Study On The Relation Of Proteomics And Small RNA In Peripheral Blood Mononuclear Ceel From Pateints With Ankylosing Spondylitis

Ankylosing spondylitis (AS) is an autoimmune disease, which ischaracterized by involving the axial joint and ligament and tendon adhesionof inflammatory changes. Since1973Brewerton reported that humanleukocyte antigen (HLA)-B27was associated with the development of AS,the pathogenesis mechanism study of AS has not been obviouslyprogressed. However, early diagnosis and interfenrence are utmostimportant for AS patints. Since it is lack of gold marker for laboratorydiagnosis, at present, the diagnosis of AS mainly depends on clinicalsymptoms and sacroiliac joint image. Therefore, the discovery andvalidation of new laboratory diagnositic biomarkers of AS has great clinicalapplication value. In this study, isobaric tags for relative and absolutequantitation (iTRAQ) and Solexa high–throughput sequencing technology,combined with bioinformatic analysis, were used to analyze the differentialexpression levels of proteomics and small RNA in patients with AS andhealth controls; the specific diagnostic biomarkers and new targets of drugaction were screen, and their possible actions in the pathogenesis of AS were explored. Furthermore, the differential expression levels of certainproteins and miRNAs were verified by Western blot and real time RT-PCR,respectivelyPARTⅠ STUDY ON PROTEIN EXPRESSION PROFILE OFBMCs IN PATEINTS WITH ANKYLOSING SPONDYLITIS BYITRAQ TECHNOLOGYObjective To identify and quantify proteins of peripheral bloodmononuclear cells (PBMCs) from patients with AS by using isobaric tagsfor relative and absolute quantitation (iTRAQ), obtaining the proteinexpression profile of PBMCs from patients with AS, to search for newdiagnostic markers of AS and to further explore the pathogenesis of AS atthe protein level.Methods Nine health controls and10cases of AS patients wererecruited, PBMCs were isolated using Ficoll-Paque plus solution, theproteins of PBMCs were extracted using cell ysate, iTRAQ coupled withmultiple chromatographic fractionation and tandem mass spectrometrywere used to screen the candidate proteins in PBMCs from AS patients andhealthy controls. Mascot software (Matrix Science) was applied to identifyand quantify the proteins, Gene Ontology (GO) analysis was used toanalyze the biological processes, and Western blot were used to furthervalidate the certain identified proteins. Results Our analysis identified1232proteins, of which183weresignificantly changed in PBMCs of AS patients compared with healthycontrols. Among these differentially expressed proteins,108proteins wereup-regulated,75proteins were down-regulated, and19proteins wereclassified as acute phase reactants. GO analysis suggested that5proteins,including the up-regulated proteins of cathepsin G (CTSG), isoform1ofserum albumin, neutrophil defensin3(DEFA3), protein tyrosinephosphatase, receptor type C (PTPRC), and the down-regulated proteinperoxiredoxin-1(PRDX1), were involved in the biological process“regulation of cell killing” in AS. Similar trends in the expression levels ofCTSG, PTPRC and PRDX1were also certified by Western blot.Conclusion The differential expression of protein profile of PBMCsfrom patients with AS are identified by iTRAQ coupled with multiplechromatographic fractionation and tandem mass spectrometry, whichprovides potential biomarkers in clinical diagnosis of AS; The differentialexpression of proteins involved in “cell killing” have some possibleimportant role in the development of AS. PARTⅡ STUDY ON miRNA EXPRESSION PROFILEOF PBMCs IN PATEINTS WITH ANKYLOSINGSPONDYLITIS BY HIGH-THROUGHT SEQUENCINGObjective To explore the differential expression of miRNA in PBMCsfrom patients with AS by next generation high-throughput sequencingtechnology, for providing new biomarkers of clinical diagnosis of AS, andto illuminate the role of the differential expression of miRNA in thepathogenesis of AS.Methods Nine health controls and10cases of AS patients wererecruited, PBMCs were isolated using Ficoll-Paque plus solution, RNAwere extracted using TRIzol reagent, sRNA libraries of10cases of ASpatients and9health controls were constructed, respectively. The samplesof two groups were sequenced simultaneously using next generationhigh-throughput sequencing technology, RNA sequence and genomesequence were compared by the software of SOAP2.0, then the matchedsequence and genome sequence repeate in GenBank and Rfam databasewere compared, miRNA were analyzed differentially with the Mireapsoftware; stem-loop qRT-PCR was used to verify certain differentialexpressions of miRNA.Results A total of7,511,859and10,178,958sRNA reads wereobtained in PBMCs from patients with AS and health controls respectively; and6,052,911(80.58%) and8,476,243(83.27%) genome-matched reads,267and231known miRNAs were identified in the library of AS and thelibrary of healthy controls, respectively. There were157known miRNAswith differential expression levels between the two libraries, includingmany cluster of miRNA and dominant miRNA such as miR-146a,miR-182,miR-155, miR-181a, miR-223et al. Real time Stem-loop qRT-PCRvalidated the similar trends with Solexa sequencing results (let-7b-3p,miR-146a-5p, miR-155-5p, let-7g-5p and miR-323a-5p).Conclusion High-throughput sequencing technology combined withbioinformatics analysis can be effectively used to study the differentialexpression of miRNA profile in PBMCs from patients with AS, thedifferential expression of miRNA can provide some specific biomarkers forthe clinical diagnosis of AS, on the other side it also provide a new idea fortargeted gene therapy of AS; the differential expression of miRNA mayplay some important immune regulation effects in the pathogenesis of AS. PART Ⅲ STUDY ON THE RELATION OFPROTEOMICS AND SMALL RNA IN PERIPHERALBLOOD MONONUCLEAR CEEL IN PATEINTS WITHANKYLOSING SPONDYLITISObjective Based on Kyoto encyclopedia of Genes and Genomes（KEGG）pathway, the aim is to explore the correlation analysis ofproteomics, sRNA combined with sRNA process analysis and thequantitative results of proteomics; To find the relationship betweensRNA–protein interaction processes in biology for validating somebiological significance.Methods Three miRNA target gene prediction database including themiRanda, TargetScan and PicTar were used to predict the differentialexpression levels of miRNA in common target genes; and then overlappedwith the differential expression analysis of genes coding for proteins toobtain some candidate miRNA and protein. The bioinformatics analysiswas applied to study the correlation of the differential expression ofproteins and predicted target gene on the same pathway.Results Two hundred and sixty-six sRNA were predicted in ASpatients, and31187target genes were found；The bioinformatics analysisshowed the corresponding target genes of miR-0121-5p were RAC2and MMP-9, the two target gene were linked to RAC2and MMP-9protein inMAPK, chemokine and leukocyte transendothelial migrationsignalpathways; the corresponding target gene of miR-0121-5p was PTPRC, thePTPRC target gene was linked to PTPRC protein in TCR and primaryimmunodeficiency signal pathway.Conclusion MAPK, TCR and chemokine signal pathways play somecritical regulation role in AS pathogenesis; RAC2, PTPRC and MMP-9have some possible biological significance in the immune regulation of ASand are expected to specific markers for the diagnosis of AS， thepathogenesis of AS may be interpreted from the RNA-protein interaction.