The Effect Of MiR-199a-5p On Th17 Cells And Its Relationship With The Immune Thrombocytopenia

Background and Objectives:Immune thrombocytopenia is one of the common autoimmune diseases in children.The main therapeutic drugs and methods are immunoglobulin,glucocorticoid,thrombopoietin,immunosuppressive agents,splenectomy and so on.The pathogenesis is not clear.A large number of studies have shown that the proportion of helper T cells(Th17)and the concentration of plasma interleukin-17(IL-17)were increased in peripheral blood of ITP patients.Th17 cells could lead to inflammatory response and play an important role in the occurrence and development of autoimmune diseases through IL-17 and other cytokines.Signal transducers and activators of transcription 3(STAT3)has major influence on the differentiation of Th17 cells.Meanwhile,many studies have shown that STAT3 could have effects of regulation in autoimmune diseases such as ITP.MicroRNA(miRNA)is a type of non-coding RNAs.Studies have shown that the expression of miR-199a-5p was abnormal in peripheral blood of ITP patients,which was suggesting that miR-199a-5p might play an important effect in the pathophysiology of ITP.But the mechanism of action on miR-199a-5p is not yet clear in the occurrence of ITP.By detecting the expressions of miR-199a-5p and Th17 in the ITP mouses model and the over-expressed miR-199a-5p cell models,we preliminarily investigate that whether miR-199a-5p is involved in the occurrence of ITP by affecting the differentiation of Th17 cells.We examined the expression of miR-199a-5p and Th17 in ITP mouse models and over-expressed miR-199a-5p cell models to investigate whether miR-199a-5p participates in the occurrence of ITP by affecting Th17 cell differentiation.Methods:1.The anti-mouse CD41 antibody was used to establish a kind of ITP mouse model by increasing the dose of monoclonal antibods,and the equal volume of PBS was used to establish the control group of mice.To detect the proportion of Th17 cells in spleen mononuclear cells by flow cytometry,serum level of IL-17 by ELISA,the expression of STAT3 in spleen mononuclear cells by Western blot,and the expression of miR-199a-5p in spleen mononuclear cells by real-time quantitative PCR from each group.2.MiR-199a-5p agomir was used to transfect the splenic mononuclear cells of healthy mice to establish a model of overexpressing miRNA-199a-5p mononuclear cells,while agomir-NC(negative control)was also used to transfect the splenic mononuclear cells of healthy mice to establish a control group cells.CD4+T cells were separated by magnetic beads and flow cytometry,and induced to differentiate into Th17 cells by in vitro culture.To detect the proportion of Th17 cells,the expression of STAT3,the expression of miR-199a-5p,the level of IL-17 in cell supernatant by using the above methods.Results:1.Compared with the control group mice,the proportion of Th17 cells and the expression of STAT3 in spleen mononuclear cells and the level of IL-17 of ITP mice were all increased,while the expression of miR-199a-5p in spleen mononuclear cells was decreased.Compared with CD4+T cells in the agomir-NC group,the proportion of Th17 in CD4+T cells and the level of IL-17 in cell supernatant were decreased in over-expressed miR-199a-5p cells,while the expression of STAT3 in CD4+T cells was decreased.Conclusions:1.ITP mouse model and over-expressed miR-199a-5p mononuclear cell model were successfully established.2.MiR-199a-5p can participate in the occurrence of mice’s ITP by negatively regulating the differentiation of Th 17 cells.