| With the development of economy and society,cardiovascular disease has brought huge health and economic burden to human beings all over the world,with high mortality and morbidity.Atherosclerosis(Atherosclerosis,AS)is one of the most common vascular lesions,and it is also the main pathological basis of cardiovascular disease.It is characterized by the formation of complex atherosclerotic plaques,which leads to arteriosclerosis and stenosis.The clinical manifestations are coronary heart disease,peripheral artery disease and ischemic stroke.Unstable atherosclerotic plaque rupture,platelet aggregation,stenosis or vascular occlusion caused by thrombosis can lead to severe acute cardiovascular disease and pose a great threat to human health.Therefore,it is particularly urgent to explore targeted therapy for atherosclerosis.In the pathogenesis of atherosclerosis,many cells and molecules are involved in its pathological and pathophysiological process,including endothelial cells,smooth muscle cells and inflammatory cells.At present,there are three main theories about the pathogenesis of atherosclerosis,namely,lipid infiltration theory,endothelial injury theory,vascularsmooth muscle cell migration and proliferation theory.Among them,the theory of endothelial injury holds that atherosclerotic plaque is the product of arterial endothelial injury,and the pathophysiology of AS comes from endothelial cell injury.Subsequently,more and more studies have pointed out that endothelial cell dysfunction is a sign of early atherosclerosis.Therefore,improving the function of endothelial cells is of great significance to the treatment of atherosclerosis.Circular RNA(CircRNAs)is a new class of endogenous non-coding RNA(NcRNA),which does not have a 5’hat and 3’poly(A)tail and exists in its characteristic covalent closed loop form.More and more studies have shown that CircRNAs exists widely in vivo,expresses stably in plasma,serum and tissues,and participates in the regulation of eukaryotic gene expression.Previous studies have found that multiple circular RNA are involved in the occurrence and development of a variety of cardiovascular diseases.CircRNA-CER is a newly discovered circular RNA,.It can regulate the expression of MMP13 in human cartilage degradation by acting as a MiR-136 "sponge",but its function in cardiovascular disease is not clear.Therefore,in this study,we explored the role of circRNA-CER in the regulation of endothelial cell function,and further studied the specific mechanism of its regulatory role.Part 1: The effects of circRNA-CER overexpression on biological behaviors such as proliferation and apoptosis of endothelial cells inducedby ox-LDL.Aim: To simulate the pathogenesis of atherosclerosis in vitro,and to observe the effects of overexpression of circRNA-CER on biological behaviors such as proliferation and apoptosis of endothelial cells in this model.Methods: The endothelial cells were treated with different concentrations of ox-LDL for different time,and the expression of circRNA-CER was detected by RT-PCR method,so as to determine the best intervention concentration and time of ox-LDL.The injury model of human umbilical vein endothelial cells treated with ox-LDL was established to simulate the pathogenesis of atherosclerosis.The effect of overexpression of circRNA-CER on the proliferation of human umbilical vein endothelial cells treated with ox-LDL was detected by CCK8 test and EdU experiment,and the effect on apoptosis was detected by flow cytometry.Furthermore,the effects of high expression of circRNA-CER on cell migration,invasion and angiogenesis were evaluated by scratch test,transwell test and tube formation test.Results: The best intervention condition of ox-LDL for follow-up experiment was as follows: the intervention concentration was 100mg/L for 24 hours.The overexpression of circular RNA circRNA-CER promotes cell proliferation,migration,invasion and angiogenesis,and inhibits cell apoptosis.Conclusion: The role of circRNA-CER in regulating the biological behavior of endothelial cells is clarified,which lays a foundation for further research.Part 2: CircRNA-CER regulates the expression of VEGF through the miR-136/MMP13 axis.Aim: To explore the specific mechanism of circRNA-CER in regulating the biological behavior of endothelial cells.Methods: The effects of silencing or overexpression of circRNA-CER on the expression of miR-136,MMP13 and VEGF were detected by western blot and RT-PCR.Furthermore,on the basis of overexpression of circRNA-CER,combined with miR-136 agonists and inhibitors,MMP13 siRNA to observe the effect on protein expression.The interaction among circRNA-CER、miR-136 and MMP13 was observed by luciferase report experiment.Results: After circRNA-CER silencing,the expression of miR-136 increased significantly,while the expression of MMP13 decreased.While silencing circRNA-CER,combined use of miR-136 inhibitors could reverse the inhibitory effect of circ-CER silencing on MMP13 expression.The overexpression of circRNA-CER can significantly reduce the expression of miR-136,while the expression of MMP13 increases significantly.When combined with miR-163 mimic,the increase of MMP13 caused by overexpression of circRNA-CER can be reversed.Theoverexpression of circRNA-CER will lead to the increase of VEGF expression,while the combination of miR-136 mimic or MMP13 siRNA can reverse the increase of VEGF caused by circRNA-CER overexpression.Luciferase assay showed that there was a direct binding between circRNA-CER and miR-136,miR-136 and MMP13.Conclusion: There is a circRNA-CER/miR-136/MMP13 regulatory axis in human umbilical vein endothelial cells,and circRNA-CER regulates the expression of VEGF through the miR-136/MMP13 axis.Part 3: CircRNA-CER regulates the biological behaviors such as proliferation and migration of endothelial cells induced by ox-LDL through miR-136/MMP13 axis.Aim: To observe the effect of miR-136/MMP13 axis regulated by circRNA-CER on the biological behaviors such as proliferation and migration of endothelial cells induced by ox-LDL.Methods: On the basis of circRNA-CER overexpression,combined with miR-136 mimic or MMP13 siRNA,the effect on the proliferation of endothelial cells was observed by CCK8 test and EdU experiment,and the effect on apoptosis was detected by flow cytometry.Furthermore,the effects on cell migration,invasion and angiogenesis were detected by scratch test,transwell test and tube formation test.Results: The results of CCK8 and EdU experiments showed that miR-136 mimic or MMP13 siRNA could significantly attenuate the effectof overexpression of circRNA-CER on cell proliferation.In the experiment of detecting apoptosis by flow cytometry,the overexpression of circRNA-CER inhibited the apoptosis of endothelial cells,and the intervention combined with miR-136 mimic or MMP13 siRNA could counteract the inhibitory effect of overexpression of circRNA-CER on apoptosis,resulting in a significant increase in the number of apoptotic endothelial cells.The ability of migration,invasion and tube formation of endothelial cells was significantly enhanced when circRNA-CER was overexpressed,while the promotion of migration,invasion and tube formation of endothelial cells induced by overexpression of circRNA-CER alone could be counteracted by the combination of miR-136 mimic or MMP13 siRNA.Conclusion: circRNA-CER regulates the biological behaviors such as proliferation,apoptosis,migration and invasion of endothelial cells through miR-136/MMP13 axis. |
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