gastric cancer ( gc ) is the second - leading cause of cancer - related death worldwide . in recent decade , even with multiple therapies , the prognosis of gc remains poor with 5-year survival rate ranging between 25% and 35% . the low survival rate is mainly due to the undetectable characteristics of gc at early stage . therefore , it is of great significance to identify the molecular biomarkers pivotal for development and progression of gc . downregulated x - linked inhibitor of apoptosis ( xiap ) in human gc cells causes apoptosis and chemotherapeutic sensitivities , and it may contribute to develop a new therapeutic strategy for gc . previous study indicates that the transcriptional silencing of deleted in liver cancer 1 ( dlc-1 ) , which is induced by epigenetic mechanism , may play a role in gastric carcinogenesis . especially , expression profiles of gc have been extensively investigated , yielding useful insights into the molecular mechanism of carcinogenesis of gc . for example , by performing a genome - wide expression analysis , junnila et al identified 58 differentially expressed genes ( degs ) between cancerous and non - neoplastic gastric tissues , among which overexpressed cxcl1 was positively associated with improved survival . lei et al screened out 260 degs in gastric tissues and further proposed serpin eptidase inhibitor , clade h1 ( serpinh1 ) and g protein - coupled receptor family c type 5a ( gprc5a ) as potential biomarkers for gc . however , a microarray - based method has those following limitations : background levels of hybridization limit the accuracy of expression measurements , particularly for transcripts with low abundance ; prior knowledge of the genome is used to design probes for hybridization with targets ( dna or rna ) , and therefore the unknown genomic regions can not be interrogated . rna - sequencing ( rna - seq ) technology emerges as a new and powerful tool superior to hybridization - based approach in measuring gene expression without those limitations mentioned earlier . this technology can provide a more detailed and precise view of the entirely expressed transcripts , including low - expressed genes , alternative splice variants , and novel transcripts . in 2012 , by performing rna - seq , kim et al obtain an global view of transcriptome in asian gc and subsequently identify the regulatory effects of amp - activated protein kinase ( ampk ) 2 on energy homeostasis . while , the obtained results are restricted and rna - seq data need deeper exploration . bioinformatics methods can help excavate more useful information from the large number of microarray data or sequencing data . based on bioinformatics methods , first , the stage - specific genes in different stage gc samples were identified , and the stage - specific genes which have transcriptional regulation function of transcription factors ( tfs ) were annotated . moreover , the underlying interactions among stage iv - specific genes were investigated by protein rna - seq data of gse36968 were downloaded from gene expression omnibus ( geo , http://www.ncbi.nlm.nih.gov/geo/ ) established at the national center for biotechnology information ( ncbi ) database , which were previously sequenced using paired - end sequencing based on the gpl9442 ab solid system 3.0 platform . gse36968 included 6 normal gastric tissue samples , 5 stage i gc samples , 5 stage ii gc samples , 8 stage iii gc samples , and 6 stage iv gc samples of asians . the tissue samples were taken from the national research resource bank program of the korea science and engineering foundation in the ministry of science and technology . and tumor stages were identified and classified by a pathologist from the gene bank at yonsei university severance hospital . the rna - seq data used in this study were downloaded from geo database , thus ethical approval and informed consent were not necessary . the author used tophat2 ( version 2.0.10 , http://ccb.jhu.edu/software/tophat/index.shtml ) to align the rna - seq reads against the university of california santa ( ucsc ) hg19 genome sequences ( http://www.genome.ucsc.edu/index.html ) with default parameters , allowing 1 or 2 base mismatches . following , the author assembled the transcripts and calculated the gene expression levels using cuffdiff and fragments per kilobase of exon model per million fragments mapped method in cufflinks , respectively . one - way analysis of variance ( anova ) was applied to screen degs among different groups with the cutoff criterion of p  <  0.05 . subsequently , the screened degs were conducted with hierarchical clustering analysis to determine stage - specific genes for