Effect Of Mir-150 On Differentiation Of Endothelial Progenitor Cells And Potential Mechanism

Deep vein thrombosis(DVT)is one of the most common peripheral vascular diseases.DVT is caused by vascular endothelial injury,hemodynamic changes and abnormal hypercoagulability.In the acute phase,there is a risk of pulmonary embolism(PE)if the lower extremity thrombus falls off,seriously threatens the life and health of the patient owing to high mortality.If the thrombus cannot be completely removed in a short period of time,thrombus organization will happen which induced post-thrombotic syndrome(PTS)in long term.PTS is characterized by painful swelling of the lower extremities,skin pigmentation,paresthesia,and severe lower extremity skin.Clinical manifestations such as repeated ulcers,seriously affect the quality of life of patients.At present,the commonly used treatment methods include simple anticoagulant therapy,surgical thrombectomy,and minimally invasive interventional therapy.Although these treatments have a good effect on acute thrombosis,they have limited therapeutic effects on chronic thrombosis and endothelial injury caused by thrombosis.Stem cells are one of the research hotspots of current ischemic diseases.Endothelial progenitor cells(EPCs),as precursors of endothelial cells,have the potential to differentiate into mature endothelial cells,which play an important role in neovascularization and endothelium repair process.Previous studies have shown that endothelial progenitor cells can migrate to the thrombus site after deep vein thrombosis,and participate in angiogenesis.However,because of the scarity of endothelial progenitor cells in peripheral circulation and less proliferative capability of endothelial progenitor cells,the clinical application of endothelial progenitor cells is limited.Therefore,recent studies focused on the feasibility for increasing the content of endothelial progenitor cells in the circulation and enhancement the biological function of endothelial progenitor cells.In addition,endothelial progenitor cells can be divided into early endothelial progenitor cells(eEPCs)and late endothelial progenitor cells(EPCs)according to their appearance time,cell morphology and surface markers.These two different cells have different stages of differentiation,proliferation,angiogenesis and paracrine ability.Therefore,early endothelial progenitor cells and late endothelial progenitor cells play different roles in the process of thrombolysis and recanalization,however,the specific role of the these two is still not very clear.Non-coding RNA(ncRNA)refers to various RNA molecules that are not translated into proteins.These RNAs can be transcribed from the genome,but without the translation process.Therefore,they can perform their functions at the transcriptional level.Non-coding RNAs mainly include rRNA,tRNA,snRNA,snoRNA,and microRNA.MicroRNAs(miRNAs)are small RNAs of about 20-24 nucleotides in length encoded by endogenous genes,which play important role in regulation of cell function.Current research indicates that approximately 30%of genes in mammals are regulated by miRNAs.miRNAs play a vital role in cell differentiation,biological development and disease development.Our previous studies also found that some miRNAs can be involved in the regulation of biological function of endothelial progenitor cells.Therefore,in this experiment,we focused on the effects of miRNA-150 on the differentiation process of endothelial progenitor cells and its effect on biological functions.We also further explored the mechanism of recanalization of thrombus underwent progenitor cell transplantation.This study enriches the theoretical basis of endothelial progenitor cell function regulation and transplantation for thrombosis,and provides an important theoretical basis for cell therapy research of deep vein thrombosis as well as other ischemic diseases.This study is mainly divided into the following five parts.Part I:Culture and identification of early and late endothelial progenitor cells from human peripheral bloodObjective:To verify the isolation,culture and identification of early and late endothelial progenitor cells from adult peripheral blood,and to provide higher purity experimental tool cells for subsequent in vivo and in vitro experiments.Methods:A total of 60 ml peripheral blood was extracted from healthy adults.Peripheral blood mononuclear cells(PBMCs)were isolated from adult peripheral blood by density gradient centrifugation.Endothelial progenitor cells were induced by differential adherence method.Obtained cells were further identified by morphology,flow cytometry,and Dil-Ac-LDL and FITC-UEA-1 double fluorescent staining.Results:Mononuclear cells obtained by density gradient centrifugation were cultured for 1 day.A small number of cells were attached to the cells,which showed oval,round,and