Two clusters did not show certain functions but basically these described as characteristic of adjacent mucosa. These final results recommend that the smaller cluster of adjacent samples was just an extreme phenotype of those samples. Interestingly, this pattern was also observed inthe validation dataset (see heatmap in Added file four: Figure S2).Transcriptional regulation of differentially expressed genes amongst adjacent and wholesome mucosaWe hypothesized that this differential expression may be triggered by a transcriptional program, activated only in adjacent FPH2 biological activity mucosa by the presence in the tumor, and commonly silenced in healthful mucosa. This hypothesis was supported by the GSEA outcomes, in which 312 transcription factors motifs were discovered to be statistically related with the adjacent mucosa phenotype (nominal p-value < 0.01) but none was found associated to healthy mucosa phenotype (Additional file 3: Table S3). To further explore this hypothesis, transcriptional networks were inferred and compared using gene expression data of adjacent and healthy mucosa (see Additional file 4: Figure S3). Venn diagram in Figure 4A shows the overlap between nodes of each network. The vast majority of healthy mucosa nodes were also active in adjacent mucosa network whereas 3120 new nodes appeared specific to the adjacent mucosa and 668 nodes disappeared from the network. As expected, DEG between adjacent and healthy mucosa were overrepresented in the new active nodes of the adjacent mucosa network (empirical p-value < 10-4) suggesting that DEG are not only performing common functions but also co-regulated in a sub-transcriptional network not active in healthy mucosa samples. Out of 895 DEG, 60 (13 ) were transcription factors (TF), and random re-sampling of genes among the complete dataset revealed that DEG were significantly enriched in TF (empirical p-value < 0.001). Among these 60 TF, 35 were specific of the adjacent mucosa transcriptional network. TF were ranked taking into account the total number of their targets (degree) and the proportion of targets in our DEG list. This rank suggested sub-networks specifically active in adjacent mucosa tissue. TF with higher PubMed ID:http://www.ncbi.nlm.nih.gov/pubmed/20704779 rank have been more distinct of adjacent mucosa, and showed larger values of eccentricity (a topological network measure of your spreading of a node in the network) and lower values of closeness centrality (Table 1). Genes from the AP-1 complex (Fosb and Jun) ranked 1st inside the TF list. The AP-1 subunits Fos, Junb, Mafb and Atf3 also appeared inside the list. Prior GSEA analysis also had revealed as most significant motive “GenesSanz-Pamplona et al. Molecular Cancer 2014, 13:46 http://www.molecular-cancer.com/content/13/1/Page six ofABCFigure 4 (See legend on next page.)Sanz-Pamplona et al. Molecular Cancer 2014, 13:46 http://www.molecular-cancer.com/content/13/1/Page 7 of(See figure on earlier page.) Figure 4 DEG analysis inside the framework of transcriptional networks. A. Venn Diagram showing the overlap amongst nodes in adjacent mucosa transcriptional network (blue) and healthful mucosa transcriptional network (green). DEG were merged together with the two transcriptional networks. B. Expression correlation in between transcription things Jun and Fos in adjacent (blue) and healthy mucosa (green). C. Gene expression levels of AP-1 subunits in healthful mucosa (green) adjacent mucosa (blue) and tumor tissue (red).with promoter regions [-2 kb,2 kb] around transcription start internet site containing the motif TGACTCANN.
