Aging related methylation influences the gene expression of key control genes in colorectal cancer and adenoma.

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.