MicroRNA-217-5p Ameliorates Endothelial Cells Apoptosis By Targeting CLIC4 In Atherosclerosis

Objective:At present,the incidence of cardiovascular and cerebrovascular diseases is increasing year by year,and atherosclerosis(AS)is a well-known important cause of vascular diseases.AS is characterized by the accumulation of lipid-rich plaques on the wall of the aorta.Pathologically,it can be manifested as proliferation of inflammatory cells in the vascular intima,endothelial dysfunction,narrowing of the lumen,and mural thrombus.Clinically common cerebrovascular diseases often develop on the basis of AS.On the basis of AS’s vascular wall disease,there will be luminal stenosis,blood flow stasis,thrombosis,and then vascular occlusion or embolism,and brain Corresponding symptoms and signs of brain parenchymal lesions.At present,the research on the mechanism of AS has gradually found that vascular endothelial dysfunction may be one of the important initial events leading to the occurrence of AS.Vascular endothelial cells are a single layer of flat cells located on the luminal surface of blood vessels.They are a very important cell population in the body and play an extremely important role in vascular permeability barrier,immune defense and inflammation.Apoptosis of vascular endothelial cells is an important process in the development of atherosclerosis.Once the endothelial cells undergo apoptosis,the vascular endothelium may lose the ability to regulate lipid homeostasis and regulate immunity and inflammation,leading to lipid deposition and forming an environment that promotes the formation of arterial plaques.In addition,the apoptosis of vascular endothelial cells is also the cause of plaque instability.The shedding of plaque may cause artery-to-artery embolism,leading to acute coronary artery blockage and sudden death.Therefore,regulating vascular endothelial cell apoptosis is a potential prevention and treatment approach for atherosclerosis.Finding the causes of vascular endothelial cell apoptosis and exploring its pathways have important theoretical value and practical significance for the prevention and treatment of atherosclerosis.Chloride Intracellular Channel 4(CLIC4),also known as mitochondrial chloride channel protein(Mitochondrial chloride channel protein,mt CLIC),is one of many family members of intracellular chloride channel protein(Cellular Chloride Channel Protein,CLIC)One,the molecular weight is about 29 KD.Current studies have shown that it can be widely expressed in a variety of cells,including tumor cells,nerve cells and vascular endothelial cells,but its biological function is not fully understood.Reported data show that CLIC4 is related to the apoptosis of a variety of cells,and a variety of stress inducers can cause the transfer of CLIC4 from the cytoplasm to the nucleus,and nuclear translocation initiates the occurrence of apoptosis.Micro RNAs(miRNAs)are 18-22 nucleotide non-coding RNAs that negatively regulate human genes during physiological and pathophysiological processes,and are closely related to the occurrence and development of many diseases.It has been reported that the level of miR-217 is related to the damage of atherosclerotic endothelial function,and this miRNA has been identified as the deepest regulatory miRNA in aging human umbilical cord endothelial cells.In the three websites of Target Scan,micro RNA.org,and mir DB,the micro RNA predicted to regulate the target gene CLIC4 all point to miR-217,but whether the two have an actual target relationship requires further verification.Endothelial cell(EC)apoptosis plays an essential role in the pathogenesis of atherosclerosis.Micro RNAs(miRNAs)and Chloride Intracellular Channels(CLICs)have been verified to participate in the EC apoptosis process,however,the underlying molecular mechanisms are still far from clear.The main aim of this study was to investigate the biological effects of Micro RNA-217-5p(miR-217-5p)and CLIC4 on EC apoptosis in atherosclerosis.Methods1.Apolipoprotein E gene knockout mice was fed with a high fat diet(HFD)for 12 weeks to construct a