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Aim: To analyze colorectal carcinogenesis and age-related DNA methylation alterations of gene sequences associated with epigenetic clock CpG sites., Methods: In silico DNA methylation analysis of 353 epigenetic clock CpG sites published by Steve Horvath was performed using methylation array data for a set of 123 colonic tissue samples [64 colorectal cancer (CRC), 42 adenoma, 17 normal; GEO accession number: GSE48684]. Among the differentially methylated age-related genes, secreted frizzled related protein 1 ( SFRP1 ) promoter methylation was further investigated in colonic tissue from 8 healthy adults, 19 normal children, 20 adenoma and 8 CRC patients using bisulfite-specific PCR followed by methylation-specific high resolution melting (MS-HRM) analysis. mRNA expression of age-related "epigenetic clock" genes was studied using Affymetrix HGU133 Plus2.0 whole transcriptome data of 153 colonic biopsy samples (49 healthy adult, 49 adenoma, 49 CRC, 6 healthy children) (GEO accession numbers: GSE37364, GSE10714, GSE4183, GSE37267). Whole promoter methylation analysis of genes showing inverse DNA methylation-gene expression data was performed on 30 colonic samples using methyl capture sequencing., Results: Fifty-seven age-related CpG sites including hypermethylated PPP1R16B , SFRP1 , SYNE1 and hypomethylated MGP, PIPOX were differentially methylated between CRC and normal tissues ( P < 0.05, Δβ ≥ 10%). In the adenoma vs normal comparison, 70 CpG sites differed significantly, including hypermethylated DKK3 , SDC2 , SFRP1 , SYNE1 and hypomethylated CEMIP , SPATA18 ( P < 0.05, Δβ ≥ 10%). In MS-HRM analysis, the SFRP1 promoter region was significantly hypermethylated in CRC (55.0% ± 8.4 %) and adenoma tissue samples (49.9% ± 18.1%) compared to normal adult (5.2% ± 2.7%) and young (2.2% ± 0.7%) colonic tissue ( P < 0.0001). DNA methylation of SFRP1 promoter was slightly, but significantly increased in healthy adults compared to normal young samples ( P < 0.02). This correlated with significantly increased SFRP1 mRNA levels in children compared to normal adult samples ( P < 0.05). In CRC tissue the mRNA expression of 117 age-related genes were changed, while in adenoma samples 102 genes showed differential expression compared with normal colonic tissue ( P < 0.05, logFC > 0.5). The change of expression for several genes including SYNE1 , CLEC3B , LTBP3 and SFRP1 , followed the same pattern in aging and carcinogenesis, though not for all genes ( e.g ., MGP)., Conclusion: Several age-related DNA methylation alterations can be observed during CRC development and progression affecting the mRNA expression of certain CRC- and adenoma-related key control genes., Competing Interests: Conflict-of-interest statement: The authors declare that no conflict of interest exists.

Aim: To characterize the regeneration-associated stem cell-related phenotype of hepatocyte-derived growth factor receptor (HGFR)-expressing cells in active ulcerative colitis (UC)., Methods: On the whole 38 peripheral blood samples and 38 colonic biopsy samples from 18 patients with histologically proven active UC and 20 healthy control subjects were collected. After preparing tissue microarrays and blood smears HGFR, caudal type homeobox 2 (CDX2), prominin-1 (CD133) and Musashi-1 conventional and double fluorescent immunolabelings were performed. Immunostained samples were digitalized using high-resolution Mirax Desk instrument, and analyzed with the Mirax TMA Module software. For semiquantitative counting of immunopositive lamina propria (LP) cells 5 fields of view were counted at magnification × 200 in each sample core, then mean ± SD were determined. In case of peripheral blood smears, 30 fields of view with 100 μm diameter were evaluated in every sample and the number of immunopositive cells (mean ± SD) was determined. Using 337 nm UVA Laser MicroDissection system at least 5000 subepithelial cells from the lamina propria were collected. Gene expression analysis of HGFR, CDX2, CD133, leucine-rich repeat-containing G-protein coupled receptor 5 (Lgr5), Musashi-1 and cytokeratin 20 (CK20) were performed in both laser-microdisscted samples and blood samples by using real time reverse transcription polymerase chain reaction (RT-PCR)., Results: By performing conventional and double fluorescent immunolabelings confirmed by RT-PCR, higher number of HGFR (blood: 6.7 ± 1.22 vs 38.5 ± 3.18; LP: 2.25 ± 0.85 vs 9.22 ± 0.65; P < 0.05), CDX2 (blood: 0 vs 0.94 ± 0.64; LP: 0.75 ± 0.55 vs 2.11 ± 0.75; P < 0.05), CD133 (blood: 1.1 ± 0.72 vs 8.3 ± 1.08; LP: 11.1 ± 0.85 vs 26.28 ± 1.71; P < 0.05) and Musashi-1 (blood and LP: 0 vs scattered) positive cells were detected in blood and lamina propria of UC samples as compared to controls. HGFR/CDX2 (blood: 0 vs 1 ± 0.59; LP: 0.8 ± 0.69 vs 2.06 ± 0.72, P < 0.05) and Musashi-1/CDX2 (blood and LP: 0 vs scattered) co-expressions were found in blood and lamina propria of UC samples. HGFR/CD133 and CD133/CDX2 co-expressions appeared only in UC lamina propria samples. CDX2, Lgr5 and Musashi-1 expressions in UC blood samples were not accompanied by CK20 mRNA expression., Conclusion: In active UC, a portion of circulating HGFR-expressing cells are committed to the epithelial lineage, and may participate in mucosal regeneration by undergoing mesenchymal-to-epithelial transition.

The latest avenue of research is revealing the existence of and role for the colonic stem cells in the physiological renewal of the mucosa and in pathological circumstances where they have both positive and negative effects. In the case of human colon, different levels of stem cell compartments exist. First, the crypt epithelial stem cells, which have a role in the normal crypt epithelial cell dynamics and in colorectal carcinogenesis. Close to the crypts, the second layer of stem cells can be found; the local subepithelial stem cell niche, including the pericryptic subepithelial myofibroblasts that regulate the epithelial cell differentiation and have a crucial role in cancer progression and chronic inflammation-related fibrosis. The third level of stem cell compartment is the immigrating bone-marrow-derived stem cells, which have an important role in wound healing after severe mucosal inflammation, but are also involved in cancer invasion. This paper focuses on stem cell biology in the context of physiological and pathological processes in the human colon.