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

doi:10.3969/j.issn.0529-1356.2013.05.006

Abstract

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

CLC Number:

R 338

HAN Song PENG Zhi-feng LI Jun-fa*. Role of microRNA-181b in oxygen-glucose deprivation -induced N2A cell ischemic injury and its regulation on HSPA5 protein levels[J]. AAS, 2013, 44(5 ): 616-620.

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