MicroRNA-99a Inhibits Insulin-induced Proliferation,Migration,Dedifferentiation,and Rapamycin Resistance Of Vascular Smooth Muscle Cells By Inhibiting Insulin-like Growth Factor-1 Receptor And Mammalian Target Of Rapamycin

Backgrounds:It has been recognized during the past few decades that diabetes mellitus(DM)has become a serious challenge to health care professionals across all nations around the world.Patients with DM,particularly those with type 2 DM(T2DM),are at higher risk for the development of neurological and cardiovascular complications,ultimately leading to morbidity and mortality for these patients.Indeed,many previous studies have indicated that patients with T2DM are vulnerable to cardiovascular diseases(CVDs),particularly coronary artery disease,as compared with those without T2DM.Atherosclerosis has been considered to be the primary pathophysiological feature of coronary artery disease,and phenotypic changes of vascular smooth muscle cells(VSMCs),including abnormal proliferation,migration,and dedifferentiation,have been suggested to play key roles in the pathogenesis and progression of T2DM-related CVDs.Furthermore,insulin resistance and subsequent upregulated serum insulin,which are common in T2DM patients,have been related to the development of insulin-induced VSMC phenotypic changes.Previous experimental studies have indicated that many signaling pathways are involved in the process of insulin-induced changes of the VSMC phenotype,such as the activation of insulin-like growth factor-1 receptor(IGF-1R)and mammalian target of rapamycin(mTOR)signaling molecules,as well as the subsequent activation of extracellular signal-regulated kinase(ERK),protein kinase B(AKT),and P70s6K signaling.However,the exact molecular pathways underlying the pathogenesis of hyperinsulinemia-induced VSMC phenotypic changes remain to be determined.A recent development in molecular biology uncovered microRNAs(miRNAs),a group of small non-coding RNAs that participate in almost all biological functions.The regulatory role of miRNAs has been found to be involved in many pathophysiological processes,including the development of metabolic diseases.Recently,some miRNAs have been demonstrated to be involved in the regulation of hyperinsulinemia-induced VSMC phenotypic changes.MiR-99a was identified as a tumor suppressor by inhibiting IGF-1R and mTOR,two important signaling molecules in high-dose insulin-induced VSMC phenotypic changes.However,to the best of our knowledge,whether miR-99a plays a regulatory role in the pathogenesis of high dose of insulin-induced VSMC phenotypic changes in T2DM has not been systematically evaluated.Objective:1.The role of miR-99a in insulin-induced abnormal proliferation,migration,and dedifferentiation of VSMCs in an in vitro model of VSMCs treatedwith insulin-2.Find the mechanism of miR-99a in regulating insulin-induced abnormal function of VSMCs.3,To find the role and mechanism of miR-99a in insulin-induced rapamycin resistance of VSMCs.Methods:1.Culture of mouse aortic VSMCs.2.miR-99a mimics and negative control(NC)were transfected to VSMC by lipofectamin2000.3.For the CCK-8 assay,VSMCs were treated with different doses of insulin.4.q-RT-PCR were used to detect the effect of different dose of insulin on miR-99a expression.5BrdU incorporation assay were used to find the role of miR-99a in insulin-induced VSMC proliferation;6.Cell cycle analysis were used to find the role of miR-99a in insulin-induced VSMC cycle progression.7.transwell assay and wound healing assay were used to evaluate the effect of miR-99a on insulin-induced VSMC migration.8.WB were used to evaluate the effect of miR-99a on insulin-induced VSMC phenotype conversion 9.Bioinformatic tools for miRNA target prediction.10.Dual luciferase assay were used to comfirm the Mtor and IGF-1R were dually blocked by miR-99a.11.q-RT-PCR and WB were used to evaluate the effect of miR-99a on IGF-1R and mTOR mRNA and protein expression.12.WB were used to evaluate the effect of miR-99a on insulin induced activation of AKT?ERK?P70S6K signaling pathway.13.CCK-8 assay were used to evaluated the effect of insulin on the inhibiton role of rapamycin on VSMC.14.BRDU assay,transwell assay and wound healing assay were used to evaluated the effect of miR-99a on instulin-induced VSCM rapamycin resistance.15.Q-RT-PCR and WB were used to find the mechanism of miR-99a ameliorate insulin-induced VSMC rapamycin resistance.Results:1.Insulin induced the proliferation of VSMCs and inhibited the expression of miR-99a.2.Exogenous miR-99a overexpression suppressed the insulin-induced proliferation,migration and phenotype conversion of VSMCs3.miR-99a dually blocked high-dose insulin-induced IGF-1R and mTOR activation.4.miR-99a ameliorated rapamycin resistance in VSMCs treated with insulin by compensating for the loss of P27kip1 regulationConclusions:1.MiR-99a plays a key regulatory role in the pathogenesis of insulin-induced abnormal functions of VSMCs via inhibiting IGF-1R and mTOR signaling.2.Exogenous miR-99a overexpression suppressed the insulin-induced proliferation,migration and phenotype conversion of VSMCs3.MiR-99a may restore the loss of regulation of P27kip1 by rapamycin seen in hyperinsulinemia-induced neovascularization.MiR-99a may be a preventative strategy for hyperinsulinemia-induced atherosclerosis and in-stent restenosis.