MIR-140-5p regulates angiogenesis following ischemic stroke by targeting VEGFA

Abstract

MicroRNA (miRNA or miR) expression profiles are altered in tissues under hypoxic-ischemic conditions. The expression of MIR-140 is downregulated >2-fold following hypoxic-ischemic brain damage, however, its role in angiogenesis subsequent to cerebral ischemia is not fully understood. The present study aimed to investigate the role of MIR-140-5p in angiogenesis and the molecular mechanism mediated by vascular endothelial growth factor A (VEGFA) in an in vitro model for brain ischemia. A rat middle cerebral artery occlusion (MCAO) model was constructed, and the results from reverse transcription-quantitative polymerase chain reaction and western blot analysis demonstrated that the expression levels of MIR-140-5p were significantly decreased, while the expression levels of VEGFA were significantly increased between 12 and 48 h in the rat cerebral following MCAO. Furthermore, human umbilical vein endothelial cells (HUVECs) were exposed to low oxygen conditions and it was demonstrated that hypoxia downregulated MIR-140-5p and upregulated VEGFA expression levels. The MIR-140-5p mimic was transfected into the normoxic and hypoxic HUVECs and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, Transwell migration and tube formation assays were performed. The results indicated that MIR-140-5p inhibited angiogenesis by decreasing cell proliferation, migration and tube formation. Additionally, in human embryonic kidney 293 cells, results from the luciferase reporter assay revealed that MIR-140-5p directly targeted the 3' untranslated region of VEGFA and that MIR-140-5p regulated the protein expression of VEGFA. To further analyze this effect, a VEGFA-pEGFP-C1 plasmid was transfected into the normoxic and hypoxic HUVECs, and it was revealed that the inhibitory effect of MIR-140-5p on angiogenesis was attenuated by the overexpression of VEGFA. In conclusion, to the best of our knowledge, the present study is the first to suggest that MIR-140-5p exerts an inhibitory effect on angiogenesis in an in vitro model of ischemia, and this effect is achieved partially by targeting VEGFA. The present study provided a novel biomarker for the treatment of cerebral ischemia.