DNA hypermethylation and decreased mRNA expression of MAL, PRIMA1, PTGDR and SFRP1 in colorectal adenoma and cancer.
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 including MAL, 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 and THBS2 showed hypomethylation both in AD and in CRC samples compared to NAT, while BCL2, PRIMA1 and PTGDR 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.


CONCLUSIONS
Hypermethylation of the selected markers (MAL, PRIMA1, PTGDR and SFRP1) can result in reduced gene expression and may contribute to the formation of colorectal cancer.
2nd Department of Internal Medicine, Semmelweis University, Budapest, Hungary. alexandra.kalmar@gmail.com.|Molecular Medicine Research Group, Hungarian Academy of Sciences, Budapest, Hungary. alexandra.kalmar@gmail.com.
Alexandra
A
Kalmár
2nd Department of Internal Medicine, Semmelweis University, Budapest, Hungary. peterfiab@gmail.com.|Molecular Medicine Research Group, Hungarian Academy of Sciences, Budapest, Hungary. peterfiab@gmail.com.
Bálint
B
Péterfia
1st Department of Pathology and Experimental Cancer Research, Semmelweis University, Budapest, Hungary. phollosi@yahoo.com.|Tumour Progression Research Group, Hungarian Academy of Sciences, Budapest, Hungary. phollosi@yahoo.com.
Péter
P
Hollósi
Molecular Medicine Research Group, Hungarian Academy of Sciences, Budapest, Hungary. orsg1@yahoo.com.
Orsolya
O
Galamb
Molecular Medicine Research Group, Hungarian Academy of Sciences, Budapest, Hungary. spisak_sandor@yahoo.com.
Sándor
S
Spisák
Molecular Medicine Research Group, Hungarian Academy of Sciences, Budapest, Hungary. wwbarna@yahoo.com.
Barnabás
B
Wichmann
Department of Physics of Complex Systems, Eötvös Loránd University, Budapest, Hungary. bodri.mh3@gmail.com.
András
A
Bodor
2nd Department of Internal Medicine, Semmelweis University, Budapest, Hungary. drtothkinga@yahoo.com.
Kinga
K
Tóth
2nd Department of Internal Medicine, Semmelweis University, Budapest, Hungary. arpad.patai@gmail.com.
Árpád V
ÁV
Patai
Molecular Medicine Research Group, Hungarian Academy of Sciences, Budapest, Hungary. valczg@yahoo.com.
Gábor
G
Valcz
2nd Department of Internal Medicine, Semmelweis University, Budapest, Hungary. nagyzsofiab@gmail.com.
Zsófia Brigitta
ZB
Nagy
1st Department of Pathology and Experimental Cancer Research, Semmelweis University, Budapest, Hungary. kubakvivien@gmail.com.
Vivien
V
Kubák
2nd Department of Internal Medicine, Semmelweis University, Budapest, Hungary. tulassay@bel2.sote.hu.|Molecular Medicine Research Group, Hungarian Academy of Sciences, Budapest, Hungary. tulassay@bel2.sote.hu.
Zsolt
Z
Tulassay
1st Department of Pathology and Experimental Cancer Research, Semmelweis University, Budapest, Hungary. koval@korb1.sote.hu.
Ilona
I
Kovalszky
2nd Department of Internal Medicine, Semmelweis University, Budapest, Hungary. molnar.bela1@med.semmelweis-univ.hu.|Molecular Medicine Research Group, Hungarian Academy of Sciences, Budapest, Hungary. molnar.bela1@med.semmelweis-univ.hu.
Béla
B
Molnár
D000236:Adenoma / Q000235:genetics* / Q000378:metabolism / Q000473:pathology; D014408:Biomarkers, Tumor / Q000378:metabolism; D045744:Cell Line, Tumor; D015179:Colorectal Neoplasms / Q000235:genetics* / Q000378:metabolism / Q000473:pathology; D019175:DNA Methylation; D015972:Gene Expression Regulation, Neoplastic; D006801:Humans; D007150:Immunohistochemistry; D036341:Intercellular Signaling Peptides and Proteins / Q000096:biosynthesis / Q000235:genetics*; D008565:Membrane Proteins / Q000096:biosynthesis / Q000235:genetics*; D062806:Myelin and Lymphocyte-Associated Proteolipid Proteins / Q000096:biosynthesis / Q000235:genetics*; D009419:Nerve Tissue Proteins / Q000096:biosynthesis / Q000235:genetics*; D016133:Polymerase Chain Reaction; D011401:Promoter Regions, Genetic; D012333:RNA, Messenger / Q000235:genetics; D011971:Receptors, Immunologic / Q000096:biosynthesis / Q000235:genetics*; D011982:Receptors, Prostaglandin / Q000096:biosynthesis / Q000235:genetics*; D000094742:Secreted Frizzled-Related Proteins
D014408:Biomarkers, Tumor; D036341:Intercellular Signaling Peptides and Proteins; D008565:Membrane Proteins; D062806:Myelin and Lymphocyte-Associated Proteolipid Proteins; D009419:Nerve Tissue Proteins; D012333:RNA, Messenger; D011971:Receptors, Immunologic; D011982:Receptors, Prostaglandin; C569026:MAL protein, human; C446290:PRIMA1 protein, human; C117636:SFRP1 protein, human; D000094742:Secreted Frizzled-Related Proteins; C036293:prostaglandin D2 receptor