基础医学与临床 ›› 2021, Vol. 41 ›› Issue (5): 698-703.

• 研究论文 • 上一篇    下一篇

miR-183靶向Bnip3l对缺氧/复氧的小鼠海马神经元细胞线粒体自噬的影响

马家玲, 高艳平*   

  1. 张家港市第一人民医院 苏州大学附属张家港医院 麻醉科, 江苏 张家港 215600
  • 收稿日期:2020-05-28 修回日期:2020-10-10 出版日期:2021-05-05 发布日期:2021-05-06
  • 通讯作者: *1564009479@qq.com
  • 基金资助:
    2019年度张家港市卫生青年科技项目(ZJGQNKJ201904); 江苏大学2018年度临床医学科技发展基金(JLY20180171)

Effect of miR-183 targeting Bnip3l on mitochondrial autophagy in hypoxia/reoxygenated hippocampal neurons in mice

MA Jia-ling, GAO Yan-ping*   

  1. Department of Anesthesiology, Zhangjiagang First People's Hospital, Zhangjiagang Hospital Affiliated to Suzhou University, Zhangjiagang 215600, China
  • Received:2020-05-28 Revised:2020-10-10 Online:2021-05-05 Published:2021-05-06
  • Contact: *1564009479@qq.com

摘要: 目的 探讨miR-183调控缺氧/复氧(H/R)的海马神经元细胞线粒体自噬的机制。方法 将小鼠源海马神经元HT-22细胞分为对照组、缺氧/复氧模型组(HR组)、miR-183过表达组(miR-183 mimics组)、阴性对照组(miR-183 NC组)。用缺氧3 h+复氧12 h方法建立缺氧/复氧模型,miR-183 mimics组及miR-183 NC组在HR组基础上转染miR-183 mimics及miR-183 NC。MTT法检测HT-22细胞存活率,透射电镜观察线粒体超微结构,RT-qPCR检测miR-183、Bnip3l mRNA水平;Western blot检测Bnip3l、beclin-1、泛素和LC3结合蛋白p62(p62)、微管相关蛋白轻链3(LC3-Ⅱ)/ LC3-Ⅰ表达。用双荧光素酶报告实验验证miR-183与Bnip3l的靶向关系。结果 与对照组相比,HR组HT-22细胞自噬体增多,细胞存活率、miR-183及p62蛋白表达降低(P<0.05),Bnip3l mRNA及蛋白、beclin-1和3(LC3-Ⅱ)/ LC3-Ⅰ蛋白表达增高(P<0.05);miR-183 mimics组除细胞存活率降低外其余上述指标均与HR组相反(P<0.05);与miR-183 NC+Bnip3l-3′UTR-WT组相比,miR-183 mimics+Bnip3l-3′UTR-WT组荧光素酶活性降低(P<0.05)。结论 miR-183过表达可靶向抑制Bnip3l表达,降低线粒体自噬,加重HT-22细胞H/R损伤。

关键词: 微小RNA-183, 海马神经元细胞, 线粒体自噬

Abstract: Objective To explore the mechanism of miR-183 regulating mitochondrial autophagy in hypoxia/reoxygenated(H/R) hippocampal neurons. Methods The mouse hippocampal neuron HT-22 cells were collected and divided into control group, hypoxia/reoxygenation(H/R) model group (HR group), miR-183 over-expression group (miR-183 mimics group), and negative control group (miR-183 NC group). The anoxia and reoxygenation models were established by the method of anoxia 3 h+reoxygenation for 12 h, miR-183 mimics group and miR-183 NC group were transfected with miR-183 mimics and miR-183 NC on the basis of HR group. The survival rate of HT-22 cells was measured by MTT method, the ultra structure of mitochondria was microscopied and the levels of miR-183 and mitochondrial autophagy related genes Bnip3l mRNA were detected by RT-qPCR; Western blot was used to detect the expression of autophagy related proteins Bnip3l, beclin-1, ubiquitin and LC3 binding protein p62 (p62), microtubule related protein light chain 3 (LC3-Ⅱ)/LC3-Ⅰ. The targeting relationship between miR-183 and Bnip3l was verified by double Luciferase Report. Results Compared with control group, the autophagy of membrane structure in HT-22 cells increased in HR group and miR-183nc group, and melanin remained after autophagy, cell survival rate, miR-183 and p62 protein expression were all decreased (P<0.05), the expression of Bnip3l mRNA and protein, beclin-1 and microtubule-associated protein 1 light chain 3-Ⅱ(LC3-Ⅱ)/LC3-Ⅰ proteins all increased(P<0.05); In the miR-183 mimics group, except for the reduced cell survival rate, all the above indicators were opposite to the HR group with statistical significance (P<0.05). Compared with miR-183 NC+ Bnip3l-3′UTR-WT group, the activity of luciferase in miR-183 mimics + Bnip3l-3′UTR-WT group decreased(P<0.05). Conclusions The over-expression of miR-183 can target at inhibiting the expression of Bnip3l mRNA, the mitochondrial autophagy of HT-22 cells, and thus aggravate the damage of HT-22 cells caused by H/R.

Key words: microRNA-183, hippocampal neurons, mitochondrial autophagy

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