RESUMO
Ischemic stroke is the major form of stroke and is accentuated by multiple comorbidities. It has been previously shown that different microRNAs (miRNAs) regulate separate aspects of ischemic stroke. Differential miRNA expression analysis in cerebrospinal fluid of stroke patients had revealed upregulation of miR-124-3p, miR-9-3p, miR-9-5p, and miR-128-3p. However, whether the overexpression is correlative or causative was not known. Here, using an in vitro oxygen-glucose deprivation/reoxygenation (OGD/R) neuronal cell model, we saw OGD/R-induced injury was associated with significant upregulation of the aforementioned four miRNAs. Target gene prediction using in situ algorithms and gene set enrichment analysis revealed significant enrichment of FOXO and Relaxin signaling pathways and regulatory processes associated with endothelial cell migration, which are all known to associate with apoptotic pathways. In situ protein-protein interaction network analysis confirmed the findings of gene set enrichment analysis. TUNEL analysis showed that OGD/R-induced injury resulted in significant apoptosis, which was significantly inhibited in neuronal cells pretransfected with inhibitors of either miR-9-5p or miR-128-3p. Further testing in an in vivo middle cerebral artery occlusion (MCAO) mouse model of ischemic stroke showed that inhibiting miR-9-5p or miR-128-3p significantly decreases MCAO-induced infraction volume and inhibited apoptotic response as revealed by decreased cleaved Caspase-3 protein expression in immunohistochemical analysis. Combined inhibition of miR-9-5p and miR-128-3p resulted in a synergistic decrease in cell death and infraction volume in vitro and in vivo, respectively. Cumulatively, our results provide critical knowledge about the mechanism by which elevated miR-9-5p and miR-128-3p causes brain damage in ischemic stroke and provides evidence of them being attractive therapeutic targets.