different sizes.The adherent cells were then cultured for 5-7 days and colony formation was observed under the microscope.The cells in the center are round,and the surrounding cells are long fusiform or irregular.After 14-21 days,the cells will be covered with the culture plate.Cell colonies will disappear and form a typical cobblestone-like appearance.Double fluorescent staining of Dil-ac-LDL with FITC-UEA-1 showed that cells could phagocytose Dil-ac-LDL and bind FITC-UEA-1.Flow cytometry detection of cell surface markers revealed that cultured cells can express CD34,VEGFR2,CD 133,CD31,CD45,CD14 and vWF.Conclusion:Endothelial progenitor cells can be obtained by density gradient centrifugation combined with differential adherence method from human peripheral blood.The obtained cells conform to the morphological characteristics of EPCs as well as Dil-Ac-LDL and FITC-UEA-1 double fluorescent staining and flow cytometry.The test further confirmed that the obtained cells have high purity and can meet the requirements of subsequent experiments.Part ? Effect of miR-150 on differentiation and biological function of endothelial progenitor cellsObjective:To investigate the effect of miR-150 on differentiation and biological function of endothelial progenitor cells.Methods:1.The endothelial progenitor cells in different culture stages were collected separately,and the expression levels of miR-150 in early endothelial progenitor cells and late endothelial progenitor cells were monitored by real-time fluorescence quantitative polymerase chain reaction.2.Regulate the expression of miR-150 in early endothelial progenitor cells and late endothelial progenitor cells,respectively,with transfection of miR-150 mimic(miR-150 agomir)or miR-150 inhibitor(miR-150 antagomir).The expression levels of miR-150 of transfected cells were detected again.3.The proliferation,migration and angiogenic ability of endothelial progenitor cells are detected after transfection.Results:1.miR-150 expression levels in early endothelial progenitor cells and late endothelial progenitor cells are different.Compared with early endothelial progenitor cells,the expression level of miR-150 is significantly increased in late endothelial progenitor cells.2.In vitro and in vitro angiogenesis experiments show that angiogenic ability is significantly increased in early endothelial progenitor cells overexpressed with miR-150,while the angiogenic ability is decreased in late endothelial progenitor cells downregulated with miR-150.3.The proliferation experiment also shows proliferative ability is significantly increased in early endothelial progenitor cells overexpressed with miR-150,while the proliferative ability is decreased in late endothelial progenitor cells downregulated with miR-150.4.IL-8 and VEGF secretion decreased after overexpression of miR-150 in early endothelial progenitor cells.Conclusions:1.The expression level of miR-150 is increased in different stages of endothelial progenitor cell differentiation.2.The expression level of miR-150 in endothelial progenitor cells can promote angiogenic ability;3.miR-150 expression in endothelial progenitor cells are associated with increased proliferative capacity.4.Elevated levels of miR-150 in endothelial progenitor cells inhibit the secretion of VEGF and IL-8 in early endothelial progenitor cells.Part ? Mechanism of miR-150 regulating differentiation of endothelial progenitor cellsObjective:To study the specific mechanism of miR-150 in regulating the differentiation of endothelial progenitor cells.Methods:1.Regulate the expression of miR-150 in early endothelial progenitor cells and late endothelial progenitor cells with miR-150 agomir or miR-150 antagomir.The total protein in different groups of cells was extracted and pSer473 Akt,total Akt,pSer256 FOXO1 and total FOXO1 in different groups was detected by western blot 2.Wortmannin,as Akt signaling pathway blocker,was added into early endothelial progenitor cells and flow cytometry was used to detect the changes of surface progenitor cells.3.In vitro angiogenesis assay was used to detect the changes of angiogenic ability of early endothelial progenitor cells treated with wortmannin.4.CCK-8 assay was used to detect early endothelial progenitor cells treated with wortmannin.5.The ELISA assay was used to detect the change of paracrine ability of early endothelial progenitor cells treated with wortmannin.Results:1.Western blot results showed that the expression level of Akt in late endothelial progenitor cells was higher than that in early endothelial progenitor cells,and the expression level of pSer473 Akt was increased after the overexpression of miR-150 in early endothelial progenitor cells.The phosphorylation level of Akt pathway was inhibited when treated with wortmannin.In addition,the expression level of FOXO1 in late endothelial