The Effect And Molecule Mechanism Of MiR-3568 On Migration And Proliferation In Vascular Smooth Muscle Cell

Objective: Atherosclerosis(Atherosclerosis,AS)is a kind of chronic inflammation disease.A long-term accumulation and a variety of factors cause the vascular damage to form cardiovascular disease.So far,the mechanism of this disease is still not completely clear.Based on the study of the pathogenesis of atherosclerosis,it is indicated that the main body of the vascular narrowed by the accumulation of lipid in the process of pathogenesis.There are many factors might cause the damage of vascular.Such as inflammatory cells and growth factors,lipid cycle and accumulation,serum and mechanical stimulation which are the influence factors of AS.And these factors lead to a series of pathological phenomena,including endothelial dysfunction,vascular endothelial cells produce chemokines,induce monocyte infiltration,and initiate proinflammatory cytokines in the intima.Then,the activation of macrophages release inflammatory mediators can produce intimal damage,and vascular smooth muscle cell migration and proliferation in the intima layer.Therefore,the maintenance of vascular smooth muscle cells(Vascular Smooth Muscle Cell,VSMC)phenotype is a research direction to block the occurrence of atherosclerosis.The research results about the mechanism of AS showed that damaged of endothelial cell can resulte the platelet adhesion to endothelial injury with plateletderived growth factor(Platelet-Derived Growth Factor,PDGF)released from the VSMC.Migration and proliferation of VSMC and a large number of deposited lipids together lead to the occurrence of atherosclerotic diseases.Micro RNA(miRNA)is a single stranded non coding RNA molecule that is expressed in both plant and animal genomes.The awareness is constantly evolving of the biological function of miRNA,which is mainly used as a negative regulator of post transcriptional gene expression.The mechanism of miRNA is selective,which can directly lead to the degradation of m RNA,or to reduce the stability of m RNA.It is play an important role on vascular function.Recently researches about microRNA in cardiovascular diseases,especially atherosclerosis,which are more and more closely related to miRNA.miR-3568 is a new kind of single stranded small molecule RNA,which has not been reported in the development of cardiovascular diseases,particularly atherosclerosis.In this study,we investigated the role of miRNA-3568 and atherosclerosis in smooth muscle cells.Methods 1.Using transient transfection technology to inhibit or over expression of miR-3568 in Vascular Smooth Muscle Cell(A7r5).2.The expression level of miR-3568 m RNA was measured by quantitative Real-time Polymerase Chain Reaction in A7r5 cells.3.Western blot assay was used to detect the expression of Akt,Fox O1 and ERK in A7r5 cells.4.The survival rate of A7r5 cells treated with miR-3568 inhibitor was measured with MTT.5.The migration of A7r5 cells treated with miR-3568 inhibitor was assessed by Boyden's Chamber assay.6.The target genes of miR-3568 was detected by double luciferase assay.Results 1.The expression of miR-3568 in A7r5 cells was significantly inhibited by transfection with Anti-miR-3568 group.Transfection of Pre-miR-3568 could increase the expression of miR-3568 in A7r5 cells.2.The expression of miR-3568 in A7r5 cells was significantly decreased by PDGFBB stimulation.3.The results of Western Blot showed that Anti-miR-3568 could increase the expression of p-Akt,p-ERK and p-Fox O1 protein in A7r5 cells under the stimulation of PDGF-BB,but decreased the expression of Fox O1.4.MTT experiments showed that Anti-miR-3568 could promote the proliferation of A7r5 cells.After BEZ-235,the proliferation was inhibited.However,Pre-miR-3568 had no significant effect on the proliferation of A7r5 cells.5.Boyden 's Chamber experiment showed that Anti-miR-3568 could promote the migration of A7r5 cells.The migration of A7r5 cells was inhibited after BEZ-235 treatment.However,Pre-miR-3568 had no significant effect on the migration of A7r5 cells.6.The results of dual luciferase reporter assay showed that miR-3568 and Fox O1 had no targeting relationship.Conclusion 1.Down-regulation of miR-3568 promotes the proliferation and migration of rat thoracic aortic smooth muscle cells.The effect of miR-3568 is not obvious.2.The effect of Anti-miR-3568 on proliferation and migration of smooth muscle cells may be mediated by Akt/Fox O1 signaling pathway.3.Down-regulation of miR-3568 can promote the expression of p-ERK protein.