Bioinformatics Analysis Of Human Nasopharyngeal Carcinoma Genome And Its Construction Of Different Gene Expression Profiling

【Background of high-thoroughly microarray data】U133 Data:The gene expression patterns of 12 microdissected naspharyngeal carcinoma(NPC) samples were compared with 4 non-neoplastic samples using the Affymetrix U133 Plus 2.0 Arrays.H80s Data:10 non-neoplastic and 23 NPC specimens were microdissected and the extracted and amplified mRNA was hybridized to H80s high-density chip.503 genes were found to be differentially expressed in nasopharyneal carcinoma tissues.TW-U133 Data:The gene expression profiles of 10 normal nasopharyngeal epithelium and 31 NPC tissues were constantly attained using Affymetrix HG U133 2.0 plus Arrays.831 probesets showed statistically significant differences.【Genome-wide gene expression profiles and differential gene expression profiles in napharyngeal carcinoma】By statistically filter of U133 Data,including P call%≥75%and probesets expression value in 75%samples each group＞20.00,normal samples and NPC samples expressed 15982 probesets,which 12555 probesets definitely expressed in both groups,2540 probesets definitely expressed in the normal group and 887 probesets.Comparison of genome-wide human gene expression profiles by unsupervised hierarchical clustering and correspondence analysis clearly distinguished normal epithelial samples from tumors,displaying the difference in summarized gene expression profiles between two group samples as their distance in a two-dimensional plane,but not provide strong statistical support for NPC subclassification with clinical stage and pathlogy classfication.A q-value/t test analysis(p＜0.05,0.01 and 0.001) estimated that 919, 550 and 192 probesets measured noticeably changed expression between tumors and normals.By unsupervised hierarchical clustering and correspondence analysis,differential gene expression profiles clearly distinguished tumors from normal epithelium.Expression levels of 13 up-regulated genes,such as E2F6,KRT15,DEPDC1,BRIP1,FLJ21901, CCT6A,FIGNL1,PUS7,KIAA1794,CSE1L,NFE2L3,IDH1 and HOXA10,and down-regulated genes,C1orf178,LTF,LXN,BEXL1, CLDN10,PIGR,WFDC2,CEACAM6,SLC44A4,C20orf114,MS4A1, FOLR1,CYP2B7P1,MEIS3P1 and MLPH,measured an at least 1.5-fold change by comparing expressed value in each NPC sample to in each normal sample in U133 Data.Three Data showed 1061 up-regulated genes and 630 down-regulated genes in NPC,among 234 up-regulated and 70 down-regulated genes in every two group data(ANY2 Data),30 up-regulated and 7 down-regulated genes superposed in three data.Especially,the change folds of increased CCT6A and IDH1 and decreased LTF and PIGR were at least 1.5-fold in each NPC vs each normal in U133 Data.【Functional categorization of differential expression genes in NPC】Four pathways,such as "Cell cycle","p53 pathway","De novo purine biosynthesis" and "p53 pathway feedback loops 2",at least p＜0.05,were examined for enrichment in up-regulated genes represented by 55 up-regulated pathways in the tumors in U133 Data.In H80s Data,the smallest P value is 0.191 of "Cell cycle" in 57 up-regulated pathways. Between 39 up-regulated pathways in TW-U133,the P value of "Integrin signalling pathway" and "p53 pathway feedback loops 2" is less than 0.05.The results of integrating analysis had shown NPC up-regulated genes involving 21 pathways coexiting with three data,which P values all more than 0.05.In ANY2 Data,the P values of "Integrin signalling pathway" and "Cell cycle" were at least 0.05 among 52 up-regulated pathways.The P values of 46 down-regulated pathways in U133 Data,36 pathways in H80s were more than 0.05,while of "Huntington disease" in 44 down-regulated pathway in TW-U133 Data is at least 0.05.The results of integrating analysis had shown the down-regulated pathways in three data and in ANY2 Data were no significant. Furthmore,there are significant 9 KEGG_PATHWAY and 5 BIOCARTA pathway of U133 Data up-regulated genes,"hsa04110:Cell cycle" of H80s Data,and 8 KEGG_PATHWAY and 3 BIOCARTA pathway of TW-U133 Data.The down-regulated genes in TW-U133 Data were involved in significant "hsa00980:Metabolism of xenobiotics by cytochrome P450","hsa05131:Pathogenic Escherichia coli infection-EPEC" and "hsa05130:Pathogenic Escherichia coli infection-EHEC".Nine GO BP categories,including "Cell cycle","DNA metabolism", "Mitosis","Nucleoside,nucleotide and nucleic acid metabolism","Cell cycle control","DNA replication","Purine metabolism","Chromosome segregation" and "DNA repair",were examined for enrichment in up-regulated genes in U133 Data.In H80s Data,"Cell cycle","Cell cycle control","DNA metabolism" and "Mitosis" were significant enrichment. Furthermore,the P values of"Cell cycle","DNA metabolism","Mitosis", "DNA