Role Of MiR-338-3p In Peripheral Immune Tolerance Of Pemphigus Vulgaris

BackgroundPemphigus vulgaris(PV)is a life-threatening and chronic mucocutaneous blistering autoimmune disease,characterized by the production of a pathogenic autoantibody against desmogleins-3(Dsg3)that causing loss of cohesion of the epidermal cells,called acantholysis.The imbalance of regulatory T cell(Treg)-mediated peripheral immune tolerance plays a vital role in the pathogenesis of PV.Treg cells suppress the overactivity of T-helper(Th)cells by expressing the signature transcription factor forkhead box P3(FOXP3),inducing immune tolerance to self-antigens.MicroRNAs(miRNAs)have frequently emerged as regulators in Treg-mediated immunosuppression in autoimmune diseases,but the role of miRNAs in the immune tolerance of PV remains to be elucidated.It has been reported that miR-338-3p was overexpressed in the peripheral blood mononuclear cells(PBMCs)of PV patients,and correlated with the production of anti-Dsg3 antibody.However,the specific role of miR-338-3p in Treg-mediated peripheral immune tolerance has not yet been explored.ObjectiveTo investigate the expression of miR-338-3p in CD4+T cells of PV patients and explore the role of miR-338-3p in Treg-mediated peripheral immune tolerance in PV.Methods1.The expression of miR-338-3p was evaluated in CD4+T cells of PV patients and healthy controls(HCs)by quantitative real-time polymerase chain reaction(qPCR).Correlation analysis between the level of miR-338-3p and disease severity was performed to explore the clinical role of miR-338-3p in PV.2.To obtain a comprehensive view of the functional and phenotypic properties of CD4+T subtypes in PV patients,we detected the expression of Treg/Th-related genes encoding transcriptional factors and cytokines in CD4+T cells of PV patients and HCs by qPCR.Additionally,we analyzed an mRNA-specific array of CD4+ T cells from PV patients and HCs,publishing in the National Center for Biotechnology Gene Expression Omnibus database(GEO).This array helped to gain further molecular insights into the mechanism of the Treg-associated immunological imbalance in PV,3.Prediction of miR-338-3p targets was performed using the TargetScan database.The predicted target genes were screened from the differentially regulated genes in CD4+ T cells of PV patients and HCs,followed by qPCR detection.Then,miRNA transfection,luciferase reporter assays,and flow cytometry were used to explore the role of miR-338-3p in Treg-mediated peripheral immune tolerance of PV.Results1.The expression of miR-338-3p was increased in CD4+T cells of active PV patients compared with those in HCs,which was positively related to the level of anti-Dsg3 antibody and the Pemphigus Disease Area Index(PDAI)score.Patients were treated with glucocorticoids for 2 weeks,and we found that the level of miR-338-3p decreased significantly in the effective treatment group while remained stable in the ineffective treatment group.2.The mRNA level of FOXP3 was decreased while the mRNA level of RORC was increased in active PV patients,suggesting impairment of Treg-mediated immunosuppression and activation of Thl7-mediated inflammation.Bioinformatics analysis of the mRNA-specific array showed that the differentially expressed genes between PV patients and HCs were largely related to immune responses,including Runt-related transcription factor 1(RUNX1)encoding an essential activator for FOXP3 expression.3.Bioinformatics prediction revealed that RUNX1 was a putative target of miR-338-3p.The mRNA level of RUNX1 was significantly decreased in CD4+T cells of PV patients,which negatively correlated with the level of miR-338-3p and positively correlated with the level of FOXP3.Luciferase reporter assays demonstrated that miR-338-3p was the target of miR-338-3p.Moreover,miRNA transfection verified that miR-338-3p attenuated the expression of RUNX1 and FOXP3,contributing to the impairment of Treg-mediated immunosuppression.Conclusion1.The expression of miR-338-3p increases in CD4+T cells of active PV patients,positively correlating with disease severity.This finding indicates that miR-338-3p is a sensitive biomarker for PV diagnosis as well as for therapeutic efficacy monitoring.2.Treg-mediated immunosuppression is supressed in PV,indicating the impairment of peripheral immune tolerance.3.Excessive expression of miR-338-3p attenuates the expression of FOXP3 by targeting RUNX1 in CD4+ T cells,leading to the impairment of Treg-mediated immunosuppression.Our findings suggest that miR-338-3p is a potential therapeutic target for repairing Treg dysfunction in PV.