MiR-93 Regulates Vascular Smooth Muscle Cell Proliferation,Migration And Neointimal Formation Through Targeting Mfn2

BackgroundCardiovascular disease is one of the most important diseases that threaten human life.Many studies have confirmed that abnormal proliferation of vascular smooth muscle plays a key role in cardiovascular disease,such as atherosclerosis and atherosclerotic restenosis.In response to injury and other stimuli,vascular smooth muscle cells(VSMCs)decreased expression of contractile proteins and increased expression of proliferating proteins,then leading to increased cell proliferation,which is the main pathogenesis of vascular restenosis after angioplasty.However,the molecular mechanism of vascular restenosis after angioplasty is not fully understood.Thus,a better understanding of these regulatory mechanisms of vascular restenosis might lead to novel strategies for suppressing cardiovascular disease.MicroRNAs(miRNAs)are a class of small non-coding RNAs that binding to the 3’untranslated region(3’UTR)of specific mRNA,resulting in degradation of the target gene mRNA.More than 2,000 miRNAs have been currently found in the human genome.MiRNAs play roles in many diseases including cancer,endocrine,nervous system,and cardiovascular.Recently,multiple lines of evidence suggest that mi RNAs play pivotal roles in the control of VSMC proliferation and migration such as miR-132,miR-22,miR-379,miR-124,etc.can inhibit the proliferation and migration of VSMCs,while miR-214,mi R-221/miR-222,miR-146 and so on can promote the proliferation and migration of VSMCs.MiR-93 was first discovered which belongs to the miR-17 microRNA cluster and is closely related to the early evolution of the vertebrate lineage.Previous studies have demonstrated that miR-93 is involved in various disease,including cancer,myocardial ischemia-reperfusion,and spinal cord neurons.However,whether miR-93 is involved in VSMC proliferation was not fully understood.Mitofusin-2(Mfn2),a GTPase present in the mitochondria,plays an important role in mitochondrial fusion and function regulation.It has been reported that Mofn2 can regulate proliferation and apoptosis in the cell.Recently,Of the study confirmed Mfn-2 can inhibit the proliferation of VSMCs,therefore,this study is intended to establish rat model of vascular stenosis after carotid artery balloon injury and explore whether miR-93 through the inhibition of Mfn2 expression and regulation of VSMCs proliferation and migration.ObjectiveTo investigate the role and mechanism of miR-93 in VSMCs by establishing animal models stimulated by platelet-derived growth factor-BB(PDGF-BB),the formation of postoperative restenosis provides a new target and theoretical basis.Methods1 Real-time reverse transcription-polymerase chain reaction(qRT-PCR)was used to detect the expression of miR-93 in vascular tissue of carotid injury rats.The expression of mi R-93 in PDGF-BB stimulated VSMCs was detected by qRT-PCR and immunofluorescence.2 The expression of mi R-93 in animals and cells was knocked down.The degree of carotid artery stenosis was detected by carotid pathological sections.The cell proliferation was detected by MTT assay and cell proliferative imaging(EDU)And double-chamber co-culture system(Transwell test)to detect the degree of cell migration.3 qRT-PCR was used to detect the mRNA expression of Cyclin D1,MMP 2 and Mfn-2 in the carotid artery and VSMCs of rats.Western blot was used to detect the protein expression of Cyclin D1,MMP and Mfn2 in rat carotid artery and VSMCs,and the phosphorylation of Raf and ERK1 / 2.Tissue and cell immunofluorescence were used to detect the expression of Mfn2 in tissues and cells.4 Dual luciferase reporter was used to identify the binding of miR-93 to the 3’UTR of Mfn-2.ResultsIn animal experiments,the expression of MiR-93 in rat vascular smooth muscle cells was increased after carotid artery injury.In cell experiments,similar results were observed in VSMCs cultured in vitro after treatment with PDGF-BB.Inhibition of miR-93 suppresses neointima formation after carotid artery injury.In addition,our results show that miR-93 inhibitors can inhibit PDGF-BB-induced proliferation and migration of VSMCs,while reducing the expression of MMP2,Cyclin D1.From the mechanism analysis,we found that Mfn2 is a direct target gene of miR-93.In addition,detecting the phosphorylation of Raf and ERK1 / 2 revealed that the proliferation and migration of miR-93-mediated VSMCs were attributed to the Raf-ERK1 / 2 pathway.ConclusionIn the present study,we identified miR-93 as a novel regulator of neointimal hyperplasia induced by VSMC proliferation,differentiation and arterial injury.We report that miR-93 promotes VSMCs proliferation and migration by targeting Mitofusin-2(Mfn2),which may be a new therapeutic target for restenosis after angioplasty.