replication","Chromosome segregation","Nucleoside, nucleotide and nucleic acid metabolism","Cell proliferation and differentiation","DNA repair" and "Cell cycle control" were at least 0.05 in TW-U133 Data.The results of integrating analysis had shown 107 NPC up-regulated GO BP categories coexisting with three data,and 102 classes in ANY2 Data."Cell cycle","Cell cycle control","DNA metabolism" and "Mitosis" in three data,and 11 BP categories in ANY2 Data were significant up-regulated."Nucleic acid binding","Synthase and synthetase","Kinase activator" and "Chaperonin" MF classes in U133 Data,"Kinase activator" and "Helicase" in H80s Data,and "Extracellular matrix structural protein", "Extracellular matrix" and "DNA helicase" in TW-U133 Data were notable up-regualted meaning.The P values of 205 MF classes coexiting in three data,among 106 classes superposed,were more than 0.05.In 127 up-regulated ANY2 Data,"Kinase activator","Synthase and synthetase", "Extracellular matrix structural protein","Kinase modulator" and "Other cytokine' showed significant meaning.The down-regulated genes in U133 Data were enrichment in "Transporter" and "Defense/immunity protein" MF classes,in H80s Data were in "Oxidoreductase",and in TW-U133 Data were in "Cytoskeletal protein","Microtubule family cytoskeletal protein" and "Serine protease inhibitor".In ANY2 Data,NPC down-regulated genes were related to "Defense/immunity protein","Oxidoreductase" and "Serine protease inhibitor".The results of Functional Annotation Clustering of three data and ANY2 Data,pathway and gene ontology classes enriched for up-regulated genes in the tumors described aspects of cell cycle,cell division and cytoskeleton-associated processes,and enriched for down-regulated genes described protease inhibitor,defense and immunity, and transporter,which were similar to the results of above pathway,BP and MF catogies analysis.【Gene map and physical map of differential expressed genes of NPC】The results of genetic pinpoint of differential expressed genes of NPC in U133 and TW-U133 Data showed that,the most differential expressed genes were exact location in chromosome 1 and 2,total to more than 20%. There were 54 significant gene proximity stretches,42 of up-regulated genes and 12 of down-regulated genes in U133 Data,while 64 stretches in TW-U133 Data.In 71 up-regulated gene proximity stretches and 47 down-regulated gene proximity stretches of U133 Data and TW-U133 Data,there were 35 overlapping stretches in human chromosomes.The 6 down-regulated gene proximity stretches in chromosomes were 1q31.3-1q42.11, 2q36.3-2q37.3,3p21.2-3p25.2,11p12-11q14.2,20q11.21-20q13.2 and Xq22.1-22.2.In ANY2 Data,234 up-regulated and 70 down-regulated genes formed 16 up and 6 down significant gene proximity stretches.There were 16 overlapping fragments,13 up-regulated and 3 down-regulated,in chromosomes by integrated U133-TW-U133 Data and ANY2 Data. Thirteen up-regulated gene stretches were 1q22-32.3,2p15-16.1, 2q23.1-35,4q21.1-21.21,4q26-27,6p22.1-24.1,8p21.1-21.1, 9q31.2-31.3,10q22.2-24.32,14q21.3-22.1,15q13.2-q26.3,18p11.1 and 18q21.2,while 3 down-regulated gene stretches were 1q32.1-32.2, 3p21.31-p21.32 and 20q13.12.According to gene proximity stretches in chromosomes,the gene map and physical map of differential expressed genes were drawn and constructed the genome/ chromosomes map of differential expressed genes.The increased gene IDH1 and decreased genes LTF and PIGR, location at overlapping differential expressed genes proximity stretches and significantly altered in NPC samples more than 2 fold,can serves as clinical molecular tumor markers of NPC.【Differential expressed genes of NPC co-exit in 3 Data and gene functional verification】There were fifteen genes associated in MHCI-mediated immunity, and ten genes in cell communication and KRAB box transcription factor. The up-regulated genes were enrichment in cell cycle,motesis and nucleoside,nucleotide and nucleic acid metabolism,down-regulated genes in ion bind.There is special functional association with LPI,LTF and PIGR.LTF is a NPC susceptibility gene;the frequency of LTF downregulated expression in lymph node metastasis of NPC was 58.42%, which was significantly higher than in primary tumor(46.36%).And LTF can inhibit cell cycle progression in G1/S by regulating cyclins/CDKs/CKI/pRb axis to suppress the NPC cells invasion and metastasis.【Differential expressed genes of NPC in different cinilical stage】The up-regulated genes of primary stage were enrichment in cell cycle,nucleotide and nucleic acid metabolism,protein modify,and transprition,which down-regulated genes in metabolism.【Putative tumor suppressor gene associated with NPC cloning in our laboratory】The fold change of expression of PRR4,PLUNC and C1orf102 in U133 Data were more than 2 fold.