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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*
解剖学报 ›› 2013, Vol. 44 ›› Issue (5) : 616-620.
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
Role of microRNA-181b in oxygen-glucose deprivation -induced N2A cell ischemic injury and its regulation on HSPA5 protein levels
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。
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 injuryin 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.
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
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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