Mechanism Research Of Fluid Shear Stress-sensitive MiR-29b-3p/CX3CL1 Axis Regulating Atherosclerosis

Atherosclerosis（AS）,the formation of fibrofatty lesions in the walls of arteries,is one of the most prevalent cardiovascular diseases worldwide,and is also an important cause of cardiovascular and cerebrovascular diseases such as ischemic heart disease,stroke and peripheral vascular disease.Atherosclerosis is considered to be a multifocal and chronic immune-inflammatory disease of large and medium-sized arteries driven by lipids,and the inflammatory response runs through the whole process of atherosclerosis development.Therefore,elucidating the pathogenic effect and molecular mechanism of inflammatory response on the occurrence and development of atherosclerosis,and searching for potential new targets have important guiding significance for the basic mechanism research and clinical prevention and treatment of atherosclerosis.Fluid shear stress is the frictional force on the vascular endothelium due to blood flow.Fluid shear stress acting on the vascular endothelium plays an important role in regulating endothelial function and the development of atherosclerosis.The incidence of atherosclerosis is lower in the straight parts of blood vessels exposed to laminar shear stress（Lss）,while the curved or bifurcated parts of blood vessels exposed to oscillatory shear stress（Oss）or turbulent flow are atherosclerosis-susceptible areas.Vascular endothelial cells convert perceived mechanical force changes into biological signals,and regulate gene expression and cell behavior through multiple pathways and mechanisms,thereby affecting vascular endothelial function and vascular physiological remodeling.Micro RNA（mi RNA）is an endogenous small RNA molecule with a size of 19～25nucleotides,which can regulate gene expression at the post-transcriptional level by inhibiting messenger RNA（m RNA）translation or promoting m RNA degradation.Mi RNA participates in the regulation of various biological processes such as cell growth,differentiation,development,and apoptosis,and plays a key role in the occurrence and development of various diseases.In this study,"the anti-atherosclerotic function of Lss"was taken as the starting point,and based on the high-throughput sequencing data of the whole transcriptome before and after Lss loading on endothelial cells,bioinformatics technology was used to systematically mine the differentially expressed m RNAs before and after Lss treatment,and to identify Lss-sensitive target m RNA and its biological functions.Combining with the upstream mi RNA predicted by the bioinformatics website,the target regulation relationship between the Lss-sensitive m RNA and the upstream mi RNA was clarified.Subsequently,the regulation of the Lss-sensitive mi RNA/m RNA axis on the biological function of vascular endothelium and the underlying molecular mechanism were explored in vitro and in vivo to clarify the impact of this regulatory axis on the development of atherosclerosis.This study aims to explore the potential mechanism of Lss against atherosclerosis from a new perspective,so as to find new targets for the prevention and treatment of atherosclerosis.PartⅠScreening and biological function identification of fluid shear stress-sensitive m RNAIn this study,differential expression analysis was performed on the whole transcriptome sequencing data of human aortic endothelial cells（HAECs）treated with steady state and Lss,and 297 up-regulated m RNAs and 155 down-regulated m RNAs were screened out.The transcriptome sequencing data of steady-state and Lss-treated human umbilical vein endothelial cells（HUVECs）included in the GEO database were analyzed using the same screening conditions,and 496 up-regulated m RNAs and 853 down-regulated m RNAs were obtained.25 key m RNAs were obtained by intersecting the down-regulated m RNAs in Lss-treated HAECs and HUVECs.Then,the target gene CX3CL1closely related to the development of atherosclerosis was screened out through GO functional annotation,KEGG pathway enrichment analysis and protein interaction network construction.And it was verified by molecular biology that the m RNA and protein expression levels of CX3CL1 were significantly down-regulated in Lss-treated HAECs and HUVECs.Biological function studies found that in tumor necrosis factorα（TNFα）-treated HAECs,Lss inhibited the expression of vascular cell adhesion molecule-1（VCAM-1）and intercellular cell adhesion molecule-1（ICAM-1）and the adhesion of monocytes to HAECs by mediating CX3CL1.PartⅡFunctional study of the fluid shear stress-sensitive mi R-29b-3p/CX3CL1axis in regulating the development of atherosclerosisIn order to further improve the mechanism network of CX3CL1 regulating vascular inflammation,this study predicted five upstream mi RNAs（mi R-424-5p,mi R-497-5p,mi R-29c-3p,mi R-29a-3p and mi R-29b-3p）with conserved binding sites with CX3CL1through the bioinformatics website.The targeting regulation relationship between mi R-29b-3p and CX3CL1 was verified by luciferase reporter gene experiments and construction of cell models overexpressing or knocking down mi R-29b-3p.Mechanistic studies revealed that Lss alleviated TNFα-induced inflammatory response in HAECs by regulating the mi R-29b-3p/CX3CL1 axis.In vivo studies showed that the inflammatory infiltration and plaque formation of the aortic intima were significantly reduced after the tail vein injection of agomir-mi RNA-29b-3p in Apo E^-/-mice fed a high fat diet（HFD）.Furthermore,tail vein injection of agomir-mi RNA-29b-3p significantly inhibited the HFD-induced upregulation of CX3CL1 and VCAM-1 in aortic tissues.Therefore,Lss alleviated vascular endothelial inflammation and atherosclerotic lesion development by regulating the mi R-29b-3p/CX3CL1 axis.Part Ⅲ Molecular mechanism of fluid shear-sensitive mi R-29b-3p/CX3CL1 axis regulating endothelial cell inflammatory responseIn order to further clarify the underlying molecular mechanism of Lss-sensitive mi R-29b-3p/CX3CL1 axis regulating endothelial inflammation and atherosclerosis formation,this study analyzed the effect of Lss-sensitive mi R-29b-3p/CX3CL1 axis on the NF-κB signaling pathway in TNFα-stimulated HAECs.The results showed that Lss-sensitive mi R-29b-3p/CX3CL1 axis alleviated the adhesion of monocytes to HAECs and the inflammatory response of endothelial cells by blocking the activation of NF-κB signaling pathway.