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中国癌症防治杂志

中国癌症防治杂志 ›› 2022, Vol. 14 ›› Issue (6): 617-623.doi: 10.3969/j.issn.1674-5671.2022.06.04

• 基础研究 • 上一篇    下一篇

miR⁃449a通过NOTCH1介导铁死亡调控非小细胞肺癌细胞增殖和凋亡 

  

  1. 内蒙古医科大学附属人民医院肿瘤内科
  • 出版日期:2022-12-25 发布日期:2022-12-30
  • 通讯作者: 温珍平 E?mail：530140391@qq.com
  • 基金资助:
    内蒙古医科大学联合项目（YKD2021LH033）

 miR⁃449a regulates proliferation and apoptosis of non⁃small cell lung cancer cells through NOTCH1⁃mediated ferroptosis

  • Online:2022-12-25 Published:2022-12-30

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摘要： 目的 探讨miR⁃449a对非小细胞肺癌细胞增殖和凋亡的调控作用及其分子机制。方法 用miR⁃499a过表达类似物Agomir⁃499a（Agomir⁃499a组）及其阴性对照（NC组）分别转染非小细胞肺癌NCI⁃H1975细胞，采用qRT⁃PCR检测转染细胞中miR⁃499a的表达水平，CCK⁃8和Ki67染色法检测细胞增殖，Annexin V染色法检测细胞凋亡。采用铁死亡抑制剂ferrostatin⁃1（Fer⁃1）和谷胱甘肽（GSH）分别处理转染Agomir⁃499a的NCI⁃H1975细胞（依次记为Agomir⁃499a+Fer⁃1组和Agomir⁃499a+GSH组），采用荧光探针法检测细胞内的Fe2+ 水平，光学比色法检测细胞内的GSH含量，qRT⁃PCR检测细胞内铁死亡和铁稳态相关基因的mRNA表达，Western blot检测NOTCH1信号通路相关蛋白的表达。结果 与NC组比较，Agomir⁃499a组NCI⁃H1975细胞的miR⁃449a表达水平明显增加（P=0.001），细胞增殖活力明显降低（P<0.05），细胞凋亡比例明显增加（P=0.004）；铁死亡相关因子PTGS2、ACSL4、SLC7A11和COX2的表达水平，Fe2+细胞比例以及铁稳态相关基因TF、STEAP3、TFRC和DMT1的表达水平均明显增加（均P<0.05），但GSH含量以及NOTCH1信号通路相关分子NOTCH1、NICD、JAG1、JAG2、DLL1和HES1的表达水平均明显降低（均P<0.05）。与Agomir⁃449a组比较，Agomir⁃449a+Fer⁃1组铁死亡相关因子PTGS2、ACSL4、COX2和SLC7A11的表达水平，Fe2+细胞比例以及铁稳态相关基因TF和STEAP3的表达水平均明显降低（均P<0.05），但GSH含量和NOTCH1信号通路相关分子NICD、JAG1和DLL1的表达水平均明显增加（均P<0.05）；Agomir⁃449a+GSH组SLC7A11、COX2和ACSL4的表达水平也较Agomir⁃449a组明显降低（均P<0.05）。 结论 miR⁃449a可诱导非小细胞肺癌NCI⁃H1975细胞凋亡和增殖抑制，其作用机制可能与抑制NOTCH1信号介导的铁死亡有关。

关键词: 非小细胞肺癌, miR?449a, NOTCH1信号通路, 铁死亡, 增殖, 凋亡

Abstract: Objective To investigate the regulatory effect of miR⁃449a on the proliferation and apoptosis of non⁃small cell lung cancer cells and its molecular mechanism. Methods The miR⁃499a overexpression analogue Agomir⁃499a (Agomir⁃499a group) and its negative control (NC group) were transfected, respectively, into non⁃small cell lung cancer  NCI⁃H1975 cells. The expression level of miR⁃499a in transfected cells was detected by qRT⁃PCR, the cell proliferation was detected by CCK⁃8 and Ki67 staining, and the apoptosis was detected by Annexin V staining. NCI⁃H1975 cells transfected with Agomir⁃499a were treated with ferrostatin⁃1 (Fer⁃1) and glutathione (GSH), respectively (denoted as Agomir⁃499a + Fer⁃1 group and Agomir⁃499a+GSH group). The Fe2+ level and the content of GSH in cells was detected by the fluorescent probe and the optical colorimetry method, respectively. qRT⁃PCR was used to detect the mRNA expression of ferroptosis and iron homeostasis related genes, and Western blot was used to detect the expression of NOTCH1 signaling pathway related proteins. Results Compared with those in the NC group, the expression level of miR⁃499a in NCI⁃H1975 cells of the Agomir⁃449a group was significantly increased (P=0.001), the proliferation activity of NCI⁃H1975 cells was significantly decreased (P<0.05), and the proportion of cell apoptosis was significantly increased (P=0.004). The expression levels of ferroptosis⁃related factors PTGS2, ACSL4, SLC7A11 and COX2 , the proportion of Fe2+ positive cells, and the expressions levels of iron homeostasis⁃related genes TF, STEAP3, TFRC and DMT1 were significantly increased (all P<0.05). However, GSH content and expression levels of NOTCH1 signaling pathway related molecules NOTCH1, NICD, JAG1, JAG2, DLL1 and HES1 were significantly decreased (all P<0.05). Compared with the Agomir⁃449a group, the expression levels of ferroptosis⁃related factors PTGS2, ACSL4, SLC7A11 and COX2, the proportion of Fe2+ positive cells, and the expressions levels of iron homeostasis⁃related genes TF and STEAP3 in the Agomir⁃449a+Fer⁃1 group were significantly decreased (all P<0.05), while the GSH content and the expression levels of NOTCH1 signaling pathway related molecules NICD, JAG1 and DLL1 were significantly increased (all P<0.05). The expression levels of SLC7A11, COX2 and ACSL4 in the Agomir⁃449a+GSH group were also significantly lower than those in Agomir⁃449a group (all P<0.05). Conclusions The miR⁃449a can induce apoptosis and proliferation inhibition of non⁃small cell lung cancer NCI⁃H1975 cells, and its mechanism may be related to the inhibition of ferroptosis mediated by NOTCH1 signaling.

Key words: Non?small cell lung cancer, miR?449a, NOTCH1 signaling pathway, Ferroptosis, Proliferation, Apoptosis

中图分类号: 

  • R734.2