MiRNA-326 Regulates Proliferation Migration And Phenotypic Switch Of Vascular Smooth Muscle Cells

ObjectiveVascular smooth muscle cells(VSMCs),located in the mesangial layer of blood vessels,is the major cellular component of arteries and a key determinant of coronary artery disease(CAD).Micro RNAs(miRNAs)are expressed in the vascular system.miR-326 is downregulated in most tumors and can inhibits tumor proliferation.However,the role of miR-326 in VSMCs is unclear.The aim of this study was to investigate the regulation of miRNA-326 in proliferation,migration and phenotypic switch in vascular smooth muscle cells,which is expected to provide potential targets for the prevention and treatment of vasoproliferative diseases.Methods(1)Expression of miR-326 was detected by q RT-PCR in normal and carotid artery balloon injury model rats.(2)VSMCs were transfected with miR-326-inhibitor,and the knockdown efficiency was detected by q RT-PCR after 24 h.(3)After endogenous knockdown of miR-326 using miR-326-inhibitor in VSMCs,contractile proteins such as α-SMA,MHC and CNN1 were detected by q RT-PCR at the m RNA expression level to observe the change of phenotypic switch in VSMCs at the m RNA level.The expression levels of proliferation proteins PCNA and Ki-67 on m RNA were detected by q RT-PCR.(4)VSMCs were transfected with miR-326-inhibitor and 24 h later by Western blot to observe how miR-326-inhibitor affects contractile proteins such as α-SMA,MHC,and CNN1,and to observe how miR-326-inhibitor regulates the phenotypic switch of VSMCs at the translational level.As well as to observe the expression of proliferative proteins such as Ki-67 and PCNA by Western blot.(5)VSMCs were transfected with miR-326-inhibitor and the effect of miR-326-inhibitor on the migration of VSMCs was analyzed by woundhealing assay after 24 h.(6)VSMCs were transfected with miR-326-inhibitor and the effect of miR-326-inhibitor on VSMCs migration was analyzed by Transwell assay after 24 h.(7)Mi R-326-mimic transfected VSMCs,and the efficiency was detected by q RT-PCR after24 h of transfection.(8)After overexpression of miR-326 in VSMCs using miR-326-mimic,the expression levels of contractile proteins such as α-SMA,MHC and CNN1 on m RNA were detected by q RT-PCR.And miR-326-mimic was observed to regulate the phenotypic switch of VSMCs at the m RNA level changes.The expression levels of proliferation proteins PCNA and Ki-67 on m RNA were also detected by q RT-PCR.(9)VSMCs were transfected with miR-326-mimic,and how miR-326-mimic regulates contractile proteins such as α-SMA,MHC and CNN1 was examined by Western blot after 24 h.Also observe how miR-326-mimic regulates the phenotypic switch of VSMCs at the translational level.As well as to observe the expression of proliferative proteins such as Ki-67 and PCNA by Western blot.(10)VSMCs were transfected with miR-326-mimic,and the effect of miR-326-mimic on VSMCs migration was observed by wound-healing assay after 24 h.(11)VSMCs were transfected with miR-326-mimic,and the effect of miR-326-mimic on the migration of VSMCs was observed by Transwell assay after 24 h.Results(1)The expression of miR-326 was down-regulated in the injury group compared to the normal group.(2)At the cellular level,inhibition of endogenous miR-326 expression promoted the proliferation and migration ability of VSMCs compared to controls,with elevated levels of Ki-67 and PCNA on protein and m RNA.Meanwhile,the levels of CNN1 and α-SMA on protein and m RNA decreased,indicating that miR-326-inhibitor also promoted the phenotypic transition of VSMCs.MHC was only down-regulated at m RNA level,while no significant difference was observed at protein level.(3)Compared to the control group,overexpression of miR-326 inhibited VSMCs proliferation and migration,and Ki-67 and PCNA levels on protein and m RNA were decreased.In addition,overexpression of miR-326 elevated the expression of α-SMA and CNN1 at the m RNA and protein levels and inhibited the phenotypic switch of VSMCs.Interestingly,MHC was only upregulated at the m RNA level,while no significant difference was observed at the protein level.ConclusionCompared to the normal group,miR-326 expression was decreased in the carotid balloon injury group.Mi R-326-inhibitor was able to downregulate contractile protein expression in VSMCs,promote phenotypic switch,and promote VSMCs proliferation and migration.Mi R-326-mimic was able to upregulate contractile protein expression,inhibit phenotypic switch,and also inhibit proliferation and migration.