Study On The Role And Mechanism Of Laminar Shear Stress-related LncRNA AF131217.1 In Atherosclerosis

Atherosclerosis?AS?is the common pathological basis of numerous cardiovascular and cerebrovascular diseases and seriously endangers human health.In general,the prevalence and mortality of cardiovascular diseases in China are still on the rise.Atherosclerosis is a chronic inflammatory vascular disease,and inflammatory response plays a crucial role in the development of atherosclerosis at all stages.However,the potential mechanism of inflammation and atherosclerosis has not been elucidated.Therefore,it is of great significance to study the pathogenesis of atherosclerosis from the perspective of inflammation for the prevention and treatment of this disease.Laminar shear stress?LSS?is the friction force of blood flow acting on endothelial cells on the surface of blood vessels.It plays an important role in anti-inflammation and anti-adhesion by affecting the expression of endothelial cells genes,thus affecting the occurrence and development of atherosclerosis.The fluid shear stress mentioned in this study is laminar shear stress.Long noncoding RNA?lncRNA?is a noncoding RNA greater than 200 nucleotides.More and more evidences show that lncRNA plays a key role in the development of cardiovascular diseases and can affect various biological processes such as vascular cell migration,proliferation,angiogenesis and inflammation.However,the expression and function of lncRNA in endothelial cells loaded by laminar shear stress remain unclear.In this study,differential expressions of lncRNA and mRNA in laminar shear stress were analyzed by high-throughput sequencing data,and miRNA and mRNA potentially related to lncRNA were predicted by bioinformatics website.LncRNA/miRNA/mRNA co-regulatory network was constructed in laminar shear stress to analyze functions of differential genes.By detecting the expression level of AF131217.1 under laminar shear stress and the targeted regulation relationship between AF131217.1/miR-128-3p/KLF4,to further explore the effect of AF131217.1/miR-128-3p/KLF4 on the biological function of endothelial cells.Part I Construction of lncRNA/miRNA/mRNA co-regulatory network in LSSHigh-throughput sequencing data of endothelial cells under LSS were downloaded from GEO database,and 52 differentially expressed lncRNAs and 373 differentially expressed mRNAs were obtained through analysis.MiRNA that may be bound by lncRNA was predicted by miRDB,miRcode,AnnoLnc and other databases,and the target genes of miRNA were predicted by Targetscan,miRDB and DIANA Tools and the results of sequencing analysis were combined to screen out the potential mRNA regulated by miRNA.According to the above results,lncRNA/miRNA/mRNA co-regulatory network was constructed.By qRT-PCR,AF131217.1 was significantly up-regulated in LSS.Part?AF131217.1 functional studyUsing the nucleoplasmic RNA separation experiment,it was found that AF131217.1was mainly located in the cytoplasm.Using sh-AF131217.1-3 to knock down the expression of AF131217.1 in endothelial cells can promote the adhesion between endothelial cells and monocytes,and the expression of adhesion-related molecules.TNF-?stimulates endothelial cell inflammatory state,knockdown the expression of AF131217.1 can aggravate the inflammation effect caused by TNF-?,suggests AF131217.1 has anti-inflammatory effects in endothelial cells.ApoE^-/-mouse atherosclerosis model was established by high-fat diet,after the lentivirus overexpression of AF131217.1 was injected into the tail vein,the aortic plaque area,lipid accumulation,VCAM-1 expression,blood lipid level,and the mRNA and protein levels of eNOS were significantly reduced,but the mRNA and protein levels of VCAM-1 were inhibited.In vivo and in vitro experiments have proved that AF131217.1 has anti-inflammatory function.Part?Mechanism research of AF131217.1 biological functionBioinformatics method combined with luciferase reporter gene assay confirmed that mir-128-3p and AF131217.1 could bind each other,and knockdown of AF131217.1could significantly up-regulate the expression level of miR-128-3p.In endothelial cells,pri-miR-128-3p was transfected,and overexpression of miR-128-3p promote the adhesion between endothelial cells and monocytes,and the expression of adhesion-related molecules.The luciferase reporter gene assay showed that KLF4 was the target gene of miR-128-3p,and overexpression of miR-128-3p or knockdown of AF131217.1 could inhibit KLF4 expression.The experimental results showed that the AF131217.1/miR-128-3p/KLF4 axis may be involved in the regulation of endothelial cell inflammation and the development of atherosclerosis.