specific gc stages . furthermore , the author annotated the stage - specific genes which have transcriptional regulation function of tfs based on the transfac database ( http://transfac.gbf.de/transfac/ ) . besides , the author defined upstream 1.5  kb region of the transcription start site as promoter region and then perform motif finding for all stage - specific genes using seqpos.p  <  0.00001 was used as the threshold . david software was employed to identify significantly enriched gene ontology functions in biological processes ( bp ) , molecular function , and cellular component ( cc ) categories for stage - specific genes . string is developed as an online database resource , which provides uniquely comprehensive information for assembling , evaluating , and disseminating ppi in a user - friendly way ( http://string-db.org/ ) . to reveal the underlying interactions among stage iv - specific genes , the author further extracted the ppi information from string database and constructed ppi network using cytoscape software ( http://cytoscape.org/ ) . rna - seq data of gse36968 were downloaded from gene expression omnibus ( geo , http://www.ncbi.nlm.nih.gov/geo/ ) established at the national center for biotechnology information ( ncbi ) database , which were previously sequenced using paired - end sequencing based on the gpl9442 ab solid system 3.0 platform . gse36968 included 6 normal gastric tissue samples , 5 stage i gc samples , 5 stage ii gc samples , 8 stage iii gc samples , and 6 stage iv gc samples of asians . the tissue samples were taken from the national research resource bank program of the korea science and engineering foundation in the ministry of science and technology . and tumor stages were identified and classified by a pathologist from the gene bank at yonsei university severance hospital . the rna - seq data used in this study were downloaded from geo database , thus ethical approval and informed consent were not necessary . the author used tophat2 ( version 2.0.10 , http://ccb.jhu.edu/software/tophat/index.shtml ) to align the rna - seq reads against the university of california santa ( ucsc ) hg19 genome sequences ( http://www.genome.ucsc.edu/index.html ) with default parameters , allowing 1 or 2 base mismatches . also , the sequence alignment should be unique for each read . following , the author assembled the transcripts and calculated the gene expression levels using cuffdiff and fragments per kilobase of exon model per million fragments mapped method in cufflinks , respectively . one - way analysis of variance ( anova ) was applied to screen degs among different groups with the cutoff criterion of p  <  0.05 . subsequently , the screened degs were conducted with hierarchical clustering analysis to determine stage - specific genes for specific gc stages . furthermore , the author annotated the stage - specific genes which have transcriptional regulation function of tfs based on the transfac database ( http://transfac.gbf.de/transfac/ ) . besides , the author defined upstream 1.5  kb region of the transcription start site as promoter region and then perform motif finding for all stage - specific genes using seqpos.p  <  0.00001 was used as the threshold . david software was employed to identify significantly enriched gene ontology functions in biological processes ( bp ) , molecular function , and cellular component ( cc ) categories for stage - specific genes . string is developed as an online database resource , which provides uniquely comprehensive information for assembling , evaluating , and disseminating ppi in a user - friendly way ( http://string-db.org/ ) . to reveal the underlying interactions among stage iv - specific genes , the author further extracted the ppi information from string database and constructed ppi network using cytoscape software ( http://cytoscape.org/ ) . based on anova and subsequent hierarchical clustering analysis , the author systematically compared the expression profiles at different gc stages , and finally obtained the heat map of stage - specific genes ( fig . 