Role And Mechanism Of Transcription Factor YY1 Involved In Differentiation Of Pathogenic Th17 Cells In Rheumatoid Arthritis

【Objective】To investigate the role of transcription factor Yin Yang1(YY1)involved in differentiation of pathogenic Th17(p Th17)cells in rheumatoid arthritis(RA),verify the regulatory mechanism of YY1 in RA,while clarify the diagnostic value of YY1 in RA.The purpose of our study was to elucidate the mechanism of action of YY1 in RA and provide the experimental and theoretical basis for RA pathogenesis.【Methods】1.Investigation of the role of YY1 in promoting p Th17 cell differentiation.Clinical information and the peripheral blood samples were collected from 46 RA patients from the Rheumatology and Hematology Department of the First Affiliated Hospital of Fujian Medical University.Thirty-six osteoarthritis(OA)patients and 46healthy people from the same period was collected from the Orthopaedics clinic and the Physical Examination Center of the First Affiliated Hospital of Fujian Medical University to use as the controls.Peripheral blood mononuclear cells(PBMCs)were obtained from subjects described above.Flow cytometry was applied to measure the number and proportion of p Th17 cells,and total RNA was isolated and quantitative real-time PCR(q PCR)was performed to determine the gene expression of YY1 and Th17-associated genes IL17A,IL-22.Subsequently,ex vivo p Th17 cell induced differentiation system was established.The YY1 lentiviral interference vector(LV-YY1-sh RNA)was performed in ex vivo p Th17 cell induced differentiation system to knockdown YY1.Then,western blot was performed to verify the expression level of YY1 in ex vivo p Th17 cell induced differentiation system and flow cytometry was conducted to assess the alterations of the number and proportion of p Th17 cells after YY1 knockdown.In the experiments in vivo,six-to eight-week-old male DBA/1J mice were used to induce the collagen-induced arthritis(CIA)model,which injected via the tail vein with lentivirus containing mouse YY1 sh RNA(LV-YY1-sh RNA 907,LV-YY1-sh RNA 1009)or empty lentiviral vectors used as control(LV-NC).Flow cytometry was performed to measure the number and proportion of p Th17 cells,HE staining was used to detect the histopathologic changes of the diseased joints,while the levels of joint inflammation were assessed using inflammation scoring system.2.Exploring the mechanism of YY1 in promoting p Th17 cell differentiation in RA.YY1 knockdown was applied in ex vivo p Th17 cell induced differentiation system.The gene expression levels of the p Th17 cell differentiation-associated key transcription factors were measured using m RNA transcriptome sequencing technology(m RNA-seq)and q PCR as well as the protein expression levels of that were verified by western blot in ex vivo p Th17 cell induced differentiation system.Moreover,the effects of T-bet overexpression were assessed in YY1 knockdown ex vivo p Th17 cell induced differentiation system.Next,online bioinformatics predictive tool JASPAR and PROMO were used for prediction of potential binding sites between YY1 with T-bet promoter region,followed by the validation using a dual-Luciferase reporter assay system and chromatin immunoprecipitation assay.Finally,dual fluorescence co-localization assay and co-immunoprecipitation assay were performed to confirm the physical interaction between YY1 protein and T-bet protein.3.Exploring the role and mechanism of miR-124-3p involved in p Th17cell differentiation through YY1 regulation in RA.Online bioinformatics predictive tool Target Scan was employed for prediction that which miRNAs could bind to YY1.The protein expression level of YY1 was measured by western blot after 293T cells were transfected with miRNA mimics or miRNA inhibitor.Last,the binding sites were further verified using a dual-luciferase reporter assay system.Flow cytometry was conducted to assess the p Th17 cell differentiation after na(?)ve CD4~+T cells were transfected with miR124-3p mimics or miR124-3p inhibitor in ex vivo p Th17 cell induced differentiation system.The expression levels of miRNAs and YY1 in PBMCs of RA patients and healthy controls were detected using q PCR and their correlation was analyzed.Clinical information about the study subjects was collected and the diagnostic value of miR-124-3p was assessed in RA.