The Regulation Role Of MicroRNA-376b-5p On Angiogenesis After Cerebral Ischemia And Mechanism

Background and Aims:Cerebral infarction is a kind of acute ischemic cerebrovascular disease with high morbidity,high disability rate and high recurrence rate, accounting for60%-80%of the total number of stroke.Isehemia irreversibly resulted in substantial neuronal degeneration and necrosis and caused severe neurological deficits.Promoting angiogenesis in ischemic brain to increase the number of new collateral circulation,will increase the blood supply and improve the ischemic brain function. The studies have found that HIF-1-VEGF-NOTCH signaling pathway was the main regulator of angiogenesis after Ischemia and hypoxia. microRNA is a kind of endogenous small single-stranded RNA molecules that are involved in the process of organism’s growth, organ formation, cell proliferation, differentiation, apoptosis and so on and are closely related to many diseases.Previous studies have found that the expression level of miR-376b-5p was decreased in ischemic brain tissue after cerebral ischemia and there was a matching relationship between the sequence of miR-376b-5p and HIF-la through biological software analysis.So we speculate that miR-376b-5p may be involved in the process of regulating angiogenesis and miR-376b-5p may regulate angiogenesis by HIF-1-VEGF-Notch signaling pathwayThis study was designed to observe the changes of miR-376b-5p, angiogenesis and HIF-la,VEGFA,Notchl in HIF-1-VEGF-Notch signaling pathway after cerebral ischemia both in vivo and in vitro and observe the effect of miR-376b-5p on angiogenesis through overexpressing and inhibiting miR-376b-5p,and explore the regulatory effect of miR-376b-5p on angiogenesis and elucidate the underlying mechanisms in vitro to provide a new strategy for the treatment of cerebral infarction. Methods:1. In vivo studies1.1we chose SD rats to establish permanent middle cerebral artery occlusion (pMCAO) model,and collect the infarct samples in the brain After duration of ischemia for1day,3days and7days.1.2Neural deficit score and TTC staining were used to identify whether pMCAO model was successfully established.1.3In order to determine angiogenesis in the infarct, immunohistochemical staining of vWF was used.1.4Expression of miR-376b-5p in the ischemia cerebral cortex was analyzed by real-time PCR. Also the expression of HIF-la mRNA, VEGFA mRNA,Notchl mRNA level were determined by real-time PCR.1.5The expression of HIF-la,VEGFA,Notchl protein level were determined by Western blot methods.2. In vitro studies2.1Establishing the human umbilical vein endothelial cells(HUVEC) hypoxia model.2.2MTT,Transwell and Matrigel experiment were used to compare cell proliferation,cell migration and tube formation between hypoxia group and normoxia group,which can determine the effect of hypoxia on angiogenesis. With the method of real-time PCR and Western blot, the expression of HIF-la,VEGFA,Notchl both in mRNA and protein level were compared between the two groups.The effect of hypoxia on the expression of miR-376b-5p was detected by real-time PCR.2.3We further transfected miR-376b-5p mimic or inhibitor into hypoxia HUVEC, angiogenesis and expression of HIF-la, VEGFA, Notch1both in mRNA and protein level were observed to determine the regulatory effect of miR-376b-5p on angiogenesis.2.4In order to further explore the regulatory mechanism of miR-376b-5p on angiogenesis, we constructed HIF-la shRNA which was transfected into hypoxia HUVEC to knockout HIF-la.By comparing the effect of miR-376b-5p mimic/inhibitor on angiogenesis and HIF-la, VEGFA, Notch1expression before and after HIF-la shRNA transfection,we could judge whether miR-376b-5p influence angiogenesis through HIF-1-VEGF-Notch signaling pathway.Results:1. In vivo studies1.1We have successfully established rats permanent middle cerebral artery occlusion (pMCAO) model.TTC staining showed that the infarction zone area was dyed white, normal brain tissue was dyed red.1.21day,3days and7days after pMCAO,the microvessel density was increased in the infarct brain tissue compared with the sham group.1.31day,3days and7days after pMCAO,the expression level of miR-376b-5p in the ischemia cerebral cortex were decreased compared with the sham group, meanwhile the expression of HIF-1α,VEGFA,Notchl both in mRNA and protein level were increased.1.41day,3days and7days after pMCAO,the expression level of HIF-1α,VEGFA,Notchl protein level were increased in the ischemia cerebral cortex compared with the sham group.2. In vitro studies2.1Compared with normoxia group,the expression level of miR-376b-5p in hypoxia group was decreased,while cell proliferation.cell migration and tube formation abilities were enhanced which suggested that angiogenesis in hypoxia were enhanced, the expression of HIF-la,VEGFA,Notchl both in mRNA and protein level were also increased. It implies that miR-376b-5p may inhibit angiogenesis in endothelial cells.2.2After transfection of miR-376b-5p mimic into hypoxia HUVEC,angiogenesis was weakened,and the expression of HIF-la,VEGFA,Notchl both in mRNA and protein level were decreased.On the contrary, after transfection of miR-376b-5p inhibitor into hypoxia HUVEC,angiogenesis was enhanced,and the expression of HIF-1α,VEGFA,Notchl both in mRNA and protein level were increased. It is identified preliminary that miR-376b-5p may regulate angiogenesis through the HIF-1-VEGF-Notch signaling pathway.2.3After transfection of HIF-la shRNA into hypoxia HUVEC,the angiogenesis indexes were different between miR-376b-5p mimic/inhibitor group and miR-376b-5p mimic/inhibitor+HIF-la shRNA co-transfected group,while the expression of HIF-la,VEGFA,Notchl both in mRNA and protein level were also changed.The part of the experiment further clarified miR-376b-5p played a negative role in regulating angiogenesis through HIF-1-VEGF-Notch signaling pathway.Conclusions:1.miR-376b-5p plays a role in inhibiting angiogenesis after cerebral ischemia.2.miR-376b-5p may be involved in the process of inhibiting angiogenesis through HIF-1-VEGF-Notch signaling pathway.3.Intervening in the expression of miR-376b-5p promotes angiogenesis,which provides a new thought for the treatment of cerebral infarction.