Peripheral Blood Gene Expression Profile Analysis In Han Chinese Patients With Rheumatoid Arthritis

Rheumatoid arthritis (RA) is a chronic inflammatory disease characterised bypersistent synovitis, systemic inflammation, and autoantibodies (rheumatoid factor andanti-cyclic citrullinated peptide antibody) with progressive disability. The cause of theinitiation and progression of RA remain unknown, though advances in understanding thepathogenesis of the disease have been made.The genome-wide assay of gene expression and genetic variation to identify geneswhich are expressed differently in affected individuals is a powerful tool to discover thethe underlying changes of the diseases. Gene expression profiling has been used to studyRA, including to predict responsiveness to therapy, to identify biomarkers for diagnosis,therapy and prognosis. Both peripheral blood mononuclear cells (PBMCs) and synovialtissues of affected joints are used in such studies. Those microarray-based studies arelimited by hybridization noise, less sensitivity and depending on the exisitng knowledgeabout genome sequence. Recently RNA-Seq is developed to be a revolutionary approch fortranscriptomics which does not rely on probe design and use tag-based sequencingtechnologies. Previous studies have documented marked differences in the prevalence ratesof rheumatoid arthritis and specific SNPs by race and ethnicity, suggesting the geneexpression profile might vary among different races. Therefore, We undertook RNA-Sequsing Illumina HiSeq2000system on PBMCs from5Han Chinese patients with active RAand5age-matched and gender-matched controls. This study identified a number ofcandidate genes which were validated by quantitative real-time PCR (qRT-PCR) in adifferent sample population later. The immunological nature of some candidate geneshighlights likely immune processes altered in the development of RA. Further,identification of gene expression patterns corresponding to disease status can be used todevelop predictive.Part I: Research on the immunogenetic mechanism of rheumatoid arthritis based onRNA-Seq technologyObjective: To identify differentially expressed genes in PBMCs from Han Chinesepatients with rheumatoid arthritis compared with healthy individuals, providing advancedclues for the pathogenesis of RA in Han Chinese patients.Methods: RNA was extracted from PBMCs collected from5patients with activedisease and5gender-matched and age-matched controls. Expression profiles of these cellswere determined using RNA-Seq technology. Cuffdiff Two Origins of Blastemal Progenitors program in cufflinks was used to obtain differential expressed genes withsignificant expression. Cluster analysis, KEGG Pathway and Gene Ontology (GO) analysiswere taken respectively for the candidate genes with differential expression over2-foldchange. In comparison with genes which have been reported, candidate genes were chosenfor next validation experiment.Results: RNA-Seq analysis identified32106transcripts in at least one sample and274transcripts were significantly differentially expressed between RA patients andcontrols with a p value <0.05and a significant fold-change>2. Of these,138transcriptswere overexpressed and136were underexpressed. Cluster analysis showed gooddelineation between patients with RA and healthy controls to confirm the validity of thisRNA-Seq experiment. GO analysis of the significant274transcripts revealed that theenrichment in immune system: antigen processing and presentation of peptide orpolysaccharide antigen via MHC class II, T cell activation, differentiation and proliferation,cellular response to molecule of bacterial origin, cellular response to lipopolysaccharide,ect; nervous system: synapse assembly, synapse structure and activity, synapseorganization, regulation of axonogenesis, central nervous system neuron development;reflex, ect; regulation of protein ubiquitination involved in ubiquitin-dependent protein.Combining GO analysis data and the genes reported,43candidate genes were identified.Conclusion: Data from GO analysis implies both innate and adaptive immunity playan important role in the pathogenesis of RA; Bacteria infection, one of the environmentfactors, may lead to a breakdown of immune tolerance; protein ubiquitination is worth tofurther study in the pathophysiology of RA.Part II: Validation on candidate genes by quantitative reverse transcription-PCR(qRT-PCR)Objective: To validate the candidate genes by quantitative reverse transcription-PCR(qRT-PCR) and find biological remarker.Methods: Peripheral blood samples were collected into EDTA tubes between6:00and8:00, Characteristics of the all study subjects were collected at the same time. Store per250ul blood sample in750ul Trizol() at-80°C. TRIzol() extraction followed by cDNAsynthesis. Candidate genes identified from the RNA-Seq study were validated byqRT-PCR in a smaller sample cohort (15cases versus15controls), then validated in alarger sample cohort (86cases versus40controls). Analyse gene expression leveldifference according to clinical characteristics in RA group. Evaluate the association between the differential gene expression profiling of RA patients with their autoimmuneresponse [anti-cyclic citrullinatedpeptide (CCP) antibodies, rheumatoid factor(RF)],disease activity [C reative protein(CRP), erythrocyte sedimentation rate (ESR), DiseaseActivity Score using28joint counts (DAS-28)], disease duration, and treatment(glucocorticoid) features.Results:11genes were downregulated significantly in patients with RA compared tohealthy controls in the smaller sample cohort：GPRIN3(р<0.01)、CXCR2(р<0.01)、FCGBP(р<0.05)、CD14(р<0.05)、RCAN1(р<0.05)、IKZF3(р<0.05)、BIRC3(р<0.05)、AFF3(р<0.05)、TSC1(р<0.05)、TYK2(р<0.05)、PADI4(р<0.05).7genes werevalidated to downregulate in patients with RA compared to healthy controls in the largersample cohort: TSC1(р<0.01)、CD14(р<0.01)、AFF3(р<0.01)、BIRC3(р<0.01)、GPRIN3(р<0.05)、FCGBP(р<0.05)、RCAN1(р<0.05). But there was no difference offollowing genes expression level between patients and healthy controls: IKZF3、CXCR2.In the context of RA, TSC1expression level was downregulated more significantly inACPA positive cases than ACPA negative cases (р<0.05) and. CD14expression levelwas downregulated more significantly in non-early RA patients than early RA patientspatients(р<0.05) and significantly associated with the stage of disease.Conclusion: TSC1and BIRC3are the first reported genes related to Han Chinesepatients with RA. Signaling pathways for validated genes may play roles in thepathogenesis of RA: CD14cooperates with Toll receptor to mediate the innate immuneresponse to bacterial lipopolysaccharide; AFF3activates transcription that may function inlymphoid development; BIRC3and TSC1mediate protein heterooligomerization; BIRC3regulates of apoptosis and anti-apoptosis.