1,25(OH)D对结直肠癌中Dickkopf-1甲基化的影响。

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

作者信息

Sun Hongyan, Yang Liehao, Li Nan, Hu Yue, Hu Qianying, Zhou Zilong, Cong Xianling

机构信息

Department of Biobank, China-Japan Union Hospital of Jilin University, Changchun, China.

Department of Dermatology, China-Japan Union Hospital of Jilin University, Changchun, China.

出版信息

Clin Epigenetics. 2025 Mar 26;17(1):52. doi: 10.1186/s13148-025-01857-5.

DOI:10.1186/s13148-025-01857-5

PMID:40140935

原文链接:https://pmc.ncbi.nlm.nih.gov/articles/PMC11948728/

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)D can induce DKK1 expression in colorectal cancer. However, whether 1,25(OH)D 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)D 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)D induced DKK1 expression in colorectal cancer cells, and pyrosequencing revealed that 1,25(OH)D 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)D upregulates DKK1 expression by inducing demethylation of the DKK1 promoter and 5'UTR in specific colorectal cancer cell lines.

摘要

背景

维生素D是一种脂溶性维生素，对结直肠癌具有保护作用。多项研究已确定维生素D与不同类型肿瘤中DNA甲基化变化之间的关联。Dickkopf-1（DKK1）抑制Wnt/β-连环蛋白信号通路，1,25(OH)D可诱导结直肠癌中DKK1的表达。然而，1,25(OH)D是否能通过调节结直肠癌中的DNA甲基化来影响DKK1的表达尚不清楚。

方法

本研究纳入了57例结直肠癌（CRC）患者和55例健康对照。分别通过酶联免疫吸附测定（ELISA）和液相色谱-串联质谱法测量血清DKK1和25(OH)D水平，并分析DKK1与临床病理特征及25(OH)D的相关性。将DKK1表达质粒转染到细胞中，通过CCK8、伤口愈合和迁移实验评估DKK1在CRC进展中的功能意义。采用亚硫酸氢盐扩增测序（BSAS）和甲基化特异性PCR检测结直肠癌细胞和组织中DKK1的甲基化状态。通过焦磷酸测序研究1,25(OH)D对DKK1甲基化的影响。进行双荧光素酶报告基因实验以研究CpG岛甲基化对DKK1转录活性的影响。

结果

血清DKK1水平降低与结直肠癌患者的神经浸润及25(OH)D状态密切相关。DKK1的过表达降低了结直肠癌细胞的增殖和迁移能力。通过BSAS检测了CRC细胞和组织中DKK1（-195至+231）的甲基化模式，包括31个CpG位点。与相邻正常组织相比，肿瘤组织中位于启动子、5'非翻译区（UTR）和外显子1的多个CpG位点的甲基化水平升高。DKK1高甲基化与结直肠癌细胞和组织中DKK1表达降低相关。1,25(OH)D诱导结直肠癌细胞中DKK1的表达，焦磷酸测序显示1,25(OH)D处理诱导位于启动子（-97至-32）和5'UTR（+39至+97）的CpG位点去甲基化。双荧光素酶报告基因实验进一步证实CpG岛甲基化（-120至+225）直接抑制DKK1转录。