1 ) . a total of 3576 genes with stage - specific expression patterns were identified . among them , 813 degs ( 23% , 813/3576 ) were specifically highly expressed in the normal gastric tissues , and 2224 degs ( 62% , 2224/3576 ; e.g. , kinesin family member c1 , kifc1 ; and septin 2 , sept2 ) which were specifically highly expressed in stage i had extremely similar expression patterns in stage ii gc samples , as well as 539 specifically highly expressed genes ( 15% , 539/3576 ; e.g. , neuropilin-2 , nrp2 ; collagen triple helix repeat containing-1 , cthrc1 ; secreted protein , acidic , cysteine - rich , osteonectin , sparc ; matrix metalloproteinase 17 , mmp17 ; and collagen , type vi , alpha 3 , col6a3 ) in stage iv gc samples . the orange , blue , and pink bars represent stage - specific genes in normal stomach tissues , stage i and ii gc samples and stage iv gc samples , respectively . , a total of 82 tfs were screened from the stage i and ii - specific genes and 22 tfs from the stage iv - specific genes . following , the author further explored the upstream regulators , which might have regulatory effects on stage - specific genes in stage i and ii gc and stage iv gc samples . a total of 9 tfs ( e.g. , high mobility group box-1 , hmgb1 ; and homeobox a13 , hoxa13 ) were identified to be significantly enriched in the promoter region of highly expressed stage i and ii - specific genes , which also experienced over - expression in stage i and ii gc and stage iv gc samples ( table 1 ) . besides , 11 tfs were screened for the stage iv - specific genes , which were up - regulated in stage iv gc samples ( table 1 ) . predicted upstream tfs for stage - specific genes . functional enrichment analysis was performed separately for the gc stage - specific genes , and the results were shown in fig . 2 . for stage - specific genes in normal tissues , functions including digestion , ion transport , and g - protein signaling were significantly enriched . besides , the stage - specific genes in stage i and ii gc samples were mainly associated with mitotic cell cycle and rna processing . additionally , the stage - specific genes in stage iv gc samples were dramatically enriched in functions , including vasculature development , skeletal system development , and urogential system development . accordingly , gc stage iv - specific genes were significantly enriched in bp such as cell migration and negative regulation of cell differentiation . functional enrichment results of normal gastric - specific genes and stage i and ii - specific genes and stage iv - specific genes . importantly , the author further focused on the involved genes which could encode proteins for cc , including extracellular matrix ( ecm ) and cell surface . as shown in table 2 , the author identified 24 , 7 , and 22 genes involved in ecm separately in normal gastric tissues , stage i and ii gc samples , and stage iv gc samples , respectively . besides , 22 , 29 , and 16 genes were highly expressed at cell surface in normal , gc stage i and ii , and stage iv tissues , respectively . extracellular matrix and cell surface - associated stage - specific genes . based on the ppi information from string database , the author further constructed the ppi network for stage iv - specific genes using cytoscape . a total of 13 nodes were identified with degree > 10 in the ppi network ( fig . 3 ) . in addition , homeo box a4 ( hoxa4 ) , gli family zinc finger 3 ( gli3 ) , runt - related tf 2 ( runx2 ) , fibroblast growth factor 2 ( fgf2 ) , high mobility group at - hook 2 ( hmga2 ) , and protein kinase c , alpha ( prkca ) were involved in 2 regulatory pathways which were considered to play key roles in stage iv gc , including hoxa4-gli3-runx2-fgf2 and hmga2-prkca ( fig . in the second pathway , hmga2 was confirmed as tf . the ppi network of stage iv - specific genes . the red nodes represent transcription factors , the green nodes represent genes with degree > 10 , and the orange nodes represent other stage iv - specific genes . based on anova and subsequent hierarchical clustering analysis , the author systematically compared the expression profiles at different gc stages , and finally obtained the heat map of stage - specific genes ( fig . 1 ) . a total of 3576 genes with stage - specific expression patterns were identified . among them , 813 degs ( 23% , 813/3576 ) were specifically highly expressed in the normal gastric tissues , and 2224 degs ( 62% , 2224/3576 ; e.g. , kinesin family member c1 , kifc1 ; and septin 2 , sept2 ) which were specifically highly expressed in stage i had extremely similar expression patterns in stage ii gc samples , as well as 539 specifically highly expressed genes ( 15% , 539/3576 ; e.g. , neuropilin-2 , nrp2 ; collagen triple helix repeat containing-1 , cthrc1 ; secreted protein , acidic , cysteine - rich , osteonectin , sparc ; matrix metalloproteinase 17 , mmp17 ; and collagen , type vi , alpha 3 , col6a3 ) in stage iv gc samples . the orange , blue , and pink bars represent stage - specific genes in normal stomach tissues , stage i and ii gc samples and stage iv gc samples , respectively . based on the transfac database , a total of 82 tfs were screened from the stage i and ii - specific genes and 22 tfs from