Moyamoya Disease Susceptibility Gene RNF213 Specifically Binds To And Regulates HIF-1α And Inhibits HCMEC/D3 Cell Proliferation,Migration And Angiogenesis Ability

Objective:Moyamoya disease（MMD）is a cerebrovascular disease characterized by progressive stenosis of the internal carotid artery or Willis ring,resulting in the formation of a compensatory"smoke like"vascular network in the basilar artery.The main clinical symptom of MMD is ischemia,which is characterized by poor prognosis,high mortality and disability rate.The etiology and pathological mechanism of MMD are not completely clear,which may be related to genetic influence,inflammation,environment and other factors.Ring finger protein 213（RNF213）,a susceptible gene of moyamoya disease,has a unique ubiquitination domain and can regulate the angiogenesis and neuromuscular movement of zebrafish.It participates in the regulation of signaling pathways such as NF-κB、Non classical Wnt/Ca²⁺、α-KGDD and MAPK/JNK,which are of great significance for maintaining the homeostasis of angiogenesis.Hypoxia is a pathological environment for cell survival,often caused by tissue ischemia.During tissue hypoxia,hypoxia inducible factor-1-α（HIF-1α）expression is the core link.However,in the current research,the regulatory mechanism of RNF213 on angiogenesis under ischemic conditions is still unclear,and the animal model of MMD has only been successfully constructed in zebrafish.Therefore,it is necessary to find a new regulatory target protein of RNF213 under hypoxic-ischemic conditions,which is of great significance to explain the pathogenesis of moyamoya disease.Methods:The expression and relationship of RNF213 and HIF-1αwere performed on the sera of patients with moyamoya disease who were treated in the Department of Neurosurgery,General Hospital of the People’s Liberation Army of China from 2004to 2018 by Elisa test kit.By using si RNA-RNF213 to inhibit the expression of RNF213gene in cells,Co Cl₂ was used to treat cells to create a chemical hypoxia model.HCMEC/D3 cells were treated and divided into four groups:negative control group（Scramble）,hypoxic negative control group（Scramble HO）,normoxic RNF213silencing group（si-RNF213）,hypoxic RNF213 silencing group（si-RNF213 HO）.The m RNA expression level of RNF213 and HIF-1αin cells were detected by q RT-PCR.The distribution and expression levels of HIF-1αprotein in cells and the expression levels of RNF213 were detected by immunofluorescence and Western Blot.The ubiquitination substrate of RNF213 was detected by mass spectrometry analysis of Co-IP products.The effects of RNF213 and hypoxia on the proliferation and migration of h CMEC/D3 cells were detected by CCK-8 method and scratch test,we detected the ability of cells to form tubes using Matrigel matrix glue.Results:1.There was significant difference between the expression of RNF213 and HIF-1αin the serum of patients with moyamoya disease and the control group.The content of RNF213 and HIF-1αin patients with RNF213 p.R4810K（G>A）mutations is significantly lower than that in patients with non-mutated moyamoya disease;2.Compared with Scramble group,after silencing RNF213,the m RNA and protein levels of RNF213 decreased significantly（P<0.05）;After hypoxic intervention on h CMEC/D3 cells,compared with normoxia group,the m RNA and protein levels of HIF-1αwere significantly up-regulated（P<0.05）;Compared with Scramble group,after silencing RNF213,The m RNA and protein levels of HIF-1αin normoxia silencing group and hypoxia group were significantly up-regulated（P<0.05）;3、HIF-1αis mainly expressed in the nucleus;After hypoxic intervention on cells,HIF-1αThe fluorescence intensity was significantly increased（P<0.05）;After silencing RNF213,The fluorescence intensity of HIF-1αwas significantly increased（P<0.05）;4.There is a mutual binding interaction between RNF213 and HIF-1α,and this interaction has specific binding;5.Compared with the Scramble group of h CMEC/D3 cells,the cell viability of the si-RNF213 group significantly increased after 48 hours of silence（P<0.05）;After 24 hours of hypoxia intervention by Co Cl₂,the cell viability of the hypoxia group and the control group did not change significantly.Compared with the normoxia group,after 12 hours of hypoxia intervention by Co Cl₂,there was no significant change in the OD value of the hypoxia group and the control group.After 48 hours of hypoxia intervention,the cell viability of the hypoxia group significantly decreased（P<0.05）;6.After RNF213 silencing intervention on h CMEC/D3 cells,the cell migration rate of si-RNF213 group was significantly higher than that of Scramble group（P<0.05）;After hypoxic intervention,the cell migration rate in the hypoxic group was significantly lower than that in the non-hypoxic group（P<0.05）.Compared with the Scramble group,after simultaneous RNF213 silencing and hypoxic intervention,the cell migration rate changed significantly（P<0.05）.7.After RNF213 silencing intervention,the number and length of endothelial cells in the si-RNF213 group were significantly higher than those in the Scramble group（P<0.05）.Conclusion:Compared with normal people,the content of RNF213 and HIF-1αin serum of patients with moyamoya disease has significant difference,and the content of RNF213 and HIF-1αin patients with RNF213 p.R4810K（G>A）mutations is significantly lower than that in patients with non-mutated moyamoya disease;RNF213and HIF-1αexists specific interaction and RNF213 can reduce the expression of HIF-1α;RNF213 restrained the proliferation and angiogenesis of h CMEC/D3 cells.