MiR-17-92 Regulates Ferroptosis In Vascular Endothelial Cells And Research On Mechanism

Vascular endothelial cell death plays a pivotal role in a wide range of pathological processes,including atherosclerosis,tissue ischemia and tumorgenesis.Cardiovascular disease is the leading cause of human death worldwide.The incidence of this disease was increasing in the past decades,in both industrialized and developing countries.The major cause of this disease was the abnormalities of vascular function and damage of vascular endothelial cells.The pathological features of this disease include thicken of arterial wall,poor vascular elacticity,and atherosclerotic plaques formation.Mechanically,oxidative damage contributes to the abnormalities of vascular function and causes pathological damage of vascular endothelium in different degrees.Ferroptosis is a newly identified cell death that which is involved in serevraldiesases.A variety of chemical and biological factors have been reported to induce the cell erroptosis.Erastin is an antitumor agent that selectively targets to RAS.Erastin could induce the iron and lipid ROS-dependenent oxidation death,mainly by avoiding the protection of anti-oxidation through inhibiting the uptake of cystine through the cystine/glutamic acid transport system and avoids.This cellular process is called ferroptosis.Ferroptosis was reported to play important roles in various diseases,but its role in vascular diseases has not been explored.In this study,we tried to clarify the role of ferroptosis in the process of vascular necrosis by using human umbilical vein endothelial cells as a model.MicroRNAs（miRNA）are a class of small endogenous noncoding RNAs,composed of 18 to 24 nucleotides,with the function of regulating gene expression by binding to the 3’-UTR of targeted mRNA.Up to date,there are more than 1000human related miRNAs have been identified.With these speficities,miRNAs are widely involved in cell proliferation,differentiation and apoptosis.The miR17-92 is an oncolytic cluster which contains miR-18a,miR-19a/b,miR-20a and miR-92a,which has been extensively studied in different tumors.In recent years,it has been reported that miR17-92 clusters played an important role in coronary heart disease and vascular ischemia injury,regulating the proliferation of endothelial cells and promoting the generation of blood vessels.However,the function and mechanism of ferroptosis in vascular endothelial cells have not been reported.Zinc lipoprotein A20,also known as tumor necrosis factor-inducible protein 3（TNFAIP3）,is a special enzyme whose n-terminal and c-terminal have ubiquitination and deubiquitinating activities respectively.A20 is also an anti-inflammatory signaling molecule that mediates multiple ubiquitin-dependent intracellular signaling cascades.Its function is to regulate ten-induced JNK（c-jun-n-terminal kinase）and NF B（nuclear transcription factor B）signals as a negative regulator,thereby inducing apoptosis and cell growth arrest in different cell models.A20 mediates TNF-induced injury of human umbilical vein endothelial cells through the tak1-dependent MAPK/enols pathway.In vivo and in vitro studies have shown that it inhibits proliferation and migration of vascular smooth muscle cells by blocking the PI3k/Act signaling pathway.A20 can inhibit pulmonary artery ECs angiogenesis by activating the nuclear transcription factor NF-kappa B.Previous studies confirmed that A20 is the target gene of miR17-92,but its role and mechanism in vascular endothelial cells have not been studied.This study aims to clarify the biological function of miR17-92 in regulating ferroptosis of vascular endothelial cells through its target gene A20,and to explore the underlying mechanisms.It will provide theoretical basis for the occurrence and development of vascular pathological injury and explores new targets and strategies for the treatment of vascular diseases.In the present study,we first found that erastin can induce ferroptosis in umbilical vein endothelial cells.The results of staining and electron microscopy showed that erastin-induced death cells were significantly different from H₂O₂-induced death cells.Erastin treated cells became global pyknosis and shrinkage of mitochondria in the cytoplasm.Intracellular ferroptosis-related lipid ROS production was detected by flow cytometry.Both the cytopathological and pathophysiological death processes induced by erastin were salvaged by ferroptosis-specific inhibitor fer-1.Meanwhile,during the induction of erastin,the expression of known ferroptosis-related protein GPX4 was downregulated in a dose-dependent manner.These morphological and biochemical experiment results were consistent with the characteristics of ferroptosis.Secondly,on the basis of the previous studies,we further showed that miR17-92overexpression promoted the proliferation of HUVEC cells and inhibited the production of ferroptosis-related lipid ROS.CCK-8 results indicated that miR17-92could protect erastin-induced ferroptosis in vascular endothelial cells.Our previous studies have shown that A20 was a target of miR17-92,and we found that the expression of A20 was up-regulated with the increase of dose in erastin-induced ferroptosis of vascular endothelial cells.At the same time,Q-PCR revealed that A20expression was down-regulated in HUEVC cells overexpressing miR17-92.Overexpression of A20 mediated by lentivirus can inhibit cell proliferation,increase production of lipid-ROS,and enhance erastin-induced ferroptosis of vascular endothelial cells.Concertedly,knockdown of A20 promoted cell proliferation and inhibited erastin-induced ferroptosis in vascular endothelial cells.Thirdly,Q-PCR and WB results showed that overexpression of miR17-92 or inhibition of A20 could lead to down-regulation of ACSL4 expression in vascular endothelial cells.In addition,we found that endogenous protein A20 directly interacts with ACSL4 in erastin-induced iron death of vascular endothelial cells by means of immunoprecipitation.Conversely,we detected A20 protein elevation after overexpression of ACSL4.In order to confirm the interaction between A20 and ACSL4,further functional verification was carried out through ACSL4 inhibitors.CCK-8 results showed that overexpression of A20 promoted ferroptosis in erastin-induced vascular endothelial cells,which was reversed by inhibiting the expression of ACSL4.In order to confirm that the expression of A20 and ACSL4 in primary vascular injury is involved in the pathological process of vascular injury,confocal immunofluorescence assay was used to detect the up-regulated expression of A20 and ACSL4 protein in clinical vascular necrosis patients compared to normal vascular sites,and the expression was co-located in tissues.In summary,We estabolished a model of ferroptosis in vascular endothelial cells.and based on the study of iron death of vascular endothelial cells,and proved the inhibitory effect of miRNA-17-92 on iron death in vascular endothelial cells.Based on the ferroptosis of vascular endothelial cells,we revealed that the A20-ACSL4 axis plays an important role in erastin-induced ferroptosis of vascular endothelial cells.This study demonstrated that miR17-92 protects vascular endothelial cells from erastin-induced ferroptosis by targeting the A20-ACSL4 axis.