Regulation Of MiR-146a On CD4+T Cells And Its Role In The Pathogenesis Of Thyroid Associated Ophthalmopathy

Background: Thyroid associated ophthalmopathy(TAO)is the most common eye disease,it is a kind of an autoimmune disease,exophthalmos,compressive optic neuropathy and other clinical manifestations caused by orbital immune and proliferative responses.In recent years,due to the change of life rhythm and social environment,Increasing incidence of thyroid associated ophthalmopathy.Because the pathogenesis of TAO is still not completely clear,there is no specific treatment,seriously affecting the patient’s work and life,and some patients due to TAO lead to blindness.It is an important research topic to explore the pathogenesis of TAO and to develop targeted therapy for TAO.At present,the research shows that the immune response of CD4+T cells plays an important role in the occurrence and development of TAO.MicroRNA(miRNA)plays an important role in the regulation of immune cell development and immune response.MiR-146 a inhibits the activation of T cells and negatively regulates the immune response.MiR-146 a regulates cell proliferation and differentiation in multiple life processes.It can be seen that miR-146 a has multiple potential targets for the immune and proliferative response of TAO.In recent years,we found that miR-146 a was low in TAO patients with orbital tissues and peripheral blood mononuclear cells(PBMC).However,the level of miR-146 a in CD4+T cells of TAO patients and its role is not clear.MiR-146 a has multiple target genes,it is a typical multi function miRNA,in the same pathway in cells under different conditions,and regulate their characteristic,participate in immune regulation,cell proliferation and differentiation,apoptosis and extracellular matrix metabolism process of life.MiR-146 a has multiple potential targets for the immune and proliferative response of TAO.MicroRNA(miRNA)is a single stranded small molecule non coding 21-25(RNA)in animals and plants.MiRNA can be combined with the 3’untranslated region of the target mRNA,the coding region or the 5’ untranslated region of the target gene,which prevents the translation process of gene transcription.Recent studies suggest that miRNA is involved in the regulation of various immune processes in humans and may serve as a biomarker or therapeutic target for the diagnosis of various diseases.The expression of miRNA has been found in a variety of autoimmune diseases,including thyroid associated ophthalmopathy,rheumatoid arthritis and multiple sclerosis.The role of miRNA in the pathogenesis of TAO needs further study.CD4+T cell involved Immune response is an important factor in thyroid associated ophthalmopathy The results showed that microRNA-146 a could inhibit the activation of T cells and negatively regulate the immune response,and participate in the process of cell proliferation and differentiation.We found that microRNA-146 a was low in TAO orbital tissue.However,the level of microRNA-146 a in CD4+T cells of patients with TAO,and their role and mechanism are not clear.We use PCR,Western blot,ELISA,RAN transfection,and method of mixed culture,clarify the molecular and cellular level: the abnormal expression of miR-146 a in CD4+T cells and its effect on the immune response to TAO,and to determine the target genes and the molecular mechanism of abnormal expression.This study will explore the mechanism of TAO from the new perspective of microRNA,and lay the foundation for the prevention and treatment of TAO.These studies revealed that the low expression of miR146 a may be an important molecular mechanism of TAO CD4+ T cells,and provide a theoretical basis for finding new targets for the treatment of TAO.Objective: to investigate the expression of miR-146 a in CD4+T cells of TAO patients.To validate NUMB is the target gene of miR-146 a by using report gene system.To investigate the effect of changing miR-146 a on the target gene and investigate expression level of miRNA-146 a target gene in TAO CD4+T cells.To investigate the effect of inhibiting miRNA-146 a expression on the immune response in normal or active TAO patients and investigate the signaling pathway of miRNA-146 a in CD4+T cells.To discuss the signaling pathway of miRNA-146 a in CD4+T cellsMethods:Peripheral blood CD4+T cells were isolated by density gradient centrifugation;Altered microRNA was confirmed by Real-time polymerase chain reaction(Real-time PCR).The use of bioinformatics software(micro RNA、Target Scan、 Mirbase)to Find potential target gene of miR-146 a.We further determined whether NUMB is the target gene of miR-146 a by double luciferase reporter experiment.Dual luciferase reporter gene system was used to detect the expression of miR-146 a gene.Luciferase reporter gene system is that the target gene 3’UTR region was cloned into the luciferase expression vector downstream of the luciferase gene sequence,so that it can express the fusion target gene 3’ UTR recombinant luciferase.The method of point mutation can cause the base mutation of the binding site of miRNA gene in the 3’UTR region of the target gene to construct mutant expression plasmid.The recombinant expression vector containing the target gene 3’UTR region and the mutant expression plasmid were transfected into miRNA cells,and miRNA and recombinant luciferase were expressed in the cells.We observe the difference of the level of miRNA between the wild-type and the mutant expression plasmid groups,and to verify the function of the target gene.Numb(WT-luciferase)and Numb(Mut-luciferase)and miR-146 amimic and Negative control were transiently transfected into 293 T cells using Lipofectamin2000;48 