MicroRNA-210 Induces Endothelial Cell Apoptosis By Directly Targeting PDK1 In The Setting Of Atherosclerosis

Background:Atherosclerosis,which is an inflammatory disease and a predominant cause of cardiovascular disorders,is a worldwide issue due to the prevalence of diets high in saturated fats and lipids.In the large arteries,atherogenic lipoproteins,especially low-density lipoprotein(LDL)cholesterol,build up to gradually form atherosclerotic plaques,which have been proven to be involved in atherosclerosis-related morbidity.Apolipoprotein E(Apo E)is a 34-k Da secreted protein that has been shown to have anti-atherosclerosis activity: it targets abundant lipoproteins in arteries and is capable of efficiently removing them.Investigations of Apo E(-/-)mice have verified the crucial role of Apo E in protection from atherosclerosis.Apo E(-/-)mice fed with high-fat diets(HFD)display high lipid levels,excessive cholesterol in the blood vessels and atherosclerotic symptoms,making this an acknowledged model for investigating atherosclerosis.The endothelium functions as an effective mediator to regulate the vascular system,with roles in processes such as hemostasis,cell cholesterol,hormone trafficking,signal transduction and inflammation.Convincing evidence indicates that dysfunction of the endothelium is associated with various vascular diseases,including diabetes mellitus,arterial thrombosis and hypercholesterolemia.Endothelial cell(EC)apoptosis and death can destroy the structure of plaques,and result in the deposition of local lipids and finally in atherogenesis.Preventing EC apoptosis has garnered considerable attention as a novel means of treating atherosclerosis.Micro RNAs(mi RNAs),are small(18–22 nucleotides)noncoding RNAs that regulate gene expression by the binding of the 3'-untranslated regions(3'-UTR)of target genes post-transcriptionally,this suppresses gene expression.Emerging evidence suggests that mi RNAs could be effective therapeutic targets for complex human diseases,including tumorgenesis,lymphopoiesis and angiogenesis.Preclinical studies have shown that mi RNAs also play pivotal roles in the pathogenesis of atherosclerosis.A recent study showed overexpression of mi R-210 in the serum samples of patients with arteriosclerosis obliterans.And further study demonstrated that mi R-210 was upregulated in human atherosclerotic plaques and might be involved in the process of atherosclerosis.However,the regulatory mechanism for mi R-210 in the setting of atherosclerosis remains unclear.Methods:A mouse model with atherosclerosis induced by a high-fat diet(HFD)was built in Apo E(-/-)mice.The levels of endothelial cell apoptosis were determined via flow cytometry and the levels of crucial pro-apoptosis proteins,such as Bax,caspase-9 and caspase-3 were detected using western blot.The expressions of mi R-210 and PDK1 in purified CD31+ endothelial cells from mouse aorta were measured via RT-q PCR and western blot.Binding between mi R-210 and the 3'-untranslated region(UTR)of PDK1 m RNA was predicted using bioinformatics analyses and confirmed with a dual luciferase reporter assay.The effects of mi R-210 were further analyzed in an in vitro model using human aortic endothelial cells(HAECs)treated with oxidized low-density lipoprotein(ox-LDL).Results:(1)We found that the HFD mice developed atherosclerosis in 12 weeks and had a significantly higher percentage of endothelial cell apoptosis detected by flow cytometry and western blot.(2)The up-regulated level of mi R-210 in the HFD mice and HAECs inversely correlated with the level of PDK1.Luciferase reporter assay determined that PDK1 is a target for mi R-210.(3)Inhibiting mi R-210 expression significantly reduced HAEC apoptosis,as evidenced by the results of the MTT and flow cytometry experiments.(4)Further analysis identified PDK1 as the target of mi R-210 and showed that PDK1 overexpression reversed the pro-apoptotic effect of mi R-210 through mediation of the PI3K/Akt/m TOR pathways.Conclusion:Our study suggests a novel role for mi R-210 in the progression of atherosclerosis through the regulation of endothelial apoptosis,and our results suggest that atherosclerosis-associated endothelial cell apoptosis might result from abundant mi R-210 that inhibits PI3K/Akt/m TOR signaling activation by directly targetingPDK1.This indicates that mi R-210 might have potential in treatment of atherosclerosis.