Fig. 1

MiRNA expression profiling and functional enrichment analysis in progressive stages of murine CRC development: a Laser capture microdissection (LCM) of pretumoral lesions and tumors. First row, standard hematoxylin-eosin staining; second row, samples before LCM; third row, samples after LCM. Original magnification × 4 (scale bar 200 μm), × 20 (scale bar 50 μm), or × 40 (scale bar 20 μm). b Clustering of phases and control samples based on the first two principal components (PC1 and PC2) in a p partial least square - discriminant analysis (sPLS-DA) model. Control: normal colon mucosa of untreated mice. c Volcano plots of differentially expressed (DE) miRNAs (P value < 0.05 and |fold change| > 2) (red dots), miRNAs with only |fold change| > 2 (orange dots), and other miRNAs (black dots). The results were obtained through comparisons between each phase and control samples. d Venn diagram representing the number of miRNAs altered in each phase and shared between different phases (numbers in Venn diagram) of CRC. Sets of well-known CRC-associated miRNAs are indicated for each CRC phase (blue: down-regulated; red: up-regulated). e Enriched biological functions regulated by the DE miRNAs for Tumor (T) vs. Normal (N) of EphA2- and EphB2-positive cell comparisons. EphA2_high tumor cells are significantly enriched in activated functions related to cell proliferation, cell viability and cell death, angiogenesis, cell migration, cell invasion and growth of tumor (P < 0.0001). EphB2_high tumor cells show instead a significant enrichment of functions related to cell viability and a decreased or no activation of cell death/apoptosis (P < 0.0001). EMT activation and cell differentiation inactivation are also observed in EphB2_high tumor cells, a behavior coherently inverted in EphB2_low tumor cells (P < 0.0001). Sizes of bubbles are proportional to the statistical significance of the enriched functions (Fisher’s exact test). Gradients of color vary from red (activation) to green (inhibition) according to the Z-scores calculated for each enriched function and for each comparison. Functions are grouped into macro-categories. f Biological functions regulated by the DE miRNAs (and their target genes) with concordant fold changes between EphB2_high cells and tissues and EphA2_high cells and tissues. The outer circle marks the biological functions that are enriched in the EphA2_high and EphB2_high cells. The heatmap represents -log(P value) for each biological function evaluated in the four tissue types of adenocarcinoma (outer), adenoma, microadenoma, and ACF (inner). Blue and red rectangles represent the predicted activation and inactivation, respectively, of biological functions. These rectangles are linked by colored edges according to the macro-categories they belong to (proliferation, differentiation, cell morphology, etc.). Statistical significance was calculated using Fisher’s exact test as implemented in IPA. Abbreviations: CLs, cell lines; CRC, colorectal cancer; ECs, epithelial cells; ECLs, EC lines; ECM, extracellular matrix; EMT, epithelial-mesenchymal transition; MSCs, mesenchymal stem cells