The Role Of MiR-22 In Curcumin Inhibition Of Vascular Smooth Muscle Proliferation,Migration And Intimal Neogenesis

BackgroundCoronary atherosclerotic heart disease(CAD)is caused by myocardial ischemia,hypoxia or necrosis.The cause of the disease is atherosclerosis of the coronary artery,which leads to stenosis or occlusion of the vascular lumen.Percutaneous coronary stent implantation(PCI)has become a widely accepted treatment for patients with coronary heart disease in China,but the problem of in-stent restenosis(ISR)after stent implantation has not been completely solved.At present,the current general view of ISR is related to neointimal hyperplasia,poor stent adhesion and endothelial repair after injury.The specific molecular mechanism of post-stent restenosis is not clear,and effective preventive measures need to be further studied.Curcumin is a bioactive polyphenolic compound isolated from turmeric plants.Current research has found that it has a wide range of pharmacological activities,such as anti-inflammatory,antioxidant,lipid-lowering,anti-atherosclerosis and anti-tumor.MiRNAs are a group of naturally occurring non-coding small molecule RNAs.Studies have suggested that miRNAs are important regulators of vascular smooth muscle cell(VSMC)proliferation,migration,apoptosis,and differentiation.Although some studies have found that miR-22 is abundantly expressed in the heart,and regulates multiple signaling pathways in cardiomyocytes,the expression of miR-22 in VSMCs is unclear.AimsIn this study,we aimed to verify the role of curcumin in regulating the expression of miR-22 in VSMCs and its effect on the function of VSMCs in vivo and in vitro.We also expounded the possible molecular mechanism of curcumin inhibiting VSMC proliferation,migration and intimal formation,so as to provide a new idea for the prevention and treatment of coronary artery stenosis or post-stent restenosis.Method1.The expression of miR-22 was detected after pretreatment of curcumin on VSMCs,and the expression of miR-22 was detected after stimulation of serum on VSMCs.2.The effect of miR-22 on the proliferation and migration of vascular smooth muscle cells was evaluated by gain/loss-of-function analyses.3.The functional target genes of miR-22 were assessed by bioinformatics analysis and molecular cloning,and were verified by Luciferase assay.4.The expression of miR-22 and target genes was assessed after pretreatment of curcumin on VMSCs,and verified the effect of curcumin on the proliferation and migration of VMSCs.5.The animal model of intimal hyperplasia after arterial balloon injury in rats was established.After the intervention of curcumin,the expression of miR-22 and the degree of intimal hyperplasia after vascular injury were detected.The effect of miR-22 on curcumin inhibiting intimal hyperplasia was verified.Result1.When curcumin at the concentration of 10 ?M interfered with VSMCs,the expression of miR-22 increased most significantly,and the expression of miR-22 decreased significantly when starved VSMCs were stimulated by serum for 24 hours2.MTS and Edu cell proliferation assay showed that overexpression of miR-22 inhibited the proliferation of VSMCs.Scratch test and transwell migration test demonstrated that overexpression of miR-22 inhibited the migration of VSMCs,while inhibition of miR-22 expression promoted the proliferation and migration of VSMCs.3.The bioinformatics website database predicted that SP1 is the target gene of miR-22.Overexpression of miR-22 in VSMCs down-regulated the expression of SP1 at both mRNA and protein levels,while inhibiting the expression of miR-22 up-regulated the expression of SP1 at mRNA and protein levels.Molecular cloning and double gene luciferase report experiments showed that the overexpression of miR-22 significantly decreased the luciferase activity,and the luciferase activity remained unchanged after the mutation of the 3'-UTR binding site of SP1.4.Curcumin inhibited the proliferation and migration of VSMCs and increased the expression of miR-22.Pretreatment with miR-22 inhibitor attenuated the effect of curcumin on the proliferation and migration of VSMCs,and increased the expression of SP1 at mRNA and protein levels.5.In the rat model of intimal hyperplasia after balloon injury,the expression of miR-22 decreased significantly during intimal hyperplasia after vascular injury.In curcumin-treated vascular injury,the expression of miR-22 increased and SP1 decreased,and the degree of intimal hyperplasia was significantly reducedConclusionOur study shows that curcumin promotes the expression of miR-22 in VSMCs,and miR-22 inhibits the proliferation and migration of VSMCs.MiR-22 can directly target at SP1 and down-regulate the expression of SP1.MiR-22/SP1 mediates curcumin in inhibiting the proliferation and migration of VSMCs.Curcumin partly inhibits intimal hyperplasia after arterial injury through miR-22/SP1.Curcumin may provide a new idea for the prevention and treatment of coronary atherosclerosis and post-stent stenosis in the future.