MiR-4284 Regulates The Apoptosis Of Human Umbilical Vein Endothelial Cells Through The Target Gene PRDX6 Under The Regulation Of Hypoxia

Arteriosclerosis obliterans(ASO)is a vascular diffuse disease,which is the main cause of morbidity and mortality of cardiovascular disease worldwide and seriously harms people's physical and mental health.In contrast,endothelial cells are involved in the important process of ASO,and play an important role in its occurrence,development and prognosis,but the molecular mechanism is not clear.miRNAs plays an important regulatory role in many vascular diseases by binding to the target genes and participating in the regulation of cell proliferation,apoptosis,migration or other functions.However,the mechanism of miR-4284 in atherosclerosis has not been studied.The purpose of this study was aim to investigate the role of miR-4284 in apoptosis of human umbilical vein endothelial cells(HUVEC)in the pathogenesis of atherosclerosis.Methods: In cell experiments,RT-qPCR was used to detect the expression level of miR-4284 under hypoxia conditions.In order to verify the effect of miR-4284 on HUVECs function,we transfected miR-42484-mimic,negative control and miR-42484-inhibitor under hypoxia stimulation.Firstly,the transfectionefficiency was detected by RT-qPCR experiment.Hoechst-333342 was used to detect apoptosis.Changes in cell viability were detected by CCK-8 assay.Then,Western blot was used to detect the expression changes of apoptosis-related proteins Bax,bcl-2,Caspase3 and Parp,so as to further investigate the molecular mechanism of the effect of miRNAs on HUVECs apoptosis.Western blot was used to detect the expression of pathway protein P-Akt and Akt to verify that miR-4284 affects the apoptotic pathway of HUVECs.Finally,we used target gene prediction software to predict miR-4284 related target genes,screened out the final target genes,and conducted target gene verification experiments.After been treated with hypoxia stimulation,HUVECs were transfected with miR-4284-mimic,negative control or miR-4284-inhibitor.PRDX6 expression was detected by RT-qPCR.PRDX6 expression was detected by western blot analysis.Results: In the cell experiment,in order to select the best hypoxia condition,we set the hypoxia time gradient condition.We concluded that miR-4284 expression was significantly up-regulated in HUVECs after hypoxic treatment,especially after 4h of hypoxic treatment.After transfection with miR-4284-mimic,the apoptosis rate of HUVECs increased significantly(P<0.05),the activity was significantly inhibited(P<0.05),and the expressions of apoptosis-related proteins Bax,Parp and Caspase3 were up-regulated,P-Akt and Akt were down-regulated(P<0.05).After transfected withmiR-4284-inhibitor,the apoptosis rates of HUVECs significantly decreased,cell viability increased,and expression of apoptosis-related proteins Bax,Parp,Caspase3 was down-regulated,and P-Akt and Akt were up-regulated(P<0.05).Results using bioinformatics analysis software showed that PRDX6 was a potential target for miR-4284.PRDX6 mRNA expression level could be decreased after miR-4284-mimic(P<0.05).miR-4284-inhibitor could induce PRDX6expression(P<0.05).The results also showed that PRDX6 protein level could be decreased in miR-4284-mimic(P<0.05),and expression level could be induced by miR-4284-inhibitor(P<0.05).Conclusion: miR-4284 is upregulated in hypoxic-treated HUVECs.The enhanced expression of miR-4284 can lead to apoptosis,while the down-regulated expression of miR-4284 can partially reduce the apoptosis induced by hypoxia.Bioinformatics analysis showed that PRDX6 may be the target gene of miR-4284 in HUVECs.Mechanism studies showed that miR-4284 targeted part of PRDX6 induced HUVECs apoptosis through the Akt pathway.This study indicated that miR-4284 may be involved in the damage of vascular endothelial cells during the occurrence and progression of atherosclerosis,providing a new potential therapeutic strategy for the study and treatment for atherosclerosis.