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