Regulation Of MicroRNA-103 On End-stage Autophagy And Proptosis In H₂O₂ Mediated Oxidative Stress Model Of Human Coronary Artery Endothelial Cells

BackgroundCoronary atherosclerosis is a kind of chronic inflammatory response accompanied by many serious complications and consequences,which is generally considered to be an important cause of global mortality and disability.Oxidative stress produces reactive oxygen species（ROS）and endothelial cell damage is widely considered as the starting factor of coronary atherosclerosis.Autophagy is a kind of eukaryotic cell life process which depends on lysosomal catabolism to damage organelles and wrong proteins.It can reverse the adverse effects of oxidative stress by inhibiting inflammatory pathway,reducing apoptosis and necrosis of cells,and inhibiting endoplasmic reticulum stress.According to the formation of autolysosomes,it can be divided into early autophagy and end-stage autophagy.Although autophagy has been widely studied in oxidative stress in recent years,its role remains controversial,especially for the end-stage autophagy.Some studies have found that pyroptosis,as a new type of programmed cell death,is closely related to cardiovascular diseases.However,after inhibition of end-stage autophagy,lysosomes can not function normally,and the accumulation of autophagy bodies in cells directly leads to the increase and aggregation of NLRP3 inflammatory bodies,and finally aggravates the inflammatory response of cells.Therefore,in the oxidative stress-mediated coronary atherosclerotic disease,the effect of end-stage autophagy on cell pyroptosis still needs further study.MicroRNAs are composed of 20-40nucleotides,which have highly conserved single stranded non coding RNA and regulate gene expression in many life processes.MicroRNA-103（miR-103）belongs to miR-103/107 family on human chromosome 5,which is widely expressed in various tissues.A large number of studies have shown that miR-103 is involved in the regulatory mechanism of cardiovascular diseases.Recently,it has been found that inhibition of miR-103 expression can block the binding of autophagosomes to lysosomes.However,whether miR-103 can regulate cell pyroptosis by end-stage autophagy remains to be studied.ObjectsThe aim of this study was to investigate whether end-stage autophagy has an effect on pyroptosis of human coronary artery endothelial cells in oxidative stress model.To clarify the change trend and the role of miR-103 under the stimulation of H₂O₂.Finally,the relationship between BNIP3 and miR-103 was verified,and whether miR-103 regulated end-stage autophagy and pyroptosis by BNIP3.Methods1.HCAEC cells were cultured in time gradient of H₂O₂ to construct the oxidative stress model of coronary artery endothelium.The survival rate of endothelial cells was detected by MTS activity test kit,the level of ROS in cells was detected by DCFH-DA probe to evaluate the degree of cell damage under the stimulation of H₂O₂at different time points,and the expression of autophagy related proteins p62,LC3B,p-mTOR/mTOR and the expression of pyroptosis related proteins NLRP3,Caspase-1 and IL-1βin cells was detected by Western bolt technology to observe the change of autophagy and pyroptosis signal pathways.2.3MA、BafA1,autophagy inhibitors,were used to inhibit the development of autophagy,MTS was used to detect cell survival rate and the level of ROS in cells was detected by DCFH-DA probe.Western blot was used to detect the expression of autophagy and pyroptosis protein,immunofluorescence staining was used to observe the dot like aggregation of LC3B and P62,and transmission electron microscope was used to observe the autophagosomes in cells,to study the role of autophagy in the process of H₂O₂ mediated oxidative stress injury.3.Real time quantitative PCR was used to detect the expression of miR-103stimulated by H₂O₂ at different time points.The control model of miR-103 with high expression,low expression and NC negative was constructed by lentivirus transfection.The results showed that miR-103 played a role in oxidative stress injury.Western blot was used to detect the expression of autophagy and pyroptosis related proteins.Immunofluorescence staining was used to observe the aggregation of LC3B and the production of autophagy bodies in cells.The regulation of miR-103 on autophagy was further studied.4.Western blot was used to detect the expression of BNIP3 in oxidative stress model and after adding bafa1.In the model of high and low expression miR-103,the regulation of miR-103 on BNIP3 was studied.Using siBNIP3 to construct BNIP3model with low expression in NC and miR-103 models respectively.At the same time,after adding H₂O₂ stimulation,the cell activity was detected and the effect of BNIP3on endothelial cells in oxidative stress model was observed.Western blot was used to detect the expression of autophagy and pyroptosis related proteins in sibnip3 cells and to study the effect of BNIP3 on autophagy.Results1.Under the stimulation of H₂O₂,the survival rate of HCAECs decreased,and the level of ROS in cells increased,suggesting that the cells were damaged under the stimulation of H₂O₂.Western blot showed that the autophagy related protein LC3BⅡ/LC3BⅠdecreased,P62 and the ratio of p-mTOR/mTOR increased,suggesting that autophagy decreased.At the same time,the expression levels of NLRP3,Caspase1and IL-1βwere increased.It is suggested that oxidative stress can promote cell pyroptosis.2.On the basis of H₂O₂ stimulation,3MA,an early autophagy inhibitor,and bafA1,an end-stage autophagy inhibitor,were added.It was found that 3MA had no significant effect on cell damage,while bafa1 increased cell death and ROS level.Subsequently,the changes of autophagy and pyrolytic death in bafa1 cells were detected.It was found that inhibition of terminal autophagy could increase the accumulation of p62 and LC3BⅡin cells and decrease the phosphorylation level of mTOR.Finally,it promotes the occurrence of inflammatory cell death.3.Under the stimulation of H₂O₂,the expression of miR-103 decreased in a time-dependent manner.After constructing the high and low expression model of miR-103,we found that high expression of miR-103 can improve the cell survival rate,inhibit the accumulation of autophagy bodies,and reduce the expression of pyrolytic protein,which can be blocked by bafA1.On the contrary,the low expression of miR-103 can increase the accumulation of autophaosomes in cells,which will aggravate the occurrence of cell pyroptosis and eventually aggravate the damage to cells.4.Western blot showed that BNIP3 expression level increased significantly under the stimulation of H₂O₂ and bafA1,while BNIP3 silencing inhibited the pyroptosis of endothelial cells by reducing autophagy related proteins such as LC3BⅡand p62.Similarly,inhibition of BNIP3 level in low expression miR-103 cells can counteract the effects of miR-103 on end-stage autophagy and ptroptosis,and finally play a protective role on endothelial cells.ConclusionsIn the model of acute oxidative stress of HCAECs mediated by H₂O₂,miR-103expression decreased,and the decreased miR-103 in endothelial cells could not play a targeted inhibition role on BNIP3 protein,thus affecting the end-stage autophagy of endothelial cells,making the autophagy related proteins LC3BⅡand P62 accumulate in the cells,inhibiting the activation of mTOR,and then increasing the level of cell pyroptosis,and finally aggravating the cell damage.On the contrary,increasing miR-103 expression can target to inhibit BNIP3,reduce the inhibition to the end-stage autophagy,and thus reduce the inflammatory cell death,and ultimately high expression of miR-103 plays a protective role on cells.Innovation and significanceThis study confirmed the effect of end-stage autophagy on the proptosis pathway in acute oxidative stress of endothelial cells.It was found that miR-103 played an important role in the oxidative stress response to end-stage autophagy and pyroptosis death.At the same time,it was found that how the target gene BNIP3 downstream of miR-103 affected end-stage autophagy and pyroptosis.This study provides a new research direction for coronary atherosclerotic disease in the cardiovascular system and a new theoretical support for the mechanism of miR-103.