miRNA124-3p was higher in the cancer tissues than in the adjacent tissue (P<0.01), and KLF4 protein was lower in the cancer tissues than in the adjacent tissue (P<0.01). The bioinformatic analysis showed there is a theoretical binding site (5’-UGCCUUAA-3’) of hsa-miRNA124-3p in 3’-UTR of KLF4. Luciferase activity assay showed that hsa-miRNA124-3p could bind to the 3’-UTR region of KLF4 gene and negatively regulate the expression of protein. The proliferation of NC-H460 cells was suppressed by transfection with pshRNA-Sponge-miRNA124 72 h after transfection (P<0.05 ). Compared with the control group, the proliferation activity of pshRNA-KLF4 transfection group was further enhanced (P<0.05) There was no significant difference in the proliferation of pshRNA-Sponge-miRNA124 and pshRNA-KLF4 cotransfection group and the control group (P>0.05). The data of cell migration assay showed that the changes of cell migration ability were the same as proliferation activity of the cells in groups 72 h after transfection.  Conclusion · Hsa-miRNA124-3p increases the proliferation and migration in NCI-H460 cells via suppressing the expression of KLF4, and reducing the content of miRNA124-3p in NC-H460 cells can inhibit cell proliferation and migration via upregulating KLF4 expression.

Key words:  lung cancer, Krüppel-like factor 4, NCI-H460, hsa-miRNA124-3p, proliferation, migration