The Effect Of MiR-15a-5p On IL-6/STAT3 Signaling Pathway Mediated CX3CL1 Promotes Endothelial Cell Proliferation

Objective:Atherosclerosis(AS)is a chronic inflammatory process that involves complex interactions between inflammatory cells,endothelial cells(ECs)and smooth muscle cells(SMCs).Early AS is characterized by recruitment of inflammatory cells and expression of inflammatory cytokines.Under the stimulation of proinflammatory cytokines,vascular cells are activated and further produce a large number of inflammatory mediators;ECs function changes and promote immature angiogenesis;the interaction of the above components eventually leads to the occurrence and development of AS plaque.Revealing the molecular regulation mechanism of AS is of great significance in preventing and treating atherosclerosis.Methods:The expression plasmid of CX3CL1 was constructed.Scratch test,MTT and EDU were used to detect the effect of CX3CL1 on endothelial cells.After micro RNA-15a-5p was transfected into endothelial cells,scratch test,MTT and EDU were used to detect the effect of micro RNA-15a-5p on endothelial cells.The 3’UTR region luciferase reporter gene plasmid pmir GLO-CX3CL1-WT-3’UTR and its sequence mutant pmir GLO-CX3CL1-MUT-3’UTR were constructed.The effects of micro RNA-15a-5p on the transcriptional regulation and expression of CX3CL1 were analyzed by luciferase activity assay,Western blotting and immunofluorescence assay.The expression plasmid of STAT3 was constructed.IL-6 was used to stimulate cells and transfect STAT3.The effects of IL-6 on proliferation and migration of endothelial cells were detected by scratch test,MTT and EDU.Human umbilical vein endothelial cells were stimulated with different concentrations of IL-6.The levels of p-STAT3,STAT3 and CX3CL1 were detected by q PCR and Western Blot assay,and the optimal time was also determined by time gradient.The luciferase reporter gene plasmid containing STAT3 binding site and mutant STAT3 binding site was constructed.The effect of STAT3 on the transcriptional activity of CX3CL1 promoter was analyzed by luciferase activity.The luciferase reporter gene plasmid p GL3-miR-15a-5p-WT and the mutation sequence of GAS region in its promoter p GL3-mir-15a-5p-MUT were constructed.The effect of STAT3 on the transcriptional activity of the promoter of micro RNA-15a-5p was analyzed by luciferase activity.Results:According to the above experimental scheme,the following results were obtained:CX3CL1 could promote the proliferation and migration of endothelial cells,while over-expression of micro RNA-15a-5p inhibited the proliferation and migration of endothelial cells.Luciferase activity assay showed that micro RNA-15a-5p affected the proliferation and migration of endothelial cells by inhibiting the expression of CX3CL1 on CX3CL1 3’UTR.Stimulating endothelial cells with IL-6 and over-expressing STAT3 can promote the proliferation and migration of endothelial cells,while stimulating cells with IL-6 can up-regulate the levels of p-STAT3 and CX3CL1,while STAT3 can promote the proliferation and migration of endothelial cells by binding to GAS region of CX3CL1 promoter.In addition,STAT3 can also promote its transcriptional activity by acting on the GAS site of the promoter of micro RNA-15a-5p,which may have a weak negative feedback effect on the regulation of CX3CL1.Conclusion:With the general recognition of the inflammatory mechanism of atherosclerosis,the relationship between chemokines and atherosclerosis has attracted more and more attention.Previous studies have shown that CX3CL1 is expressed in atherosclerotic lesions,but not in normal arteries.This study demonstrated that micro RNA-15a-5p could interfere with the expression of CX3CL1 mediated by inflammatory factors by binding to CX3CL1 3’UTR.It is of great significance to actively study the regulation mechanism of atherosclerosis,search for new regulatory genes and molecular markers,and to early diagnosis,prognosis and search for new therapeutic targets of atherosclerosis.