| Part1 Expression of miR-155 carried by microparticles in aGVHD after allo-HSCT and their relationship with clinical significanceAim:To investigate the expression levels of microRNA-155(miR-155)carried by microparticles(MPs)in acute graft-versus-host disease(aGVHD)patients and assess their relationship with clinical significance.Methods:We firstly compared dynamic changes of miR-155 in MPs and plasma from peripheral blood of 28 aGVHD patients and 30 non-aGVHD patients(pre-HSCT,+7d,+14d,+21 d,+28d and aGVHD point)using quantitative real-time polymerase chain reaction(qRT-PCR).T cell separation kit was used to isolate T cells from the peripheral blood of aGVHD and non-aGVHD patients at the above time points and qRT-PCR was used to detect the expression levels of miR-155 in T cells.The expression differences of miR-155 in MPs,plasma and T cells in two groups at different time points were compared.The expression differences of miR-155 in MPs,plasma and T cells in aGVHD patients at different time points were compared.Results:Compared with non-aGVHD patients,the miR-155 expressions in MPs and plasma from the peripheral blood of aGVHD patients were significantly elevated at+7d,+14d,+21d and +28d,and the elevation of miR-155 in MPs was more obvious.For aGVHD patients,the miR-155 expressions of MPs from peripheral blood on+7d,+14d,+21d,+28d and aGVHD point were significantly higher than that of pre-HSCT,and the miR-155 expressions of plasma on +7d,+28d and aGVHD point were also significantly higher than that of pre-HSCT.The expression differences of miR-155 in T cells between non-aGVHD and aGVHD patients were not remarkable pre-HSCT,at +7d,+14d,+21d and +28d,and the miR-155 level in T cells at aGVHD point from aGVHD patients was remarkably higher than pre-HSCT level.Conclusion:Our data indicated that the miR-155 levels of MPs in the peripheral blood could forecast the occurrence of aGVHD before the aGVHD symptom,and the miR-155 in MPs could be considered as the specific biological indicator for diagnosing and predicting aGVHD.Part 2 In vitro study of the effect of endothelial microparticles-delivery miR-155 on T cells function and correlated mechanismsAim:To explore the effects of microRNA-155(miR-155)carried by endothelial microparticles(EMPs)on the function of T cells and correlated mechanisms in in vitro study.Methods:Quantitative real-time polymerase chain reaction(qRT-PCR)analysis was used to compare the expressions of miR-155 in EMPs derived from TNF-α-stimulated human umbilical vein endothelial cells(HUVECs)line EA.hy926 cells and untreated EA.hy926 cells.Confocal laser-scanning microscopy was used to observe the fusion of EMPs delivering miR-155(double fluorescently-labeled EMPs)with T cells.Endothelial cell protector simvastatin was pre-treated to EA.hy926 cells and antagomir-155 was designed and transfected into EA.hy 926 cells to down-regulate the expression of miR-155.Then TNF-a was used to stimulate EA.hy926 cells to produce EMPs.The experiments were divided into control group,EMPs group,simvastatin-EMPs group,antagomir-155-EMPs group,antagomir-NC-EMPs group and five kinds of EMPs were added into T cells for 72 hours.qRT-PCR was used to detect the expressions of miR-155 in T cells.CFSE was used to detect the proliferation of T cells.Annexin V-FITC/PI flow cytometry analysis was used to detect the apoptosis event of T cells and western blot was used to test the expressions of apoptosis-related proteins including Bcl-2,Bax and Caspase-3.The concentrations of cytokines in the T cells supernatant were detected using enzyme-linked immunosorbent assay(ELISA),and flow cytometry analysis was used to test the differentiation of T cells subpopulations.Results:①The miR-155 expressions in EMPs were significantly higher than those in EA.hy926 cells.② Confocal laser-scanning microscopy revealed that double fluorescently-labeled EMPs could enter into the cytoplasm of T cells.③Simvastatin could significantly reduce the levels of EMPs derived from TNF-a-stimulated EA.hy926 cells,and subsequently change the expressions of miR-155 in T cells.④Inhibition of miR-155 in EMPs could remarkably reduce the expressions of miR-155 in T cells.⑤Suppression of miR-155 in EMPs did not influence the proliferation of T cells.⑥Suppression of miR-155 in EMPs did not affect the apoptosis and the expressions of apoptosis-related proteins including Bcl-2,Bax and Caspase-3.⑦Inhibition of miR-155 in EMPs could suppress the differentiation of T cells toward Thl,Th9 and Thl7 cells,while facilitate the differentiation toward Th2 and Treg cells。