the stage iv - specific genes . following , the author further explored the upstream regulators , which might have regulatory effects on stage - specific genes in stage i and ii gc and stage iv gc samples . a total of 9 tfs ( e.g. , high mobility group box-1 , hmgb1 ; and homeobox a13 , hoxa13 ) were identified to be significantly enriched in the promoter region of highly expressed stage i and ii - specific genes , which also experienced over - expression in stage i and ii gc and stage iv gc samples ( table 1 ) . besides , 11 tfs were screened for the stage iv - specific genes , which were up - regulated in stage iv gc samples ( table 1 ) . functional enrichment analysis was performed separately for the gc stage - specific genes , and the results were shown in fig . functions including digestion , ion transport , and g - protein signaling were significantly enriched . besides , the stage - specific genes in stage i and ii gc samples were mainly associated with mitotic cell cycle and rna processing . additionally , the stage - specific genes in stage iv gc samples were dramatically enriched in functions , including vasculature development , skeletal system development , and urogential system development . accordingly , gc stage iv - specific genes were significantly enriched in bp such as cell migration and negative regulation of cell differentiation . functional enrichment results of normal gastric - specific genes and stage i and ii - specific genes and stage iv - specific genes . importantly , the author further focused on the involved genes which could encode proteins for cc , including extracellular matrix ( ecm ) and cell surface . as shown in table 2 , the author identified 24 , 7 , and 22 genes involved in ecm separately in normal gastric tissues , stage i and ii gc samples , and stage iv gc samples , respectively . besides , 22 , 29 , and 16 genes were highly expressed at cell surface in normal , gc stage i and ii , and stage iv tissues , respectively . based on the ppi information from string database , the author further constructed the ppi network for stage iv - specific genes using cytoscape . a total of 13 nodes were identified with degree > 10 in the ppi network ( fig . homeo box a4 ( hoxa4 ) , gli family zinc finger 3 ( gli3 ) , runt - related tf 2 ( runx2 ) , fibroblast growth factor 2 ( fgf2 ) , high mobility group at - hook 2 ( hmga2 ) , and protein kinase c , alpha ( prkca ) were involved in 2 regulatory pathways which were considered to play key roles in stage iv gc , including hoxa4-gli3-runx2-fgf2 and hmga2-prkca ( fig . hoxa4 , gli3 , and runx2 were identified as tfs . and in the second pathway , hmga2 was confirmed as tf . the red nodes represent transcription factors , the green nodes represent genes with degree > 10 , and the orange nodes represent other stage iv - specific genes . moreover , those genes were mainly associated with mitotic cell cycle and rna processing , implying their overexpression might promote cancer cells proliferation . for example , kifc1 , a c - terminal kinesin motor , belongs to the minus - end - directed kinesin-14 family in human cells . several studies have shown that kifc1 plays an essential role in mitosis and meiosis by organizing and stabilizing spindles using its sliding activity along microtubules . in addition , sept2 has also been proved to play an important role in carcinoma cell division and proliferation through affecting cytoskeletons , which could be suggested as a promising target for gc therapy . therefore , the author speculated that the overexpressed of kif14 and sept2 in stage i and stage ii gc might contribute to the aggravated cell proliferation . on the other hand , 539 stage iv - specific genes were screened , which were significantly enriched in functions , including vasculature development , skeletal system development , neuron differentiation , and urogenital system development . for instance , nrp2 is a transmembrane glycoprotein receptor for vascular endothelial growth factor - c , which is an important lymphangiogenic factor and exerts crucial role in lymph node metastasis of various human cancers including gc . several studies have reported the up - regulation of cthrc1 in various human solid cancers and associated it with cancer cell adhesion to ecm in the progression of cancer invasion and metastasis . moreover , the author identified several ecm - related genes with up - regulation in stage iv of gc , including sparc , mmp17 , and col6a3 . the relative expression level of the sparc was shown to be higher in gc tissues than in adjacent normal mucosae , leading to lower 5-year overall survival . the down - regulation of sparc