The effect of 1,25(OH)2D3 on Dickkopf-1 methylation in colorectal cancer.

Abstract

Background: Vitamin D is a fat-soluble vitamin that has a protective role in colorectal cancer. Several studies have identified the association between vitamin D and changes in DNA methylation in different types of tumours. Dickkopf-1 (DKK1) inhibits the Wnt/β-catenin signalling pathway, and 1,25(OH)₂D₃ can induce DKK1 expression in colorectal cancer. However, whether 1,25(OH)₂D₃ can affect DKK1 expression by regulating DNA methylation in colorectal cancer is not known.

Methods: Fifty-seven colorectal cancer (CRC) patients and fifty-five healthy controls were included in this study. Serum DKK1 and 25(OH)D levels were measured via ELISA and liquid chromatography‒tandem mass spectrometry, respectively, and the associations of DKK1 with clinicopathological characteristics and 25(OH)D were analysed. A DKK1 expression plasmid was transfected into cells to assess the functional significance of DKK1 in CRC progression via CCK8, wound healing and migration assays. BiSulphite Amplicon Sequencing (BSAS) and methylation-specific PCR were used to detect the DKK1 methylation status of colorectal cancer cells and tissues. The effect of 1,25(OH)₂D₃ on DKK1 methylation was investigated by pyrosequencing. A dual-luciferase reporter assay was performed to investigate the influence of CpG island methylation on DKK1 transcriptional activity.

Results: A decreased serum DKK1 level was closely associated with nerve infiltration and 25(OH)D status in patients with colorectal cancer. Overexpression of DKK1 reduced the proliferative and migratory capabilities of colorectal cancer cells. The methylation patterns of DKK1 (- 195 to + 231), including 31 CpG sites, were assayed via BSAS in CRC cells and tissues. Compared with those in adjacent normal tissues, the methylation levels of multiple CpG sites located in the promoter, 5'UTR and exon 1 were increased in tumour tissues. DKK1 hypermethylation was associated with decreased DKK1 expression in colorectal cancer cells and tissues. 1,25(OH)₂D₃ induced DKK1 expression in colorectal cancer cells, and pyrosequencing revealed that 1,25(OH)₂D₃ treatment induced demethylation of CpG sites located in the promoter (- 97 to - 32) and 5'UTR (+ 39 to + 97). The dual-luciferase reporter assay further confirmed that CpG island methylation (-120 to + 225) directly represses DKK1 transcription.

Conclusion: DKK1 functions as a tumour suppressor in colorectal cancer, and 1,25(OH)₂D₃ upregulates DKK1 expression by inducing demethylation of the DKK1 promoter and 5'UTR in specific colorectal cancer cell lines.