Conclusion:Our data suggested that EMPs-delivery miR-155 could enter into the cytoplasm of T cells.The levels of miR-155 in EMPs did not influence the proliferation and apoptosis of T cells.EMPs-delivery miR-155 could promote the differentiation of T cells toward Thl,Th9 and Thl7 cells,but suppress the differentiation to Th2 and Treg cells.Part 3 In vivo study of the effect of endothelial microparticles-delivery miR-155 in aGVHD and correlated mechanismsAim:To explore the effects of microRNA-155(miR-155)carried by endothelial microparticles(EMPs)in acute graft-versus-host disease(aGVHD)and correlated mechanisms in in vivo study.Methods:The aGVHD mouse model of allogeneic hematopoietic stem cell transplantation(allo-HSCT)was established,and flow cytometry analysis was used to detect the dynamic changes of EMPs in peripheral blood from BM mice and aGVHD mice at different time points(pre-HSCT,+4d,+8d,+12d,+16d and aGVHD point).Quantitative real-time polymerase chain reaction(qRT-PCR)was used to detect the miR-155 expressions in MPs,plasma and T cells isolated from peripheral blood of BM and aGVHD mice at the above time points.Then the experiments were divided into eight groups:①BM group:the recipient BALB/c mice received total body irradiation(TBI)was administered with bone marrow cells isolated from C57BL6 donors,② aGVHD group:the recipient BALB/c mice received TBI was administered with bone marrow cells and spleen cells isolated from C57BL6 donors,③ EMPs group:the aGVHD mice were administered with EMPs derived from TNF-a-stimulated mouse aortic endothelial cells(MAECs),④simvastatin-EMPs:the aGVHD mice were administered with EMPs derived from TNF-a-stimulated MAECs previously protected by 2.5 μmol/L simvastatin,⑤antagomir-155-EMPs group:aGVHD mice were treated with EMPs derived from TNF-α-stimulated MAECs previously transfected with antagomir-155,⑥ antagomir-NC-EMPs:aGVHD mice were treated with EMPs derived from TNF-a-stimulated MAECs previously transfected with antagomir-NC,⑦ antagomir-155:aGVHD mice were treated with antagomir-155,⑧ antagomir-NC group:aGVHD mice were treated with antagomir-NC.Antagomir-155 or antagomir-NC was given at +7d with a dose of 25mg/kg,followed by 5mg/kg intravenously twice weekly up to +21d.All kinds of EMPs(60μg/d)were administered to mice intravenously on dayO and +7d.Survival rates of mice were calculated and clinical signs of aGVHD were determined in each group.Sections of organs were stained with hematoxylin&eosin and the histopathology changes in each group were observed.The concentrations of Thl(IFN-γ,TNF-α),Th2(IL-4,IL-6,IL-10),Th9(IL-9)and Th17(IL-17A)related cytokines were quantified in the mouse serum by enzyme-linked immunosorbent assay(ELISA).Flow cytometry analysis was used to test the differentiation of T cells subpopulations in the mice peripheral blood of each group.Results:①Flow cytometry analysis demonstrated that EMPs from peripheral blood of aGVHD mice were signifcantly elevated at +8d,+12d,+16d and slightly decreased at aGVHD point compared with BM mice.②The miR-155 levels in MPs from peripheral blood of aGVHD mice were signifcantly higher on +4d,+8d,+12d,+16d and aGVHD point compared with BM mice.As for aGVHD mice,the expression of miR-155 in MPs isolated from peripheral blood of aGVHD mice was remarkably higher than that in plasma.③The expression of miR-155 in T cells isolated from peripheral blood of aGVHD mice was significantly higher than BM mice at +16d and aGVHD point.Moreover,the rising peak of miR-155 expression in MPs from peripheral blood was remarkably earlier than that in T cells.④ Systemic EMPs administration via tail-vein caused more aggressive aGVHD with significantly shorter survival,enhanced mortality and higher histological injury compared with aGVHD group.Suppression of miR-155 in EMPs could reverse the above abnormal appearances.The administration of EMPs derived from TNF-a-stimulated MAECs previously protected by simvastatin via tail-vein caused more ameliorative aGVHD with significantly longer survival,lower mortality and improved histological injury compared with aGVHD group.⑤The characteristic cytokines produced by Th1,Th9 and Th17 cells were elevated in the serum from high concentration of EMPs-treated aGVHD group.The proportions of CD4+/CD8+T cells and Th17 cells were remarkably elevated and the ratio of Treg was reduced.Administration of miR-155-deficient EMPs could reverse the abnormal appearances of cytokines and T cell subpopulations.Conclusion:The in vivo study indicated that the expression of miR-155 carried by MPs could serve as an early predictive index as well as an early intervention target of aGVHD.EMPs-delivery miR-155 could promote the differentiation of T cells toward Thl,Th9 and Th17 cells,but suppress the differentiation to Th2 and Treg cells,which subsequently participate in and promote aGVHD. |
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