inhibits invasion and growth of human gc cells.mmp-17 belongs to the membrane type - mmps subfamily anchored to the plasma membrane via a glycosyl - phosphatidyl inositol anchor , which is highly expressed in human cancers and associated with cancer progression . besides , col6a3 has also been found to participate in gc progression by regulating ecm - receptor interaction pathway . thus , the author could speculate that these up - regulated and stage iv - specific genes may contribute to a gradual disappearance of tissue specificity and diverse cell differentiation potentials via affecting various bp . besides , by performing motif scanning , the author also screened out some key tfs for different stage - specific genes , which can bind the promoter sequence of most stage - specific genes . herein , 9 tfs were obtained in stage i and ii gc , such as hmgb1 and hoxa13 , which also experienced over - expression in stage i and ii gc and stage iv gc samples . in the previous study , serum hmgb1 level is significantly associated with invasion , lymph node metastasis , tumor size , and poor prognosis of gc.hmgb1 silencing significantly decreases cell proliferation by regulating cell cycle level and cell cycle - related gene cyclin d1 expression , and inhibits cellular metastatic ability by down - regulating mmp-9 expression in gc mgc-803 cells . besides , homeobox ( hox ) gene family is known to play crucial roles in tumorigenesis . recent study indicates that up - regulated hoxa13 may be a novel prognostic marker in gc and correlation with tnm stage , histological differentiation , relapse , and overall survival and disease - free survival rate . the up - regulation of tfs hmgb1 and hoxa13 might contribute to the overexpression of genes in stage i and stage ii gc . furthermore , by constructing ppi network for the stage iv - specific genes , 2 interaction pathways were obtained , including hoxa4-gli3-runx2-fgf2 and hmga2-prkca . among these interaction networks , the first pathway indicated that activation of runx2 was based on hoxa4gli3 interaction and gli3runx2 interaction , which may subsequently regulated fgf2-mediated expression activation of downstream genes in stage iv gc samples . the second regulatory pathway hmga2-prkca suggested that the hmga2 might regulate the downstream genes via interacting with prkca in stage iv gc samples . also , a total of 9 and 11 up - regulated tfs were identified for the stage i and ii - specific genes and stage iv - specific genes , respectively . besides , kif14 , sept2 , nrp2 , cthrc1 , sparc , mmp17 , col6a3 , hmgb1 , hoxa13 , hoxa4 , and hmga2 might play important roles in pathogenesis of gc in asians . the present study provided a novel insight into the molecular mechanisms underlying different gc stages . however , the results were obtained by bioinformatics methods and the sample sizes were small , thus further experimental validations were still needed .
abstractbackground : to identify gastric cancer ( gc)-associated genes and transcription factors ( tfs ) using rna - sequencing ( rna - seq ) data of asians.materials and methods : the rna - seq data ( gse36968 ) were downloaded from gene expression omnibus database , including 6 noncancerous gastric tissue samples , 5 stage i gc samples , 5 stage ii gc samples , 8 stage iii gc samples , and 6 stage iv gc samples . the gene expression values in each sample were calculated using cuffdiff . 
 following , stage - specific genes were identified by 1-way analysis of variance and hierarchical clustering analysis . 
 upstream tfs were identified using seqpos . 
 besides , functional enrichment analysis of stage - specific genes was performed by david . in addition , the underlying protein  protein interactions ( ppis ) information among stage iv - specific genes were extracted from string database and ppi network was constructed using cytoscape software.results:a total of 3576 stage - specific genes were identified , including 813 specifically up - regulated genes in the normal gastric tissues , 2224 stage i and ii - specific genes , and 539 stage iv - specific genes . also , a total of 9 and 11 up - regulated tfs were identified for the stage i and ii - specific genes and stage iv - specific genes , respectively . 
 functional enrichment showed sparc , mmp17 , and col6a3 were related to extracellular matrix . 
 notably , 2 regulatory pathways hoxa4-gli3-runx2-fgf2 and hmga2-prkca were obtained from the ppi network for stage iv - specific genes . in the ppi network , 
 tfs hoxa4 and hmga2 might function via mediating other genes.conclusion:these stage - specific genes and tfs might act in the pathogenesis of gc in asians .