mouse model of atherosclerosis.After taking the aorta,use HE staining method to observe the pathological changes of aortic sclerosis tissue.Oil red O staining method was used to observe the lipid deposition of atherosclerosis.The expression of miR-217-5p in cells was detected by Real-time PCR.Western blot and Immunofluorescence was used to detect protein level of CLIC4 in cells.Oxidized low-density lipoprotein(ox-LDL;50μg/m L)was applied to human aortic endothelial cells for 24 h to establish a model of atherosclerosis in vitro.The expression of miR-217-5p was detected by Real-time PCR,and the level of CLIC4 protein was detected by Western blot and immunofluorescence.2.To study the effects of miR-217-5p and CLIC4 on aortic endothelial cells treated with ox-LDL,human aortic endothelial cells were transfected with miR-217-5p minics to up-regulate the level of miR-217-5p.Real-time PCR was used to detect the level of miR-217-5p in the cells to verify the transfection effects.Flow cytometry and Hoechst staining were used to detect the cell apoptosis.Use western blot to detect the protein content of pro-caspase-3,cleaved-caspase-3,pro-caspase-9,cleaved-caspase-9,Bax,and Bcl-2 proteins in cells,and the levels of cytochrome C in cytoplasm and mitochondria.JC-1 method detects mitochondrial membrane potential.Intracellular transfection of CLIC4 siRNA down-regulated the level of CLIC4,and Western blot detectd the level of CLIC4 in the cells to verify the transfection effect.Flow cytometry and Hoechst staining method detected cell apoptosis,and Western blot detected the levels of pro-caspase-3,cleaved-caspase-3,pro-caspase-9,cleaved-caspase-9,Bax,and Bcl-2 in cells,and Cytochrome C levels in cytoplasm and mitochondria.JC-1 method was used to detect mitochondrial membrane potential.3.To further research the target relationship between miR-217-5p and CLIC4,human aortic endothelial cells were transfected with miR-217-5p minics.The levels of CLIC4 in the cells were detected by Real-time PCR and Western blot.The targeting between miR-217-5p and CLIC4 was verified by Dual-Luciferase Reporter Assay System.To observe whether the effect of miR-217-5p could be reversed after CLIC4 overexpressioncell,apoptosis was detected by flow cytometry,and pro-caspase-3,cleaved-caspase-3,pro-caspase-9,and cleaved-caspase-9 in cells were detected by Western blot.Results1.The successful establishment of mouse arteriosclerosis model was confirmed by Sudan staining,HE staining,and oil red staining.In mouse aortic endothelial cells,compared with the Normal diet(ND)group,the expression level of miR-217-5p in the HFD group significantly reduced,and the expression level of CLIC4 protein in the HFD group increased significantly.Immunofluorescence staining showed that the expression of CLIC4 was significantly up-regulated in the HFD group.In the human aortic sclerosis cells,the levels of miR-217-5p in the ox-LDL group was obviously reduced compared with the control group,which were detected by real-time PCR.Western blot and immunofluorescence detection results showed that the level of CLIC4 in the ox-LDL group was significantly higher than that in the control group.2.Human aortic endothelial cells were transfected with miR-217-5p minics,and real-time PCR was used to detect the levels of miR-217-5p in the cells.Compared with the ox-LDL+NC group,the expression level of miR-217-5p in the ox-LDL+ miR-217-5p mimics group increased significantly,confirming the successful transfection.Flow cytometry was used to detect cell apoptosis.After overexpression of miR-217-5p,the proportion of apoptotic cells in the ox-LDL+miR-217-5p mimics group was lower than that in the ox-LDL+NC group.The JC-1 method detected the mitochondrial membrane potential.After ox-LDL treatment,the mitochondrial membrane potential decreased,but the mitochondrial membrane potential was restored after the overexpression of miR-217-5p.Hoechst staining method was used to detect cell apoptosis.After ox-LDL treatment,the number of apoptotic cells increased significantly,while after transfection with miR-217-5p mimics,the number