Effect of EBV miR-BART17-3p on Treg/Th17 Balance in Children with Primary Immune Thrombocytopenia
Objective To investigate the mechanism of EBV miR-BART17-3p affecting the Treg/Th17 balance in children with primary immune thrombocytopenia(ITP).Methods The peripheral blood from ITP children(ITP group,20 cases)and healthy children(control group,20 cases)was collected and the CD4+T cells were isolated.The real-time fluorescence quantitative polymerase chain reaction,Western blot,and enzyme-linked immunosorbent assay were used to detect the mRNA,protein expression levels and contents of EBV miR-BART17-3p,T cell immunoglobulin domain and mucin domain-3(Tim-3),forkhead box protein P3(FoxP3),interleukin-17A(IL-17A),and transforming growth factor-β(TGF-β).The dual-luciferase reporter gene assay was used to explore the effect of EBV miR-BART17-3p on the expression level of Tim-3.Fifteen BALB/C mice were randomly divided into the blank control group,the model group and the observation group,with five mice in each group.Mice were intraperitoneally injected with antiplatelet antibody MWReg30 to construct the ITP models.After 4 d of modeling,the mice in the observation group were injected with adenovirus vector carrying EBV miR-BART17-3p inhibitor via the tail vein.Cells were stained for morphological observation;the TGF-β,IL-17A contents and platelet count in peripheral blood were detected;the flow cytometry was used to measure the Th17 and Treg levels in CD4+T cells,and their percentage was calculated.Results Compared with those in the control group,the expression level of EBV miR-BART17-3p in peripheral blood in the ITP group significantly increased,while the expression levels of Tim-3 and TGF-β mRNA significantly decreased(P<0.05);the expression level of Tim-3 mRNA was significantly negatively correlated with the expression level of EBV miR-BART17-3p(r=-0.732,P<0.001).Tim-3 lentivirus vector carrying pLKO.1-sh-Tim-3(sh-Tim-3)could significantly decrease the Tim-3,FoxP3 and TGF-β levels(P<0.05).The miR-BART17-3p mimic significantly increased the expression levels of miR-BART17-3p in CD4+T cells,and significantly decreased the mRNA and protein expression levels of Tim-3,FoxP3,and TGF-β(P<0.05);the miR-BART17-3p mimic could significantly decrease the TGF-β content,and the overexpression of Tim-3+miR-BART17-3p reversed the inhibitory effect of miR-BART17-3p mimic on the TGF-β.The animal experiment showed that silencing EBV miR-BART17-3p could promote the Treg differentiation and decrease the megakaryocyte count in spleen and bone marrow tissues,and significantly increase the platelet count in peripheral blood.Conclusion EBV miR-BART17-3p can regulate the immune imbalance of Treg/Th17 in ITP children through the FoxP3/Tim-3 pathway.
EBV miR-BART17-3pT cell immunoglobulin domain and mucin domain-3forkhead box protein P3helper T cell 17primary immune thrombocytopenia
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维普
