MiR-126Regulates Endothelial Progenitor Cells Via Its Target PIK3R2and Promotes Deep Venous Thrombosis Recanalization

Objective: This research is to study of miR-126regulating endothelial progenitor cells（EPCs）and promoting venous thrombosis resolution,organization and recanalization.And these findings indicate a potential therapeutic intervention of deep venous thrombosis.Methods: Firstly, rat bone marrow-derived EPCs were isolated, cultivated andidentificated. Secondly,EPCs were transfected with control oligoes and miR-126mimicsor inhibitor by electroporation. Proliferation capacity and cell cycle were analyzed by MTTand flow cytometry.Cell migration analysis was done by wound healing and transwellassay.And tubulogenic activity test was performed by matrigel tube formation assay.Thirdly,by gain-of-function examination, its putative target genes were searched for usingonline search tool. PIK3R2was selected as the candidate target gene of rno-miR-126inEPCs.And further Luciferase reporters were constructed containing either a wild-typePIK3R23’UTR sequence (pMIR/PIK3R2/wt), or a mutated PIK3R23’UTR(pMIR/PIK3R2/mut). Luciferase activity was assessed by co-transfecting the luciferasereporter vectors with the miR-126mimics, inhibitor or NC. The luciferase activity ofreporter was observed. PIK3R2expression was detected by Western blot assays,and PIK3,Akt and p-Akt proteins in PI3K/AKT signal channel by Western blot assays,too.Fourthly,to stably express miR-126in EPCs, the lentiviral expression vectorpLVX-IRES-ZsGreen-miR-126was constructed.They was then transfected into293Tcells. The supernatant containing the lentivirus was harvested at72h. EPCs were infectedwith1ml lentivirus suspension. Green fluorescence was observed to indicate thetransduction efficiency at48h post transduction. miR-126expression in EPCs was detected by real-time quantitative PCR (qPCR).Forthermore, Experimental rat models ofdeep vein thrombosis were obtained by complete ligation of inferior vena cava below therenal veins. When deep venous thrombosis was formed after IVC ligation, the alive ratswere divided as3groups for cells transplantion via tail intravenous injection: A (n=12),blank control group (blank control), which received1ml PBS; B (n=12),EPCs/pLVX-IRES-ZsGreen Vector group (EPCs/vector), which received1.0×106EPCstransfected with lentivirus particle of pLVX-IRES-ZsGreen vector; C (n=12),EPCs/pLVX-IRES-ZsGreen-miR-126group (EPCs/miR-126), which received1.0×106EPCs transfected with lentivirus particle of pLVX-IRES-ZsGreen-miR-126. On the7th and14th day post operation, the rats were sacrificed and the thrombus segment of inferior venacava and thrombus were acquired,then were weighed. The homing of EPCs was shown byGFP expression and observed using fluorescence microscope. And the samples weretreated by dimethybenzene and embedded in paraffin. In order to observe thrombusorganization and recanalization, hematoxylin and eosin (HE) staining andimmunohistochemical staining for CD34and were performed. All statistical analyses wereperformed using SPSS15.0software. A two-tailed value of P <0.05was consideredstatistically significant.Results: Rat bone marrow-derived EPCs were isolated, cultivated and identificatedsuccessfully.After EPCs were transfected by electroporation successfully, proliferationcapacity of EPCs was enhanced and cell cycle was changed in early stage when transfectedwith miR-126mimics.The relative wound size of EPCs transfected with NC or miR-126oligonucleotides was analyzed at0,24,48and72h post wounding. Compared with NCmimics, miR-126mimics significantly enhanced the migration of EPCs across the woundspace at24,48and72h. In contrast, miR-126inhibitor significantly suppressed themigration of EPCs at the same time. Consistent with the wound healing assay, migrationchamber assay also showed that miR-126mimics significantly promoted while miR-126inhibitor significantly inhibited EPCs migration into the chamber membrane comparedwith control group. EPCs transfected with miR-126mimics showed a significant improvement of capillary tube formation compared with NC by matrigel tube formationassay. In contrast, miR-126inhibitor-infected cells showed a significant impairment ofcapillary tube formation compared with NC. After an extensive review of onlinemicroRNA database (that is, TargetScan, Microrna.org and miRanda), we selected PIK3R2as the candidate target gene of rno-miR-126in EPCs. Luciferase reporter assays firstperformed to verify a direct interaction between miR-126and the3’UTR of PIK3R2.Luciferase reporters were constructed containing either a wild-type PIK3R23’UTRsequence (pMIR/PIK3R2/wt), or a mutated PIK3R23’UTR (pMIR/PIK3R2/mut).Luciferase activity was assessed by co-transfecting the luciferase reporter vectors with themiR-126mimics, inhibitor or NC. Luciferase activity of pMIR/PIK3R2/wt was markedlydecreased in cells transfected with miR-126mimics, compared to luciferase activity ofpMIR/PIK3R2/mut. Conversely, the luciferase activity of reporter plasmid was notinterfered after transfection with miR-126inhibitor. miR-126mimics decreased thePIK3R2expression compared to NC mimics, while miR-126inhibitor increased PIK3R2protein levels. PI3K and phospho-Akt (p-Akt) levels were significantly enhanced in theEPCs transfected with miR-126mimics compared to negative control mimics. A reverseresult was observed when miR-126was knocked down in EPCs transfected with miR-126inhibitor compared to negative control inhibitor.Restriction enzyme digestion andsequencing results suggest that lentiviral vector pLVX-IRES-ZsGreenl-miR-126wasconstructed correctly.And EPCs were effectively transfected by them.Aftertransfection,the miR-126expression in EPCs was higher216times than the control group.On the7thand14thday after EPCs transplantation, Compared with control group, theweight of thrombus decreased in EPCs/vector and EPCs/miR-126groups. Furthermore, thethrombus weight of EPCs/miR-126group was even lower than that of EPCs/vectorgroup.EPCs homing were observed in section by using fluorescence microscope. Andtransplanted EPCs were recruited into the thrombus in both EPCs/vector andEPCs/miR-126group. In contrast, No GFP was observed in control group. We can alsofind that the larger number of GFP positive cells was appeared in EPCs/miR-126group compared with EPCs/vector group. HE staining indicated there were more degree oforganization in EPCs/vector and EPCs/miR-126group,even higher in EPCs/miR-126group. Compared with control group, more neovascularition were formed in EPCs/vectorand EPCs/miR-126group,and were maximum in EPCs/miR-126group.All werestatistically significant.Conclusions: MiR-126enhances the ability of proangiogenic properties of EPCs,including proliferation, migration and tubulogenic activity, by targeting PIK3R2directly.The mechanism of miR-126effecting on EPCs is through PI3K/Akt signal pathway. EPCsvia upregulation of miR-126expression can promote venous thrombosisresolution,organization and recanalization. Based on the results in this research,regulationof miR-126in EPCs may represent a potential therapeutic intervention in EPCs-mediatedtherapy for deep venous thrombosis.