Study The Role Of Myocardin/miR-206/Connexin43 Pathway On Vascular Smooth Muscle Cell Proliferation And Atherosclerosis

While connexin43(CX43)plays an important role in regulating the proliferation of vascular smooth muscle cell(VSMC),myocardin is now considered to play an important role of regulating VSMC differentiation.MiR-206 is an important regulator of the proliferation of smooth muscle cells.This paper mainly focused on three moleculars:myocardin,miR-206 and CX43.But the relationship among them and the VSMCs phenotype conversion to microRNA206(miR-206)is still unclear.In this thesis,the applications of myocardin/miR-206/connexin43 pathway to vascular smooth muscle cell proliferation and atherosclerosis disease model were studied.The contents are as follows:1L To evaluate the correlation between miR-206 and CX43 on vascular smooth muscle cell proliferation and phenotypic transformation by proliferation test.The results showed that the proliferation rate of HA-VSMCs accompanying phenotype decreased by miR-206 mimics significantly.The proliferation rate of HA-VSMCs accompanying phenotypic was decreased by inhibiting CX43 expression.The luciferase report experiment verifies that miR-206 can bind CX43-3'UTR to lower the level of CX43 expression.2.To evaluate the overexpression myocardin on proliferation,phenotype conversion and expression of CX43 and miR-206 of VSMCs by proliferation test.The results show that the proliferation rate of HA-VSMCs accompanying phenotype decreased by myocardin overexpression obviously.The expression of CX43 protein was significantly decreased after overexpression of myocardin,but the mRNA level was not significantly changed.However miR-206 expression increased by myocardin overexpression significantly.The results of luciferase report showed that myocardin could not affect the transcriptional activity of CX43 promoter.The luciferase report experiment and ChIP experiment prove that myocardin can be directly binding with miR-206 promoter,and myocardin can not be directly binding with miR-206 promoter with mutation CArG binding site.Myocardin regulates miR-206 expression of binding with CArG binding site on miR-206 promoter.3.To construct and package lentivirus vectors(mmu-miR-206-3p and mmu-miR-206-control),group treatment of ApoE-/-atherosclerosis modelled mice.After treatment,there was no significant difference in weight changes of the mices,triglyceride level and low density lipoprotein level.We have confirmed that the total cholesterol level of mices infected with miR-206 lentivirus decreased slightly and HDL level increased significantly,suggesting that serum HDL could be increased after miR-206 virus infection.By using HE staining and masson staining,it was found that the lesion area and collagen fiber content of arterial plaque in mices infected with mmu-miR-206-3p virus decreased significantly compared with that of miR-206-control group,but still higher than normal level.The level of CX43 expression of the vascular tissue of mice infected with miR-206 was significantly decreased by Western blot test CX43 expression.Using HE staining,Masson staining and immunofluorescence assay to detect the CX43 expression positive cells in the blood vessels of mice found that CX43 expression mainly in the vascular smooth muscle layer,ApoE-/-mouse plaque lesion region expression increased,infection miR-206 virus particles after CX43 expression decreased significantly.miR-206 suppresses CX43 expression and the proliferation level of vascular smooth muscle cells,inhibit the conversion to vascular smooth muscle cells to proliferation phenotype.miR-206,The expression of CX43 was reduced by complementary distribution of miR-206 and CX43 3'UTR region,which inhibited the proliferation and phenotype conversion of vascular smooth muscle cells.Overexpression of myocardin can effectively reduce the proliferation level of vascular smooth muscle cells and inhibit the conversion to proliferation phenotype.We discovered that the role of myocardin/miR-206/CX43 pathway on vascular smooth muscle cells proliferation and phenotypic transformation.The experimental results show that miR-206 can effectively reduce the area of atherosclerotic plaque and collagen fiber deposition,and could delay the progression of the disease.Overexpression of miR-206 can effectively lower the level of CX43 protein in vascular tissue.It is associated with the narrowing of atherosclerotic plaques.The overexpression of miR-206 in the atherosclerotic mouse model decreased the serum total cholesterol level of mice and raised the level of high-density lipoprotein,which had the potential value in preventing and curing atherosclerotic diseases.Myocardin and miR-206 play important role in regulating CX43 expression on vascular smooth muscle cells proliferation,phenotypic transformation,and provide research basis and new research direction for the prevention and treatment of vascular proliferation-related diseases.