MicroRNA-493 Regulates Angiogenesis In A Rat Model Of Ischemic Stroke By Targeting MIF

Objective:To investigated the role and mechanism of miR-493 in regulating post-stroke angiogenesis.Methods:SD rats middle cerebral artery occlusion (MCAO) model were established, then a real-time quantitative PCR to detect the expression of miR-493 in the ischemic boundary zone at 3rd,7th day respectively. Intracerebroventricular injection agomir-493, agomir control, PBS, then a FITC perfusion used to detect the blood vessel in the IBZ. Primary cultured rat brain microvascular endothelial cells were subjected to oxygen-glucose deprivation (OGD) to establish in vitro model of ischemia and 6 hours later to detect miR-493 expression by PCR. Up-regulation or down-regulation of miR-493 by transfection of miR-493 mimic or miR-493 inhibitor, then PCR used to detect the transfection efficiency. We used matrigel experiment, transwell experiment and wound healing assay, CCK8 experiment to detect cells tube formation, migration, proliferation ability after transfection of miR-493 mimic or miR-493 inhibitor. To detect MIF mRNA and protein level expression in IBZ of rats subjected MCAO on 3rd and 7th day, we used PCR and western-blot technique. In vitro, we used PCR, western-blot, ELISA to test MIF mRNA and protein level expression in RBMECs subjected to OGD for 6 hours or transfection by miR-493 mimic or miR-493 inhibitor. Dual-luciferasereporter systemwas used to confirm that MIF is a direct target of miR-493. A lentiviral vectors expressing small interfering RNA (siRNA) against the MIF gene (MIF-siRNA) was designed, and the effects of MIF-siRNA were detected by RBMECs'tube formation, migration and proliferation ability after transfection of miR-493 inhibitor. Exogenous rMIF was administrated in the same condition to confirm that the role of miR-493 in angiogenesis depends on MIF. Western blot was used to detect AKT, p-AKT, ERK and p-ERK expressions in RBMECs transfected with miR-493 mimic/mimic control under OGD for 6h or miR-493 inhibitor/inhibitor control under normoxia. Intracerebroventricular injection antagomir-493 and controls, then TTC assay to test the infarct volume, a FITC perfusion used to detect the blood vessel in the IBZ.Results:1) The expression of miR-493 was down-regulated on 3rd,7th day after MCAO, and the miR-493 expression was also down-regulation in RBMECs under OGD for 6 h.2) After injection of agomir-493, Vascular perfusion in IBZ was reduced compared with agomir control.3) After transfection of miR-493 mimic, the tube formation, migration and proliferation of RBMECs were decreased, while by transfection of miR-493 inhibitor, these cell abilities were increased.4) By using miRwalk, miRanda and RNAhybrid to search the possible targets of miR-493. The predicted target MIF was identified. MIF expression was increased in ischemia condition and was negative regulation by miR-493. Dual-luciferasereporter system confirmed MIF was miR-493 direct target.5) In MIF-siRNA transfection RBMECs, miR-493 inhibitor transfection couldn't increase the tube formation migration and proliferation. But once given rMIF, these cell abilities could be rescued. These results indicated that the effect of miR-493 on angiogenesis relied on MIF.6) AKT, ERK pathway was the downstream of MIF.7) After intracerebroventricular injection of antagomir-493, the infarct volume decreased, neurological function score decreased and vascular perfusion in IBZ increased.Conclusion:Our findings highlight the importance of miR-493 in regulating angiogenesis after MCAO, the miR-493 expression is down-regulated after ischemia both in vivo and in vitro, the pro-angiogenesis function of miR-493 relies on MIF. The downstream of MIF is AKT, ERK path. Our findings indicate miR-493 as a potential therapeutic target in treatment of stroke.