The Mechanism Of MiRNA-27b Targeting EPAS1 To Regulate Angiogenesis In Endothelial Progenitor Cells

Objective: Exploring the mechanism of miRNA-27 b targeting EPAS1 to regulate angiogenesis in endothelial progenitor cells,providing theoretical basis for the study of distraction osteogenesis mechanism.Methods: 1.Extracting the puppy bone marrow,the EPCs were isolated and cultured by density gradient centrifugation.Morphological observation and Dil-Fitc double fluorescence staining were carried out to identify EPCs.Then detecting the proliferation,migration,and angiogenesis abilityof EPCs.2.miRNA-27 b mimics,inhibitors and negative control lentivirus vectors were constructed,and EPCs were divided into 5 groups: The normal EPCs without transfection of lentivirus(CTRL group),the EPCs transfected with miRNA-27 b mimics lentivirus(mimicsgroup),the EPCstransfected with miRNA-27 bmimics negative control lentivirus(mimics NC group),the EPCs transfected with miRNA-27 b inhibitor lentivirus(inhibitorgroup),and the EPCs transfected with inhibitor negative control lentivirus(inhibitor NC group).The optimal MOI of lentivirus infecting EPCs was determined,and the overexpression and downregulation efficiency of miRNA-27 b gene was detected by qRT-PCR,establishing canine EPCs models of miRNA-27 b overexpression and downregulation.3.The effects of overexpression and downregulation of miRNA-27 b gene on the proliferation,migration and angiogenesis of EPCs were respectively detected by flow cell cycle,CCK-8,transwell membrane crossing assay and matrigel tubule formation assay.4.qRT-PCR and western blot were used to detect the expression of vasogenic factors VEGF-A and bFGF in CTRL group EPCs,mimics group EPCs,mimics NC group EPCs,inhibitor group EPCs,inhibitor NC group EPCs.5.The expression of AGGF1,EPAS1(HIF-2α)and BMPR2 in the five groups of EPCs were detected by qRT-PCR and western blot.6.Luciferase reporter vectors of wildtype BMPR2 3’UTR and mutant BMPR2 3’UTR were constructed to detect the regulatory effect of miRNA-27 b on BMPR2.7.Luciferase reporter vectors of wildtype EPAS1 3’UTR and mutant EPAS1 3’UTR were constructed to detect the regulatory effect of miRNA-27 b on EPAS1.Results: 1.Puppies bone marrow-derived endothelial progenitor cells were successfully cultured and identified.EPCs can engulf Dil-ac-LDL and bind FITC-UEA-1.2.The miRNA-27 b mimics,inhibitor and negative control lentivirus vector were constructed,and the lentivirus was transfected into the endothelial progenitor cells.After 72 hours,the fluorescence expression efficiency of EPCs was about 80% and the cells grew well.The miRNA-27 b gene overexpression and downregulation efficiency were detected by qRT-PCR.The expression of miRNA-27 b in mimics group EPCs was increased compared with CTRL group and mimics NC group EPCs.The expression of miRNA-27 b in inhibitor group EPCswas decreased compared with CTRL group and inhibitor NC group EPCs(P<0.05).3.Flow cytometry results showed that the proportion of cells at DNA replication and cell division phase(S/ G2-M phase)was decreased in the mimics group,while the proportion of cells at DNA replication and cell division phase was increased in the inhibitor group.The results of CCK-8experiment showed that the overexpression of miRNA-27 b could inhibit the proliferation capacity of EPCs,and the downregulation of miRNA-27 b could promote the proliferation capacity of EPCs.Transwell membrane penetration test confirmed that miRNA-27 b mimics weaken the migration ability of EPCs,while miRNA-27 b inhibitors enhanced the migration ability of EPCs.Matrigel tubule formation experiment showed that the overexpression of miRNA-27 b gene inhibited the lumen structure formation of EPCs in vitro,while the downregulation of miRNA-27 b gene promoted the lumen structureformation of EPCs.4.The expression of vascularization marker VEGF-A and bFGF in 5groups of EPCs were detected by qRT-PCR and western blot.The results showed that the expression levels of VEGF-A and bFGF decreased after the expression level of miRNA-27 b gene in EPCs was upregulated.After downregulating the expression level of miRNA-27 b gene in EPCs,the expression levels of VEGF-A and bFGF increased,and all of the above differences were statistically significant(P<0.05).5.The expressions of AGGF1,EPAS1 and BMPR2 in 5 groups of EPCs were detected by qRT-PCR and western blot.The results showed that the expression levels of AGGF1 in each group were not significantly different(P>0.05).After upregulating the expression of miRNA-27 b,the expression levels of EPAS1 and BMPR2 were decreased,and the expression levels of EPAS1 and BMPR2 were increased after the downregulation of miRNA-27 b.All of the above differences were statistically significant(P<0.05).6.Luciferase reporter vectors of wild-type BMPR2 3’UTR and mutant BMPR2 3 ’UTR were successfully constructed and identified.After co-transfection of miRNA-27 b mimics and wild-type BMPR2 3’UTR luciferase reporter vectors,the luciferase activity showed no differences campared to the NC group(P>0.05).Furthermore,co-transfection of miRNA-27 b mimics and mutant BMPR2 3’UTR luciferase reporter vectors also did not affect luciferase activity(P>0.05),indicating that there was no binding effect between miRNA-27 b and BMPR2 in this experiment.7.Luciferase reporter vectors of wild-type EPAS1 3’UTR and mutant EPAS1 3’UTR were successfully constructed and identified.After cotransfection of miRNA-27 b mimics and wild-type EPAS1 3’UTR luciferase reporter vectors,luciferase activity significantly decreased campared to the NC group(P<0.05).And co-transfection of miRNA-27 b mimics and mutant EPAS1 3’UTR luciferase reporter vectors did not affect luciferase activity(P>0.05),indicating that there is binding effect between miRNA-27 b and EPAS1,and miRNA-27 b can target on EPAS1.Conclusion: The EPCs cultured in vitro possess strong ability of proliferation,migration and neovascularization.The overexpression and downregulation of miRNA-27 b in EPCs by lentiviral plasmids suggesting that miRNA-27 b inhibits the angiogenesis of EPCs by negatively regulating its target gene EPAS1,and the mechanism may be related to the activation of the HIF/VEGF signaling pathway.