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中国癌症防治杂志

中国癌症防治杂志 ›› 2018, Vol. 10 ›› Issue (3): 193-198.doi: 10.3969/j.issn.1674-5671.2018.03.06

• 基础研究 • 上一篇    下一篇

miR-140-5p调控Nrf2影响结肠癌细胞5-FU耐药性

  

  1. 福建省立医院胃肠外科
  • 出版日期:2018-06-25 发布日期:2018-07-02
  • 通讯作者: 黄良祥 E-mail:rainpine@163.com
  • 基金资助:

    福建省自然科学基金资助项目（2014J01284）

miR-140-5p attenuates 5-FU-resistance of colon cancer cells by targeting Nrf2

  • Online:2018-06-25 Published:2018-07-02

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摘要：

目的 观察miR-140-5p对结肠癌耐药细胞株SW480/5-FU及其亲本细胞株SW480 5-FU敏感性的影响及其与核因子NF-E2相关因子2（NF-E2-related factor 2，Nrf2）的关系，探讨miR-140-5p调控结肠癌细胞5-FU耐药的可能机制。 方法 用结肠癌细胞株SW480构建结肠癌耐药细胞株SW480/5-FU，qRT-PCR法检测两株细胞中miR-140-5p的表达，CCK-8法和细胞集落形成实验评估miR-140-5p对两株细胞5-FU敏感性的影响；采用qRT-PCR法、Western blot法双荧光素酶报告基因验证miR-140-5p与Nrf2的关系，CCK-8法、细胞集落形成实验探讨miR-140-5p调控结肠癌细胞5-FU敏感性与Nrf2的关系。 结果 成功构建结肠癌耐药细胞株SW480/5-FU，其miR-140-5p表达水平显著低于亲本细胞株SW480（P＜0.05）；CCK-8法、细胞集落形成实验结果显示，miR-140-5p可增强SW480/5-FU细胞对5-FU的敏感性，而降低SW480对5-FU的耐药性；qRT-PCR法、Westren blot法、双荧光素酶报告基因实验共同证实miR-140-5p可结合并抑制Nrf2的表达；CCK-8、细胞集落形成实验表明，Nrf2过表达能逆转miR-140-5p对结肠癌耐药细胞SW480/5-FU的5-FU敏感性调控结果。结论 miR-140-5p可能通过结合并抑制Nrf2表达，增加结肠癌耐药细胞株SW480/5-FU对5-FU的敏感性。

关键词: 结肠肿瘤, miR-140-5p, 核因子NF-E2相关因子2, 5-FU, 耐药性

Abstract:

Objective To evaluate the effect of miR-140-5p on the 5-FU sensitivity of SW480 cells and 5-FU-resistant SW480 cells，and to explore whether the effects of miR-140-5p involve Nrf2. Methods We constructed the 5-FU-resistant cell line SW480/5-FU from SW480 colon cancer cells. Expression of miR-140-5p was detected using qRT-PCR，and sensitivity of the two cell lines to 5-FU was measured using the CCK-8 and cell colony formation assays. Possible relationships between miR-140-5p and Nrf2 expression were explored using qRT-PCR，Western blotting and a dual-luciferase reporter gene assay. The involvement of Nrf2 in regulation of 5-FU-resistance by miR-140-5p was tested in CCK-8 and cell colony formation assays. Results SW480/5-FU cells showed strong 5-FU resistance，which qRT-PCR linked to much lower miR-140-5p expression. CCK-8 and cell colony formation assays showed that miR-140-5p expression correlated with significantly higher 5-FU sensitivity in SW480/5-FU cells. The combination of qRT-PCR，Western blotting and dual-luciferase reporter gene assays suggested that miR-140-5p binds directly to Nrf2 and inhibits it. Overexpression of Nrf2 in SW480/5-FU cells rescued 5-FU sensitivity，based on CCK-8 and cell colony formation assays. Conclusion It appears that miR-140-5p expression is associated with higher 5-FU sensitivity in SW480 cells，and that miR-140-5p sensitizes cells by binding and inhibiting Nrf2.

Key words: Colon neoplasms, miR-140-5p, Nrf2, 5-FU, Resistant