【Results】1.YY1 promoted p Th17 cell differentiation in RA patients and CIA mice.Flow cytometry results showed that the number and proportion of p Th17 cells were elevated in RA patients compared to OA patients and healthy controls(P<0.05).q PCR results demonstrated that YY1 expression was positive orrelated with the expression of IL-17A(R=0.434,P=0.0046)and IL-22(R=0.351,P=0.0246)in PBMCs of RA patients.In ex vivo p Th17 cell induced differentiation system,we examined the success of lentiviral targeting YY1 using western blot and found that the number and proportion of p Th17 cells were decreased after YY1 knockdown(P<0.05).In CIA mice model,the inflammation score in the diseased joints were assessed and the results showed the scores were significantly decreased in CIA mice which knockdown YY1.Moreover,HE staining results suggested that the cartilage and bone destruction of the joint as well as the synovial inflammation also attenuated after YY1 knockdown.In addition,the number and proportion of p Th17 cells also showed reduction after YY1knockdown(P<0.05).2.YY1 can promote transcription factor T-bet transcription and interact with T-bet protein to make effect on p Th17 cell differentiation.Both m RNA-seq and PCR results showed that the expression level of key p Th17cell differentiation regulatory transcription factor T-bet were decreased in YY1knockdown ex vivo p Th17 cell induced differentiation system(P<0.05).Western blot also confirmed that the protein level of T-bet was increased in ex vivo p Th17 cell induced differentiation system.Moreover,T-bet overexpression in YY1 knockdown ex vivo p Th17 cell induced differentiation system could partily rescue the number and proportions of differentiated p Th17 cells.Next,The potential YY1-binding sites on the promoter region of T-bet gene were inspected by online bioinformatics predictive tool JASPAR and PROMO,then a dual-luciferase reporter assay system and chromatin immunoprecipitation assay were performed and verified that YY1 could bind to the T-bet promoter region located at-346 to-341 site and promoted its transcription.Dual fluorescence co-localization assay were applied and the pictures demonstrated that T-bet co-localized with YY1 in nucleus of p Th17 cells in ex vivo p Th17 cell induced differentiation system.Then,co-immunoprecipitation assay further validated that there was a physical interaction between YY1 protein and T-bet protein.3.miR-124-3p was involved in p Th17 cell differentiation through YY1 regulation in RA.Online prediction programs Target Scan results showed candidate miRNAs let-7a-5p,miR-124-3p and miR-218-5p may be bind to the 3’untranslated region(UTR)of YY1 m RNA.After 293T cells were separately transfected with the corresponding miRNA mimics and miRNA inhibitor,western blot showed that miR-124-3p could inhibit YY1 protein expression while no significant inhibition of YY1 protein was observed when transfected with let-7a-5p or miR-218-5p.Dual-luciferase reporter assay system also validated miR-124-3p could bind to the YY1 m RNA located at 1825to 1831 site and regulate YY1 expression.Next,q PCR was performed in PBMCs of RA patients and a significant negative correlation between the gene expression level of YY1 and miR-124-3p was observed(R=-0.4781,P=0.0075).After further assessing the diagnostic value of miR-124-3p,we found that there was a significant negative correlation between miR-124-3p and rheumatoid factor(R=-0.4445,P=0.0177),while no obvious correlations were found between miR-124-3p and C-reactive protein or erythrocyte sedimentation rate(P>0.05).【Conclusions】The number and proportion of p Th17 cells were increased in the peripheral blood of RA patients,and associated with YY1 expression.Blocking of YY1 could decreased the number and proportion of p Th17 cells in in the peripheral blood of CIA mice and ameliorate the inflammatory reaction as well as joint destruction.Mechanically,YY1promoted pathogenic Th17 cell differentiation in RA through promoting T-bet transcription and interacting with T-bet protein.Additionally,miR-124-3p negatively regulated YY1 by binding 3’UTR of YY1 m RNA and participated in p Th17 cell differentiation.In summary,YY1 and miR-124-3p are expected to become new targets for RA diagnosis and treatment,and facilitate the development of new means of that.