Abstract 422: DNA hypermethylation or upregulated miRNA21 expression potentially leads to decreased mRNA expression of COL1A2, SFRP2, SOCS3, BCL2, MAL and PTGS2 in left-sided colorectal adenoma and cancer
Authors: Molnar Bela , Peterfia Balint , Kalmar Alexandra , Hollosi Peter , Nagy Zsofia Brigitta , Wichmann Barnabas , Kovalszky Ilona , Tulassay Zsolt 
Published: May 2015

Background: It is well known that beside genetic factors such as mutations, epigenetic mechanisms can also contribute to colorectal cancer formation and progression. Although several genes influenced by DNA methylation and miRNA expression alteration have been identified, our knowledge of the background of gene expression alterations during CRC development remains incomplete.

Aims: Our aims were to identify DNA methylation markers and miRNAs playing role in left-sided CRC development on the basis of gene expression alterations along the adenoma-carcinoma formation.

Materials & methods: Whole genome expression profiling was performed by using HGU133 Plus 2.0 microarrays (Affymetrix) on healthy colonic (n=49), colorectal adenoma (n=49) and left-sided CRC (n=49) biopsy samples and also on laser microdissected (LCM) epithelial and stromal cells from healthy (n=6) and CRC (n=6) samples. Methylation status of genes with gradually decreasing or increasing expression along the adenoma-carcinoma sequence were analyzed on macrodissected (n=10) and LCM (n=5) healthy colonic, adenomatous biopsy (n=10) and LCM (n=5), macrodissected (n=10) and LCM (n=5) left-sided colorectal cancer samples using bisulfite-sequencing PCR (BS-PCR) followed by pyrosequencing. In silico miRNA prediction for the selected genes with miRWALK algorithm, miRNA expression was analyzed on colorectal cancers (n=3), adenomas (n=3) and normal tissue adjacent to tumor (NAT)(n=3) samples using the Human Panel I + II with the miRCURY Universal RT microRNA PCR protocol (Exiqon). In order to investigate the potential impact of DNA methylation on the protein levels PTGDR and SFRP1 immunohistochemistry experiments were performed.

Results: A set of transcripts (18 genes including MAL, SFRP1, SULT1A1, PRIMA1, PTGDR) showed decreasing expression (p≤0,01) in the biopsy samples along the adenoma-carcinoma sequence. COL1A2, SFRP2, SOCS3 showed hypermethylation and THBS2 showed hypomethylation both in the adenomas and tumor samples compared to NAT, while BCL2, PRIMA1 and PTGDR showed hypermethylation only in the CRC group. miR-21 was found to be significantly (p&lt;0,01) upregulated in the adenoma and tumor samples compared to the healthy colonic tissue controls that can potentially influence the expression of genes without remarkable DNA methylation alteration (e.g. BCL2, MAL, PTGS2). Decreasing protein levels of PTGDR and SFRP1 could be observed along the adenoma-carcinoma sequence.

Conclusion: Genome-wide gene expression-based screening was found to be a suitable approach for the identification of genes, that can be potentially downregulated by DNA hypermethylation or miRNA upregulation. Hypermethylation of the selected markers (COL1A2, SFRP2, SOCS3) or miR21 upregulation might result in reduced expression and may contribute to the formation of colorectal cancer.

Citation Format: Bela Molnar, Balint Peterfia, Alexandra Kalmar, Peter Hollosi, Zsofia Brigitta Nagy, Barnabas Wichmann, Ilona Kovalszky, Zsolt Tulassay. DNA hypermethylation or upregulated miRNA21 expression potentially leads to decreased mRNA expression of COL1A2, SFRP2, SOCS3, BCL2, MAL and PTGS2 in left-sided colorectal adenoma and cancer. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 422. doi:10.1158/1538-7445.AM2014-422