hours after transfection,cells were collected,we use dual luciferase reporter assay system and Renilla luciferase as control for detection of firefly luciferase activity.Peripheral blood CD4+T cells were isolated by density gradient centrifugation;Transfection:The miRNA-146 a mimic,miRNA-146 a inhibitor,BLANK or Negative control using INTERFERin non liposomal cationic amphoteric molecules transiently transfected into normal human CD4+T cells;The miRNA-146 a mimic,miRNA-146 a inhibitor,BLANK or Negative control using INTERFERin non liposomal cationic amphoteric molecules Transiently transfected into CD4+T cells with active TAO patients.Altered microRNA was confirmed by Real-time polymerase chain reaction(Real-time PCR).NUMB protein expression levels was detected by Western blot.Peripheral blood CD4+T cells and B cell were isolated by density gradient centrifugation;Transfection;ELISA was used to detect the expression of CD40L;CD4+T proliferation assay was detected to using the proliferation kit;ELISA method was used to detect the expression of TGF and IL10 cytokines;ELISA method was used to detect the concentration of IgG antibody.Transfection;Real time quantitative polymerase chain reaction(real-time PCR)detection of tumor necrosis factor receptor associated factor 6(TRAF6),interleukin-1 receptor associated kinase-1(IRAK1)mRNA expression level.The use of western blot detection of necrosis factor receptor associated factor 6(TRAF6)and interleukin-1 receptor associated kinase-1(IRAK1)protein expression level.Results: miR-146 a was significantly downregulated in CD4+T cells of patients with active TAO.The bioinformatics software(Microrna/ Targetscans/ Mirbase)was used to predict the potential target genes of miR-146 a,and the NUMB is a predicted target of miR-146 a.Transfection of miR-146 a mimics into 293 T cells can inhibit the expression NUMB WT-luciferase activity,but failed to inhibit NUMB MUTluciferase activity.The expression of miR-146 a in normal human CD4+T cells transfected with miR-146 a mimic was increased and the level of NUMB protein was decreased.The expression of miR-146 a in normal human CD4+T cells transfected with miR-146 a inhibitor was decreased and the level of NUMB protein was increased.The level of miR-146 a expression in CD4+T cells was increased and the level of NUMB protein was decreased in the active of TAO transfected with miRNA-146 a mimics.The expression of miR-146 a in CD4+T cells was decreased and the level of NUMB protein was increased in the active TAO transfected with miRNA-146 a inhibitor.Compared with the healthy control group,the expression of NUMB protein in CD4+T cells was increased in patients with active TAO.After transfection of miRNA-146 a mimic,the expression of CD40 L in normal human CD4+T cells was decreased,The activity of cell proliferation was decreased,the expression of TGF and IL10 decreased,and the level of IgG antibody decreased;After transfection of miRNA-146 a mimic;the expression of CD40 L was decreased in active TAO CD4+T cells,the proliferation activity of cells decreased,the expression of TGF and IL10 decreased,and the level of IgG antibody decreased;After transfection of miRNA-146 a inhibitor,the expression of CD40 L in normal human CD4+T cells was increased,The activity of cell proliferation was enhanced,the expression of TGF and IL10 increased,and the level of IgG antibody increased;After transfection of miRNA-146 a inhibitor;the expression of CD40 L was increased in active TAO CD4+T cells,the proliferation activity of cells increased,the expression of TGF and IL10 increased,and the level of IgG antibody increased;After transfection of miRNA-146 a mimics normal CD4+T cell tumor necrosis factor receptor associated factor 6(TRAF6)and interleukin-1 receptor associated kinase-1(IRAK1)had no significant difference in the expression level of mRNA,but the protein level decreased;After transfection of miRNA-146 a mimics,active TAO CD4+T cell tumor necrosis factor receptor associated factor 6(TRAF6)and interleukin-1 receptor associated kinase-1(IRAK1)had no significant difference in the expression level of mRNA,but the protein level decreased.After transfection of miRNA-146 a inhibitor normal CD4+T cell tumor necrosis factor receptor associated factor 6(TRAF6)and interleukin-1 receptor associated kinase-1(IRAK1)had no significant difference in the expression level of mRNA,but the protein level increased;After transfection of miRNA-146 a inhibitor,active TAO CD4+T cell tumor necrosis factor receptor associated factor 6(TRAF6)and interleukin-1 receptor associated kinase-1(IRAK1)had no significant difference in the expression level of mRNA,but the protein level increased.Conclusion: miR-146 a was significantly downregulated in active TAO CD4+T cells.NUMB is the target gene of miR-146 a,miR-146 a can repress mRNA translation of target gene by binding to the 3’-UTR.Overexpression of miRNA-146 a can decrease the expression level of NUMB protein in normal human CD4+T cells;inhibition of miRNA-146 a expression can increase the expression level of NUMB protein in normal human CD4+T cells;overexpression of miRNA-146 a can decrease the expression of NUMB protein in CD4+T cells of TAO patients in active TAO;inhibition of miRNA-146 a expression can increase the expression of NUMB protein in CD4+T cells in patients with active TAO;The expression of NUMB protein was higher in CD4+T cells of patients with active TAO compared to healthy control group.Overexpression of miRNA-146 a may play an inhibitory role in NF-KB signaling phathways in CD4+Tcells.Inhibiting of miRNA-146 a may play an active role in NF-KB signaling pathways in CD4+Tcells.