progenitor cells was higher than that in early endothelial progenitor cells,and the phosphorylation level of FOXO1 was increased after overexpression of miR-150 in early endothelial progenitor cells.Conversely,the phosphorylation level of FOXO1 was decreased after inhibiton of Akt pathway.2.Flow cytometry showed that the surface markers were changed after adding wortmannin to early endothelial progenitor cells.3.In vitro angiogenesis experiments showed that blocking the Akt pathway can reduce the angiogenic ability of endothelial progenitor cells and CCK-8 showed that wortmannin can offset the proliferative effect of miR-150 on early endothelial progenitor cells.5.Paracrine experiments show that wortmannin can counteract the inhibitory effect of miR-150 on the paracrine ability of early endothelial progenitor cells.Conclusion:1.Akt/FOXO1 signaling pathway is involved in the regulation of differentiation of endothelial progenitor cells 2.Blocking Akt/FOXO1 signaling pathway can inhibit the biological regulation of miR-150 on early endothelial progenitor cells.Part IV Prediction and validation of target gene of miR-150Objective:To predict and validate possible target genes for miR-150 in regulating endothelial progenitor cell differentiation.Methods:1.Bioinformatics methods was used to predict possible target genes of miR-150.2.Western blot was used to detect protein expression levels of target genes in early and late endothelial progenitor cells.3.Luciferase reporter gene assay,qPCR and western blot were performed to verify target gene of miR-150.4.Western blot was further performed to analyze the effect of inhibition of miR-150 target gene on Akt/FOXO1 signaling pathway in endothelial progenitor cells.Results:1.Bioinformatics predicts that c-Myb is a possible target gene of miR-150.2.Western blot results show that c-Myb expression in early endothelial progenitor cells is higher than that in late endothelial progenitor cells.3.Luciferase reporter assay shows miR-150 can reduce the fluorescence intensity by binding to the plasmid containing c-Myb 3'UTR region.qPCR and western blot results reveal increased expression of miR-150 could decrease expression of c-Myb.4.Western blot results further show that c-Myb is involved in the regulation of Akt/FOXO1 signaling pathway in endothelial progenitor cells.Conclusion:1.c-Myb is the target gene of miR-150.2.c-Myb is involved in the regulation of Akt/FOXO1 signaling pathway in endothelial progenitor cells.Part V Effect of early endothelial progenitor cells and late endothelial progenitor cells on venous thrombolysis recanalizationObjective:To study the effect of endothelial progenitor cells on the recanalization of venous thrombosis in different stages of differentiation.Methods:1.Animal models of deep vein thrombosis were established in nude rats and divide into five groups:group A(n=10),blank control group,1 ml PBS was injected through the tail vein;group B(n=10),eEPCs group,1 ml of cell suspension containing 1×106 early endothelial progenitor cells was injected via tail vein;group C(n=10),eEPCs/miR-150-+lEPCs group,1 ml mixed cell suspension with 1×106 late endothelial progenitor cells and early progenitor cells with up-regulated miR-150 was injected via tail vein;group D(n=10),eEPCs/NC+lEPCs group,1 ml mixed cell suspension with 1×106 late endothelial progenitor cells and early endothelial progenitor cells was injected through the tail vein;Group E(n=10),lEPCs group,1 ml cell suspension of late endothelial progenitor cells was injected through the tail vein.2.Specimens were collected 7 days after cell transplantation.Inflammatory cell aggregation and neovascularization were detected by HE staining.Thrombus weight was detected in each group and microvascular formation was detected by immunohistochemical staining.Finally,venous thrombolysis was determined by digital subtraction angiography.Results:1.HE staining results show that animals in eEPCs/miR-150+lEPCs group have the most newborn blood vessels,which indicates the best dissolution and recanalization.2.The weight of thrombus in each cell transplantation group was reduced compared to that in blank control group,among which eEPCs/miR-150+lEPCs group had the lightest weight.3.Immunohistochemical staining show the highest intensity of CD34 in eEPCs/miR-150+lEPCs group.4.Digital subtraction angiography results show that compared with the blank control group,blood flow was restored in each cell transplantation group.The eEPCs/miR-150+lEPCs group had the most blood flow recovery.Conclusion:1.Endothelial progenitor cells in different stages of differentiation can promote the dissolution and recanalization of venous thrombosis;2.Mixed transplantation of early endothelial progenitor cells with overexpression of miR-150 and late endothelial progenitor cells could achieve the best thrombolysis and recanalization.