Two clusters did not show precise functions but essentially these described as characteristic of adjacent mucosa. These benefits recommend that the smaller cluster of adjacent samples was just an intense phenotype of these samples. Interestingly, this pattern was also observed inthe validation dataset (see heatmap in Additional file 4: Figure S2).Transcriptional regulation of differentially expressed genes among adjacent and wholesome mucosaWe hypothesized that this differential expression could possibly be triggered by a transcriptional system, activated only in adjacent mucosa by the presence of your tumor, and usually silenced in wholesome mucosa. This hypothesis was supported by the GSEA final results, in which 312 transcription things motifs had been found to be statistically associated 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 had been additional certain of adjacent mucosa, and showed higher values of eccentricity (a topological network measure of the spreading of a node in the network) and lower values of closeness centrality (Table 1). Genes from the AP-1 complicated (Fosb and Jun) ranked 1st inside the TF list. The AP-1 subunits Fos, Junb, Mafb and Atf3 also appeared within the list. Preceding GSEA evaluation also had revealed as most important motive “GenesSanz-Pamplona et al. Molecular Cancer 2014, 13:46 http://www.molecular-cancer.com/purchase PAC-14028 content/13/1/Page 6 ofABCFigure 4 (See legend on subsequent page.)Sanz-Pamplona et al. Molecular Cancer 2014, 13:46 http://www.molecular-cancer.com/content/13/1/Page 7 of(See figure on preceding web page.) Figure 4 DEG analysis inside the framework of transcriptional networks. A. Venn Diagram showing the overlap between nodes in adjacent mucosa transcriptional network (blue) and healthier mucosa transcriptional network (green). DEG were merged using the two transcriptional networks. B. Expression correlation between transcription factors Jun and Fos in adjacent (blue) and healthier 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] about transcription start out site containing the motif TGACTCANN.
