| Background＆ObjectivesUlcerative colitis(UC),is a form of chronic,relapsing and nonspecific inflammation of colon with uncertain etiology.The widely accepted pathogenesis of UC is a interactive result of environmental factors as well as immune and genetic components.It has been demonstrated that peripheral blood mononuclear cells (PBMCs) playing a vital role in the immune disorder of UC by extensive research. We carried out high-flux microarray to clarify the changes of gene expression profiles of PBMCs in patients with ulcerative colitis,in order to identify disregulated genes that may contribute to the pathogenesis of UC.Those molecular can be expected to be potential therapeutic targets and biomarkers of the disease.Materialas＆MethodsPBMCs were collected from 12 UC patients in active stage(half males and half females,mean age 36.25±10.98 y;range 19～60 y) that have been diagnosed based on a combination of clinical features,endoscopy and pathology findings using standard diagnostic criteria and 6 healthy controls(3 females,mean age 35.17±14.88 y;range 19～60 y).All the individals have been ruled out autoimmune related diseases,infection,malignancies and other diseases that would be an apparent factor afecting gene expression in these cells.The total RNA was isolated from PBMCs according to the manual of Trizol kit(Invitrogen) and then Punfied based on the guide of Rneasy mini kit(QIagen).The iniensity ratio of 28s to 18s rRNA in total RNA samples was examined by 1%agarose gel electroPhoresis(100V,o.5h) to estimate the integrity of total RNA.According to the protocal of Phalanx,cRNA labeled with biotin were synthesized and then digested into fragments.2 ug of RNA from each individual was converted into cyanine labeled target cRNA then hybridized to Human OneArray microarray(Phalanx Biotech Group,Taiwan,China) containing 32,050 oligonucleotides:30,968 human genome probes,and 1082 experimental control probes.The followed procedure was washing,scanning(DNA Microarray Scanner G2565B,Agilent,USA) and extracting gene expression results according to the manufacturer's protocol,raw intensity values were normalized and log-transformed by GeneSpring GX 10 software(Agilent Technologies,USA).The RVM t-test with a threshold of false discovery rate(FDR)＜0.5%and p-value＜0.01 was applied to select differentially expressed genes,and the entire data were were analyzed by principal component analysis(PCA) and to reduces data complexity in a rational way without any prior knowledge of categories so as to determine if any intrinsic clustering or outliers existed within the data set.Gene Ontology(GO) analysis was carried out to organize disregulated genes into hierarchical categories based on biological process.Results1.The integhty of RNA isolated in each group was fine and the ratio of absorbance of total RNA at OD260/280nm was between 1.8～2.0.The high quality of RNA have been demonstrated by the results of electrophoresis.2.The large expression profiles difference of PBMCs between UC and control group has been confirmed by PCA.3.Comparative analysis revealed that 4438 probes(4188 genes) are differentially expressed between two groups,of which 3689 probes(3590 genes) were down-regulated whereas 749 probes(598 genes) were up-regulated.4.A p-value＜0.01 and FDR＜0.05 in the Fisher's exact test were selected as the significant criteria.Using this threshold,A total of 67 high-enrichment GOs targeted by over-expressed gene and one term corresponding to underexpressed genes were identified,segregation of genes that were overexpressed into functional classes revealed genes that encode products involved in many critical biological processes and three main classes of genes including those involved in:(1) immune and inflammatory response(immune response,inflammatory response,innate immune response,positive regulation of natural killer cell mediated cytotoxicity),(2) cell cycle and proliferation(cell cycle,cell proliferation, natural killer cell proliferation,NK T cell proliferation,cell differentiation,cell division,cell cycle arrest),(3) DNA metabolism and repair(response to DNA damage stimulus,DNA repair,DNA recombination,double-strand break repair via homologous recombination,DNA replication,positive regulation of DNA repair)Conclusions1.The gene expression profiles of PBMCs in UC patients is significantly different from that of control individuals,and the disregulated genes mainly involved in the critical biological processes of immune and inflammatory response,cell cycle and proliferation,DNA metabolism and repair,which indicate the pathogenesis of UC is a complicated pathophysiological procedure.2.The disregulated moleculars involved into those biological processes mentioned above are promising to be targets for therapy and markers to facilitate diagnosis.3.Microarray technique is a high thoughput and low waste tool for screening of differentially expressed gene in PBMCs of UC with excellent efficiency.In conclusion,we used PBMCs to obtain genome-wide gene expression profiles for active UC and the results has highlighted many disregulated genes involved in various biological processes that may contribute to the pathogenesis of UC.Further examination for those disregulated moleculars are promising to provide targets for therapy and diagnoses of UC.
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