Abstract
BackgroundThe aberrant methylation of CpG islands in the promoter is associated with colorectal cancer (CRC) carcinogenesis. In our previous study, the promoter of alcohol dehydrogenase, iron containing, 1 (ADHFE1) was most highly methylated in CRC compared to normal colorectal mucosa. In this study, we examined the expression and function of the ADHFE1 in CRC.MethodsWe examined the promoter methylation and mRNA expression of ADHFE1 with 5-aza-2′-deoxycytidine (5-Aza-2-dC) in 12 CRC cell lines, 124 paired CRC and adjacent normal mucosa, and 59 advanced adenomas. To confirm methylation of ADHFE1, we performed bisulfite genomic sequencing in 3 CRC cell lines, 6 paired CRC and adjacent normal mucosa. ADHFE1 protein expression was studied using western blot and immunohistochemistry, respectively in the 36 and 243 paired CRC and adjacent normal tissue. We transfected the DLD-1 with pcDNA3.1 vector containing ADHFE1 and examined the expression of differentiation marker, such as ALP, CEA and Cdx2. We examined the ADHFE1 expression at distinct developmental stages in mouse embryos.ResultsThe ADHFE1 promoter was hypermethylated in all CRC cell lines, 81.8% in CRCs, and 84.7% in advanced adenomas, with reciprocal change by 5-Aza-2-dC. The expression of ADHFE1 mRNA was down-regulated in all CRC cell lines and 96.3% in CRC tissues. The expression of ADHFE1 protein was down-regulated in 91.7% of CRC tissues. In the immunohistochemistry, normal epithelial cells at the crypt top showed very strong ADHFE1 expression, whereas they were much weaker at the crypt base. In CRC, the good differentiation was significantly associated with high ADHFE1 expression. The activity of differentiation marker, such as ALP and CEA, was higher in pcDNA3.1-ADHFE1 transfected CRC cells with consistent correlation with ADHFE1 protein than control. In mouse embryos, ADHFE1 in the large intestine was the first detected at E15.5. At E18.5, ADHFE1 was predominantly expressed in the top of the mature crypt epithelium.ConclusionsIt showed that the hypermethylation of ADHFE1 promoter in CRC is concordance with down-regulation of ADHFE1 mRNA and ADHFE1 protein. ADHFE1 has an important role of differentiation in CRC, as well as normal colorectal mucosa and embryonic developmental processes.
Highlights
The aberrant methylation of CpG islands in the promoter is associated with colorectal cancer (CRC) carcinogenesis
Correlation between Alcohol Dehydrogenase (ADHFE1) promoter methylation and ADHFE1 down-regulation in CRC cell lines To assay the aberrant DNA methylation of ADHFE1, we examined the 12 CRC cell lines by methylation-specific PCR
All 12 CRC cell lines showed more than 10% percentage of methylated reference (PMR) (Figure 1)
Summary
The aberrant methylation of CpG islands in the promoter is associated with colorectal cancer (CRC) carcinogenesis. The promoter of alcohol dehydrogenase, iron containing, 1 (ADHFE1) was most highly methylated in CRC compared to normal colorectal mucosa. Methods: We examined the promoter methylation and mRNA expression of ADHFE1 with 5-aza-20-deoxycytidine (5-Aza-2-dC) in 12 CRC cell lines, 124 paired CRC and adjacent normal mucosa, and 59 advanced adenomas. ADHFE1 protein expression was studied using western blot and immunohistochemistry, respectively in the 36 and 243 paired CRC and adjacent normal tissue. The most common epigenetic alteration in CRC is aberrant DNA methylation, in which a methyl group is added to the cytosine base in the dinucleotide sequence CpG islands, which are often associated with the promoter [1,2]. Several genes involved in CRC carcinogenesis found to be silenced by DNA methylation include: MCC, MLH1, MGMT, APC, hMLH1, GATA-4, GATA-5, TFPI2, and SOX17 [2,3,4,5,6,7,8]
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