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

IF 4.8 2区医学 Q1 GENETICS & HEREDITY

Clinical Epigenetics Pub Date : 2025-03-26 DOI:10.1186/s13148-025-01857-5

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

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引用次数: 0

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.

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1,25(OH)2D3对结直肠癌Dickkopf-1甲基化的影响。

背景：维生素D是一种脂溶性维生素，对结直肠癌有保护作用。几项研究已经确定了维生素D与不同类型肿瘤中DNA甲基化变化之间的联系。Dickkopf-1 （DKK1）抑制Wnt/β-catenin信号通路，1,25(OH)2D3可诱导DKK1在结直肠癌中的表达。然而，1,25(OH)2D3是否通过调节结直肠癌DNA甲基化影响DKK1表达尚不清楚。方法：选取57例结直肠癌患者和55例健康对照者作为研究对象。分别采用ELISA和液相色谱-串联质谱法检测各组血清DKK1和25(OH)D水平，分析DKK1与临床病理特征和25(OH)D的相关性。将DKK1表达质粒转染到细胞中，通过CCK8、伤口愈合和迁移试验来评估DKK1在结直肠癌进展中的功能意义。采用BiSulphite Amplicon Sequencing （BSAS）和甲基化特异性PCR检测结直肠癌细胞和组织的DKK1甲基化状态。采用焦磷酸测序法研究了1,25(OH)2D3对DKK1甲基化的影响。采用双荧光素酶报告基因法研究CpG岛甲基化对DKK1转录活性的影响。结果：大肠癌患者血清DKK1水平降低与神经浸润和25(OH)D水平密切相关。DKK1过表达降低结直肠癌细胞的增殖和迁移能力。通过BSAS检测CRC细胞和组织中DKK1的甲基化模式（- 195至+ 231），包括31个CpG位点。与邻近正常组织相比，肿瘤组织中位于启动子、5'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)2D3诱导结直肠癌细胞中的DKK1表达，焦磷酸测序显示，1,25(OH)2D3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。结论：DKK1在结直肠癌中具有抑瘤作用，125 (OH)2D3通过诱导特定结直肠癌细胞系中DKK1启动子和5'UTR的去甲基化上调DKK1的表达。

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来源期刊

Clinical Epigenetics ONCOLOGY-

自引率

5.30%

发文量

150

期刊介绍： Clinical Epigenetics, the official journal of the Clinical Epigenetics Society, is an open access, peer-reviewed journal that encompasses all aspects of epigenetic principles and mechanisms in relation to human disease, diagnosis and therapy. Clinical trials and research in disease model organisms are particularly welcome.