of apoptotic cells decreased.Use western blot to detect the expression of apoptosis-related proteins in the cells.The results showed that after ox-LDL treatment,the expression of Bcl-2 protein was significantly reduced,while the protein expression of Bax,cleaved-caspase-3,and cleaved-caspase-9 were significantly increased,Mi R-217-5p overexpression could reverse these changes.Western blot was used to detect the level of cytochrome C in the mitochondria.The expression of cytochrome C in the mitochondria of the ox-LDL group was lower than that in the control group.The expression of cytochrome C in the mitochondria of the ox-LDL+miR-217-5p mimics group was obvious higher than the control group.The expression of CLIC4 gene in human aortic endothelium was silenced by CLIC4 siRNA,and then apoptosis was observed by Hoechst staining.The results showed that after ox-LDL treatment,apoptotic cells increased significantly,but after transfection of CLIC4 siRNA,the number of apoptotic cells decreased.Meanwhile,the detection of apoptosis by flow cytometry also found that the proportion of apoptotic cells in the ox-LDL+NC siRNA group was reduced after transfection of CLIC4 siRNA.The JC-1method detected the mitochondrial membrane potential.The results showed that the mitochondrial membrane potential decreased after ox-LDL treatment,while the mitochondrial membrane potential recovered after the overexpression of miR-217-5p.Western blot was used to detect the expression of apoptosis-related proteins in the cells.The results showed that after ox-LDL treatment,the expression of Bcl-2 protein was significantly reduced,while the protein expression of Bax,cleaved-caspase-3,and cleaved-caspase-9 were significantly increased,CLIC4 siRNA can reverse the above changes.Western blot was used to detect the levels of cytochrome C in the mitochondria.Compared with the ox-LDL+NC siRNA group,the expression of cytochrome C in the mitochondria of the cells in the group of transfected with the CLIC4 siRNA was significantly increased.3.Real-time fluorescence quantitative PCR and Western blot were used to detect the levels of CLIC4 in human aortic endothelial cells.After ox-LDL treatment,compared with the NC mimics group,the expressions of CLIC4 m RNA and protein in the miR-217-5p mimics group were significantly reduced.Dual-Luciferase Reporter Assay System verified the targeting between miR-217-5p and CLIC4.Compared with NC mimics+CLIC4-wt and miR-217 mimics+CLCI4-mut group,the luciferin Enzyme activity in miR-217 mimics+CLIC4-wt group was decreased significantly.Flow cytometry was used to determine cell apoptosis in each group.After overexpression of miR-217-5p,the proportion of apoptotic cells in the ox-LDL+miR-217-5p mimics group was lower than that in the ox-LDL+NC group,The number of apoptotic cells in miR-217mimics+ CLIC4-wt group was higher than that in the miR-217-5p overexpression group.Use western blot to detect the protein levels of cleaved-caspase-3 and cleaved-caspase-9.The expression of cleaved-caspase-3 and cleaved-caspase-9 in the miR-217-5p overexpression group was lower than that in the ox-LDL treatment group.The levels of the above apoptosis-related proteins in the miR-217 mimics+CLIC4-wt group were higher than those in the miR-217-5p overexpression group,which was confirmed that overexpression of CLIC4 can reverse the effects of miR-217-5p.Conclusion:1.Through the atherosclerosis mouse model and in vitro experiments,it was confirmed that the expression of miR-217-5p decreased and the expression of CLIC4 increased in atherosclerotic aortic endothelial cells.2.CLIC4 can induce the apoptosis of aortic endothelial cells through the mitochondrial pathway,and the overexpression of miR-217-5p can inhibit the apoptosis of aortic endothelial cells induced by the mitochondrial pathway.3.miR-217-5p can target to CLIC4,overexpression of miR-217-5p can reduce the expression of CLIC4,overexpression of CLIC4 can reverse the effect of miR-217-5p,the two are negatively regulated,and jointly regulate the mitochondrial pathway Induced apoptosis of aortic endothelial cells.