解剖学报

解剖学报 ›› 2013, Vol. 44 ›› Issue (5 ): 616-620.doi: 10.3969/j.issn.0529-1356.2013.05.006

• 神经生物学 • 上一篇    下一篇

miRNA-181b在氧-糖剥夺致N2A细胞缺血损伤中的作用及对热休克蛋白A5表达的调节

韩松 彭志锋 李俊发*   

  1. 首都医科大学神经生物学系 北京脑重大疾病研究院，北京 100069
  • 收稿日期:2013-02-21 修回日期:2013-03-28 出版日期:2013-10-06 发布日期:2013-10-06
  • 通讯作者: 李俊发 E-mail:junfali@ccmu.edu.cn
  • 基金资助:

    国家自然科学基金资助项目

Role of microRNA-181b in oxygen-glucose deprivation -induced N2A cell ischemic injury and its regulation on HSPA5 protein levels

HAN Song  PENG Zhi-feng  LI Jun-fa*   

  1. Department of Neurobiology and Beijing Institute for Brain Disorders,Capital Medical University, Beijing 100069, China
  • Received:2013-02-21 Revised:2013-03-28 Online:2013-10-06 Published:2013-10-06

摘要：

目的 探讨miR-181b在氧糖剥夺(OGD)致N2As神经瘤细胞缺血损伤中的作用，及其对热休克蛋白（HSP）A5表达的调节。方法 应用N2A细胞OGD模型模拟神经细胞缺血损伤，MTT比色法检测N2A细胞生存率，免疫印迹法检测HSPA5蛋白表达水平，实时定量PCR法检测miR-181b和HSPA5 mRNA表达水平，荧光素酶报告基因技术检测miR-181b对HSPA5 mRNA的直接调控作用。 结果 miR-181b在OGD致N2A细胞缺血损伤中表达水平明显降低(n=5)；在OGD致N2A细胞缺血损伤过程中，通过上调或抑制miR-181b的表达水平可以显著影响N2A细胞的生存率(n=6)；而在非OGD条件下，miR-181b表达水平的改变对N2A细胞活力无影响；miR-181b表达水平的改变可显著影响HSPA5蛋白表达水平(n=3)，而非HSPA5的mRNA水平；共转染miR181b前体（pre-miR-181b）或miR-181b抑制剂（anti-miR-181b）可显著抑制或增高含有HSPA5 mRNA 3’-UTR的荧光素酶报告基因的活性(n=5)。 结论 miR-181b通过负性调节HSPA5的蛋白表达水平，在OGD致N2A神经细胞缺血性损伤中发挥重要作用。

关键词: 氧-糖剥夺, 缺血性损伤, miR-181b, 热休克蛋白A5, 免疫印迹法, 实时定量PCR, 双荧光素酶报告基因分析, N2A细胞

Abstract:

Objective To explore the role of microRNA-181b(miR-181b )in oxygen-glucose deprivation (OGD)-induced N2A cell ischemic injury and its regulation on HSPA5 protein levels. Methods Using N2A cell OGD model to mimic ischemic injuryin vitro, the extent of N2A cell survival rate was assessed by thiazolyl blue tetrazolium bromide (MTT) assay. The heat shock protein A5 (HSPA5 ) levels and the expression levels of miR-181b and HSPA5 mRNA were determined by using Western blotting and Real-time PCR, respectively. Luciferase reporter assay was performed to identify the direct binding of miR-181b with 3’-UTR of HSPA5 mRNA. Results The miR-181b expression level decreased significantly (P<0.05, n=5 per group) in OGD-treated N2A cells. Under the condition of OGD but not in non-OGD condition, alteration of miR-181b expression level by transfection with pre-miR-181b or anti-miR-181b significantly affected N2A cell survival rate(-n=6). Accordingly, the changes of miR-181b levels significantly altered HSPA5 protein levels(n=3), but not the expression levels of HSPA5 mRNA. In addition, the results of luciferase reporter assay indicated that co-transfection of the luciferase reporters with pre-miR-181b or anti-miR-181b resulted in the inhibition or enhancement of the luciferase activities of luciferase expressing plasmid containing 3’-UTR of HSPA5 mRNA(n=5). Conclusions miR-181b plays an important role in N2A cell ischemic injury through negatively regulating HSPA5 protein level, which may provide a potential therapeutic target for ischemic stroke in miRNA levels.

Key words: Oxygen-glucose deprivation, Ischemic injury, Micro RNA-181b, Heat shock protein A5, Western blotting, Real-time PCR, Dual luciferase reporter assays, N2A cell
 

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