Mechanism Of Targeted Regulation Of Robo4gene Expression By Mir-140-5p In Vascular Smooth Muscle Cells

Background:MicroRNAs have been established as essential regulators of atherosclerosisdevelopment.Despite previous reports showing the pathogenic role of miR-140-5p in angiogenesis process and glioma cell proliferation and invasion,the imformation of pathogenic roles of miR-140-5p in the development of atherosclerosis is limited.Objective:This aim of this study is to investigated the cellular roles of miR-140-5p on human vascular smooth muscle cells(VSMCs),as well as its target gene.Materials and Methods: QRT-PCR was used to detect the expression of miR-140-5p andROBO4 mRNA in atherosclerotic plaque tissue and vascular tissue of patients with non-vascular disease,and Western blotting method was used to detect the expression level ofROBO4 protein in atherosclerotic plaque tissue.Furthermore,the expression of miR-140-5p andROBO4 was compared in these the two groups.Smooth muscle cells were treated with ox-LDL and transfected with miR-140 inhibitor andROBO4 overexpression vectors in vitor,respectively,.Then we applied CCK8 method,Transwell cell experiment,flow cytometry to decect the proliferation and migration ability and apoptosis levelof vascular smooth muscle cells Detection of invasive ability and apoptosis level.The dual luciferase reporter gene detection method was used to detect the interaction between miR-140-5p andROBO4.Finally,we performed rescue experiments to detect the cell proliferation,apoptosis,migration and invasion ability,and to evaluate the changes of miR-140-5p /ROBO4 on vascular smooth muscle cell phenotype.In the experiment,NLRP3 inflammatory bodies were detected;NLRP3 expression was intervened to evaluate the regulatory relationship between miR-140-5p /ROBO4 and NLRP3 in vascular smooth muscle cells.Results: miR-140-5p is highly expressed in arterial plaque tissues of patients with atherosclerosis;while the expression level ofROBO4 protein is opposite to the trend of miR-140-5p expression.Ox-LDL can promote the expression of miR-140-5p in human vascular smooth muscle cells,.Inhibit the expression ofROBO4 was promote smooth muscle cell proliferation,migration,invasion and inhibit apoptosis.Further results show that miR-140-5p inhibitor can reverse the regulation of Ox-LDL vascular smooth muscle cell proliferation,migration,invasion and apoptosis.The role ofROBO4 in smooth muscle cells as same as that of miR-140-5p inhibitor.Dual luciferase results show that miR-140-5p can bind to the 3'-UTR sequence ofROBO4 gene.Finally,the rescue experiment showed thatROBO4 siRNA and NLRP3 overexpression can block the regulation of miR-140-5p inhibitor on the proliferation,migration,invasion and apoptosis of human vascular smooth muscle cells after OxLDLtreatment.Conclusion: miR-140-5p can promote the proliferation,migration and invasion of vascular smooth muscle cells by inhibiting the expression ofROBO4 gene,inhibit the apoptosis of vascular smooth muscle cells,and promote the mechanism of NLRP3 inflammatory corpuscles induced by Ox-LDL in vascular smooth muscle cells,thereby promoting atherosclerosis.This study provides new ideas for the prevention and treatment of atherosclerosis.