MLCK Regulates The Proliferation And Migration Of Vascular Smooth Muscle Cells Through MiRNA-92a

Objective:Atherosclerosis(AS)is a chronic blood vascular wall diseases based on abnormal lipid metabolism,which was induced by multiple factors.AS is the primary cause leading to cardiovascular disease,the number of annual deaths due to AS-related diseases account for more than half of total global deaths.Exploring the pathogenesis of AS has a great significance for prevention and treatment of cardiovascular and cerebrovascular diseases.During atherosclerotic vascular remodeling,it is important that vascular smooth muscle cells(VSMCs)migrate from the media into the intima,and proliferate in response to mediators such as platelet-derived growth factor(PDGF).Myosin light chain kinase(MLCK)is a key enzyme in the regulation of VSMCs contraction.MLCK inhibition can protect blood vessels and reduce the formation of AS plaque.However,the mechanism of the proliferation and migration of VSMCs regulated by MLCK is not clear during AS formation.Recent studies suggest that microRNAs play a key role in the regulation of function and plasticity of VSMCs.This study aims to explore the miRNAs related to the MLCK-incuced AS vascular remodeling and its mechanism by building atherosclerotic mouse models and culturing Human aortic smooth muscle cells(AoSMCs),guinea pig basilar artery smooth muscle cells(GbaSM-4)and MLCK knockdown type GbaSM-4(MLCK-/Gba).Method:(1)Atherosclerotic mouse model was built with apolipoprotein E-deficient mice(Apolipoprotein E,ApoE-/-),the formation of plaques in aortas were analyzed by HE(hematoxylin-eosin)staining.The changes of blood lipids levels were detected with kits.(2)The expression level of MLCK mRNA and miR-92a were measured by quantitative Real-time Polymerase Chain Reaction in thoracic aortas.(3)Using transient transfection techniques to suppress or over express miR-92a in VSMCs.(4)The migration of VSMCs treated with miR-92a or MLCK inhibitor was assessed by Boyden Chamber assay.(5)The survival rate of VSMCs treated with miR-92a or MLCK inhibitor was measured with MTT.(6)Stress fiber remodeling and mammalian transcription factor 4(Kruppel-like factor 4,KLF4)distribution in VSMCs treated with miR-92a were analyzed by immunofluorescence.(7)Western blot assay was used to detect the expression of MLCK and KLF4 in VSMCs.(8)The high-fat diet mouse model was built with C57 mice,and ML-7(C15H17IN202S·HC1,ML-7),a selective inhibitor of MLCK,or normal saline was injected into mice tail vein.The expression level of miR-92a in thoracic aortas and VSMCs treated with MLCK inhibitor were detected by quantitative Real-time Polymerase Chain Reaction.Results:1.In the atherosclerotic mouse model:(1)In high-fat diet group,the levels of T-CHO(Total cholesterol).TG(Triglyceride)and LDL(Low density lipoprotein cholesterol)were upregulated,but HDL(High density lipoprotein cholesterol)was significantly decreased in 9th weeks.(2)In normal diet group,the blood vessel walls had no significant changes in the third to the 9th weeks.The vessel walls were thickened in 12th weeks but plaques were not generated.In high-fat diet group,the vessel walls had no significant change in the third to the 6th weeks.The walls were thickened in 9th weeks with a small volume aortic plaque.The atherosclerotic plaques were completed formed in 12th weeks.(3)In the thoracic aortas,the expression of MLCK mRNA was increased and peaked in the 6th to 9th weeks and then reduced in 12th weeks.Also,the expression of miR-92a had a similar trends with mRNA expression of MLCK.2.In VSMCs:(1)The proliferation and migration of AoSMCs could be reduced by MLCK inhibitor(ML-7)or miR-92a inhibitor.(2)miR-92a inhibitor reduced the proliferation and migration of GbaSM-4.However,miR-92a mimics promoted the proliferation and migration of MLCK-/Gba.(3)Upon the stimulation of PDGF-BB,GbaSM-4 projected large number of filopodia observed by microscope while MLCK-/Gba had no significant formation of filopodia.In GbaSM-4,miR-92a inhibitor reduced the formation of filopodia and promoted KLF4 protein expression.In MLCK-/Gba,by contrast,miR-92a mimics partially restored the formation of filopodia and inhibited KLF4 protein expression.3.ML-7 suppressed expression of miR-92a in AoSMCs and the thoracic aortas of high fat diet mice.In additon,Compared with GbaSM-4,the expression of miR-92a was lower in MLCK-/Gba.Conclusion:(1)In the atherosclerotic disease process,the expression of MLCK mRNA and miR-92a were both increased.(2)MLCK and miR-92a are both involved in the regulation of proliferation and migration of VSMCs.(3)MLCK regulated the proliferation and migration of VSMCs via miR-92a and thus promoted the formation of AS.