DNA hypermethylation and decreased mRNA expression of MAL, PRIMA1, PTGDR and SFRP1 in colorectal adenoma and cancer. Issue 1 (December 2015) - Kalmár, Alexandra
Abstract Background Colorectal cancer (CRC) development is accompanied by changes in expression for several genes; but the details of the underlying regulatory procesess remain unknown. Our aims were to assess the role of epigenetic processes in tumour formation and to identify characteristic DNA methylation and miRNA alterations in the colorectal adenoma-carcinoma sequence. Methods Whole genome expression profiling was performed on colonic biopsy samples (49 healthy normal, 49 colorectal adenoma (AD), 49 CRC); on laser capture microdissected (LCM) epithelial and stromal cells from 6 CRC-normal adjacent tissue (NAT) samples pairs, and on demethylated human CRC cell lines using HGU133 Plus 2.0 microarrays (Affymetrix). Methylation status of genes with gradually altering expression along the AD-CRC sequence was further analysed on 10–10 macrodissected and 5–5 LCM samples from healthy colon, from adenoma and from CRC biopsy samples using bisulfite-sequencing PCR (BS-PCR) followed by pyrosequencing.In silico miRNA prediction for the selected genes was performed with miRWALK algorithm, miRNA expression was analysed on 3 CRC-NAT sample pairs and 3 adenoma tissue samples using the Human Panel I + II (Exiqon). SFRP1 immunohistochemistry experiments were performed. Results A set of transcripts (18 genes includingMAL, SFRP1, SULT1A1, PRIMA1, PTGDR ) showed decreasing expression (p < 0.01) in the biopsy samples along the adenoma-carcinoma sequence. Three of those (COL1A2, SFRP2, SOCS3 ) showed hypermethylation andTHBS2 showed hypomethylation both in AD and in CRC samples compared to NAT, whileBCL2, PRIMA1 andPTGDR showed hypermethylation only in the CRC group. miR-21 was found to be significantly (p < 0.01) upregulated in adenoma and tumour samples compared to the healthy colonic tissue controls and could explain the altered expression of genes for which DNA methylation changes do not appear to play role (e.g.BCL2, MAL, PTGS2 ). Demethylation treatment could upregulate gene expression of genes that were found to be hypermethylated in human CRC tissue samples. Decreasing protein levels of SFRP1 was also observed along the adenoma-carcinoma sequence. Conclusion Hypermethylation of the selected markers (MAL, PRIMA1, PTGDR andSFRP1 ) can result in reduced gene expression and may contribute to the formation of colorectal cancer.