title: Downregulation of miR-181b in mouse brain following ischemic stroke induces neuroprotection against ischemic injury through targeting heat shock protein A5 and ubiquitin carboxyl-terminal hydrolase isozyme L1.
abstract: Understanding the molecular mechanism of cerebral hypoxic preconditioning (HPC)-induced endogenous neuroprotection may provide potential therapeutic targets for ischemic stroke. By using bioinformatics analysis, we found that miR-181b, one of 19 differentially expressed miRNAs, may target aconitate hydratase (ACO2), heat shock protein A5 (HSPA5), and ubiquitin carboxyl-terminal hydrolase isozyme L1 (UCHL1) among 26 changed protein kinase C isoform-specific interacting proteins in HPC mouse brain. In this study, the role of miR-181b in oxygen-glucose deprivation (OGD)-induced N2A cell ischemic injury in vitro and mouse middle cerebral artery occlusion (MCAO)-induced cerebral ischemic injury in vivo, and its regulation of ACO2, HSPA5, and UCHL1 were further determined. We found that miR-181b expression levels significantly decreased in mouse brain following MCAO and in OGD-treated N2A cells. Up- and downregulation of miR-181b by transfection of pre- or anti-miR-181b could negatively regulate HSPA5 and UCHL1 (but not ACO2) protein levels as well as N2A cell death and programmed cell death in OGD-treated N2A cells. By using a T7 promoter-driven control dual luciferase assay, we confirmed that miR-181b could bind to the 3'-untranslated rergions of HSPA5 and UCHL1 mRNAs and repress their translations. miR-181b antagomir reduced caspase-3 cleavage and neural cell loss in cerebral ischemic cortex and improved neurological deficit of mice after MCAO. In addition, HSPA5 and UCHL1 short interfering RNAs (siRNAs) blocked anti-miR-181b-mediated neuroprotection against OGD-induced N2A cell injury in vitro. These results suggest that the downregulated miR-181b induces neuroprotection against ischemic injury through negatively regulating HSPA5 and UCHL1 protein levels, providing a potential therapeutic target for ischemic stroke.
authors.affiliation: Department of Neurobiology and Beijing Institute for Brain Disorders, Capital Medical University, Beijing, People's Republic of China; Department of Physiology, School of Medicine, Shanxi Datong University, Datong, People's Republic of China.
authors.forename: Zhifeng
authors.initials: Z
authors.lastname: Peng
authors.forename: Jiefei
authors.initials: J
authors.lastname: Li
authors.forename: Yun
authors.initials: Y
authors.lastname: Li
authors.forename: Xuan
authors.initials: X
authors.lastname: Yang
authors.forename: Sujuan
authors.initials: S
authors.lastname: Feng
authors.forename: Song
authors.initials: S
authors.lastname: Han
authors.forename: Junfa
authors.initials: J
authors.lastname: Li
mesh_terms: D000818:Animals; D015153:Blotting, Western; D001921:Brain / Q000378:metabolism* / Q000473:pathology; D002545:Brain Ischemia / Q000235:genetics* / Q000378:metabolism; D004195:Disease Models, Animal; D015536:Down-Regulation; D000091342:Endoplasmic Reticulum Chaperone BiP; D006360:Heat-Shock Proteins / Q000235:genetics / Q000378:metabolism*; D020287:In Situ Nick-End Labeling; D019194:Ischemic Preconditioning; D008297:Male; D051379:Mice; D008807:Mice, Inbred BALB C; D035683:MicroRNAs / Q000235:genetics* / Q000378:metabolism; D060888:Real-Time Polymerase Chain Reaction; D020133:Reverse Transcriptase Polymerase Chain Reaction; D020521:Stroke / Q000235:genetics / Q000473:pathology; D043222:Ubiquitin Thiolesterase / Q000235:genetics / Q000378:metabolism*
chemical_list: D000091342:Endoplasmic Reticulum Chaperone BiP; D006360:Heat-Shock Proteins; C000717248:Hspa5 protein, mouse; D035683:MicroRNAs; C529100:mirn181 microRNA, mouse; D043222:Ubiquitin Thiolesterase; C496976:Uchl1 protein, mouse
keywords: hypoxia/ischemia; hypoxic preconditioning; miRNA; protein kinase C; stroke