MiR-140 Regulates Angiogenesis Under Oxidative Stress By Targeting TLR4

Objective:Atherosclerosis is a common aortic disease.Acute cardiovascular events caused by atherosclerosis are the leading cause of death in the global population.Angiogenesis runs through the entire process of atherosclerotic vulnerable plaque formation and is a key factor in promoting plaque instability and inducing serious complications.Oxidative stress can directly cause endothelial damage and induce angiogenesis.MicroRNAs are a class of short-chain non-coding small RNAs that regulate cell biological functions such as cell growth,development,differentiation,inflammation,stress response,and apoptosis by inhibiting mRNA synthesis or promoting degradation.Researches show that microRNAs are closely related to the formation of atherosclerotic angiogenesis,which are widely involved in the regulation of angiogenic factors and are potential targets for the study and prevention of atherosclerotic plaque instability.Our previous research found that TLR4 promotes endothelial oxidative damage and induces angiogenesis.We used bioinformatics methods to predict microRNAs that regulate TLR4 and found that miR-140 significantly expressed in oxidative stress endothelial cells.In this study,human umbilical vein endothelial cells?HVUE-12 cells?and ApoE^-/-mice were used to study the mechanism of miR-140 targeting TLR4 in angiogenesis under oxidative stress.Our results would lay the foundation for new clinical diagnostic targets and treatment of atherosclerotic angiogenesis.Methods:1.Screening the optimal LPC concentration and establishing an oxidative stress HUVE-12 cell model;2.HUVE-12 cells stably expressing or silencing TLR4 were established after Lentiviral vector overexpressing and silencing TLR4was prepared and transfected into HUVE-12 cells.The expression efficiency of TLR4 in HUVE-12 cells was verified by RT-qPCR.The oxidative stress was induced by adding with 30?M LPC in the HUVE-12cells for 24h.3.The plasmid vector overexpressing and silencing expression of miR-140 was prepared,transiently transfected into HUVE-12 cells,and the expression efficiency of miR-140 was verified by RT-qPCR.The oxidative stress was induced by treatment with 30?M LPC for 24 h.4.LDH and CCK8 kits were used to detect oxidative stress damage and to observe the effects of TLR4 and miR-140 on oxidative stress injury in HUVE-12 cells;5.WB,IF and RT-qPCR were used to detect the expression of TLR4,VEGFA and miR140,and to observe the effects of TLR4 and miR-140 on the expression of TLR4,VEGFA and miR140 in oxidative stress HUVE-12cells;6.Matrigel tubule formation and CAM angiogenesis assay to detect angiogenesis in HUVE-12,and to observe the effects of TLR4 and miR-140 on oxidative stress cell angiogenesis;7.HUVE-12 cells overexpressing or silencing TLR4 were simultaneously transfected with miR-140 and treated with 30?M LPC for 24h to induce oxidative stress.We observed miR-140 targeting TLR4to regulate oxidative stress induced angiogenesis;The dual luciferase reporter gene system verifies the target relationship between miR-140 and TLR4;8.6-to 7-week-old SPF-class ApoE^-/-mice and TLR4^-/-mice were crossed to establish ApoE^-/-/TLR4^-/-mice,and the mouse genotypes were screened and identified.AS model mice were established by high-fat-feed for 16 weeks;9.Detected the blood lipid levels of AS mice.The plaque formation on the aortic wall of mice was observed by stereomicroscope.The pathological structure of the plaque was observed by HE staining.The lipid infiltration in the plaque was detected by oil red O staining.To observe the effect of TLR4 on blood lipid levels and lipid plaque formation in AS mice;10.IHC and IF were used to detect the expression of VEGFA,VEGFAR2 and vWF and to observe the effect of TLR4 on plaque angiogenesis in AS mice;11.RT-qPCR and WB assays were used to observe the expression levels of TLR4,VEGFA and miR-140 in AS mice;12.Masson staining was used to detect the content of collagen fibers in plaques,and to observed the effect of TLR4 on the plaque stability of AS mice;13.The association between miR-140,TLR4,and VEGFA was analyze by Pearson correlation analysis.Results:1.30?M LPC is the best modeling concentration of oxidative stress cells;2.The expression of TLR4 and VEGFA was increased and the expression of miR-140 was decreased in HUVE-12 cells with oxidative stress;3.Oxidative stress HUVE-12 induces angiogenesis enhancement;4.The fluorescence expression rate of transfected cells was over90%under fluorescence microscope.Compared with the vehicle group,the expression of TLR4 was increased by about 70-fold in overexpressing TLR4 cells,the expression of TLR4 was decreased by about 50-fold in cells that silent expression of TLR4;5.Compared with the vehicle group,the expression of miR-140 was increased by about 20-fold in overexpressing miR-140 cells,the expression of miR-140 was decreased by about 10-fold in cells that silent expression of miR-140;6.Overexpression of TLR4 promotes oxidative stress injury,tubule formation and CAM angiogenesis,increases VEGFA expression,and decreases miR-140 expression in oxidative stress cells;Silencing expression of TLR4 reduces oxidative stress injury,tubule formation and CAM angiogenesis,and decreases the expression of VEGFA,and increases miR-140 expression in oxidative stress cells;7.Overexpression of miR-140 reduces oxidative stress injury,tubule formation and CAM angiogenesis,and decreases the expression of TLR4and VEGFA in oxidative stress cells.Silencing expression of miR-140promotes oxidative stress,tubule formation and CAM angiogenesis,and enhance the expression of VEGFA,TLR4 in oxidative stress cells;8.Overexpression of miR-140 reduced oxidative stress injury,tubular formation and CAM angiogenesis,and decreased expression of TLR4 and VEGFA in oxidative stress cells that overexpressing TLR4;overexpression of miR-140 were no significant differences on oxidative stress injury,tubule formation,CAM angiogenesis,and VEGFA expression in oxidative stress cells that silencing expression TLR4;9.The dual luciferase reporter system showed that miR-140significantly inhibited the fluorescent signal of the TLR4 reporter gene,and the TLR4 reporter gene was up-regulated after the corresponding binding site mutation;10.Agarose gel electrophoresis results showed that ApoE^-/-and ApoE^-/-/TLR4^-/-mice were developed at 254 and 140 bp bands,respectively;11.The mice were fed with high fat for 16 weeks,the plaque formation and varying degrees of lipid infiltration were observed on the aortic wall,and blood lipid levels were elevated in mice;12.Compared with ApoE^-/-group,ApoE^-/-/TLR4^-/-group mice had less atherosclerotic plaque formation,lipid infiltration,and blood lipid levels,the expression of VEGFA,VEGFR2 and vWF was decreased,the expression level of miR-140 was increased,and the content of collagen fibers in the plaque was increased;13.Correlation analysis showed that TLR4 was positively correlated with VEGFA expression,and miR-140 was negatively correlated with TLR4 and VEGFA expression in AS mice.Conclusion:1.TLR4 promotes oxidative stress-induced angiogenesis.2.miR-140 inhibited the oxidative stress-induced angiogenesis by targeting the TLR4.