| ObjectiveThe repair process of acute myocardial infarction(AMI)is closely associated with whether there is rapid formation of blood vessel or reopening of the occluded blood vessels in the infarcted zone.It is of great significance to rapidly establish a good collateral circulation following AMI to improve the blood supply and to promote the survival of myocardium in the infarcted zone.The study was designed to investigate the effect of microRNA-101(miR-101)on angiogenesis in AMI model as well as to explore the relevant regulatory mechanism.Method(1)The expression of mi R-101 in human umbilical vein endothelial cells(HUVECs)under hypoxia was detected by mi RNA qRT-PCR.(2)The effect of mi R-101 on proliferation,migration and angiogenesis of HUVECs was examined by Ed U assay,scratch assay,and tubule formation assay,respectively.(3)In addition,12 bioinformatics software,such as miRanda and miRMap were employed to predict the target genes of mi R-101 and the binding sites of 3’-untranslated region(3’-UTR)of eukaryotic translation initiation factor 4E3(EIF4E3).Dual luciferase reporter assay was used to detect the direct binding of miR-101 to EIF4E3 and the suppressive effect of miR-101 on EIF4E3 expression.(4)The expression of EIF4E3,EIF4E1,HIF-1α and VEGF-A was determined by Western blot after transfection of miR-101,agomi R-101,antagomiR-101,scrambled RNA and EIF4E3 siRNA in HUVECs.(5)Moreover,purified Adv-miR-101 interference and empty vector adenovirus were injected into C57BL/6 mouse AMI model,followed by detection of the proliferation of vascular endothelial cells(CD31/Ki67)and microvessel density(CD31/α-SMA)by immunofluorescence staining,as well as the assessment of changes in mouse heart function by M-mode ultrasound.Results(1)The expression of mi R-101 in HUVECs under CoCl2-induced hypoxia was increased within 5 days compared to that without hypoxia(p < 0.05).(2)Under hypoxia,the proliferation,migration and reticular formation of HUVECs were significantly enhanced in the agomi R-101 group,in comparison with those in the antagomiR-101 and scrambled RNA groups(p < 0.05).(3)Bioinformatics software prediction and dual luciferase reporter assays confirmed that miR-101 could target and bind the 3’-UTR of EIF4E3,thereby inhibiting the expression of EIF4E3(p < 0.05).After siRNA interference of EIF4E3 expression,the expression of HIF-1α and VEGF-A was significantly up-regulated than that in the control group,as indicated by Western blot(p < 0.05).(4)The growth of HUVECs was interfered with agomiR-101,antagomiR-101 and scrambled RNA,respectively.The expression of EIF4E3 in the agomimiR-101 group was significantly down-regulated compared to those in the antagomiR-101 and scrambled RNA groups,while the expression of HIF-1α and VEGF-A in the agomi R-101 group was significantly up-regulated than that in the scrambled RNA group(p < 0.05).(5)In addition,Adv-miR-101 interference was applied in the C57BL/6 mouse MI model,which could promote the proliferation of vascular endothelial cells and increase vessel density,subsequently improving heart function(p < 0.05).ConclusionMiR-101 could promote the expression of HIF-1α and VEGF-A,regulate the proliferation,migration and angiogenesis of endothelial cells by inhibiting the expression of EIF4E3,which might provide a novel approach for the treatment of ischemic heart disease. |
