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Methylation of WT1, CA10 in Peripheral Blood Leukocyte Is Associated with Breast Cancer Risk

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Methylation of WT1, CA10 in Peripheral Blood Leukocyte Is Associated with Breast Cancer Risk Ge et al. BMC Cancer (2020) 20:713 https://doi.org/10.1186/s12885-020-07183-8 RESEARCH ARTICLE Open Access Methylation of WT1, CA10 in peripheral blood leukocyte is associated with breast cancer risk: a case-control study Anqi Ge1, Song Gao2, Yupeng Liu1, Hui Zhang1, Xuan Wang1, Lei Zhang1, Da Pang2* and Yashuang Zhao1* Abstract Background: Studies have shown that abnormal changes of specific-gene DNA methylation in leukocytes may be associated with an elevated risk of cancer. However, associations between the methylation of the zinc-related genes, WT1 and CA10, and breast cancer risk remain unknown. Methods: The methylation of WT1 and CA10 was analyzed by methylation-sensitive high-resolution-melting (MS- HRM) in a case-control study with female subjects (N = 959). Logistic regression was used to analyze the associations, and propensity score (PS) method was used to adjust confounders. Results: The results showed that WT1 hypermethylation was associated with an increased risk of breast cancer, with an odds ratio (OR) of 3.07 [95% confidence interval (CI): 1.67–5.64, P < 0.01]. Subgroup analyses showed that WT1 hypermethylation was specifically associated with an elevated risk of luminal A subtype (OR = 2.62, 95% CI: 1.11– 6.20, P = 0.03) and luminal B subtype (OR = 3.23, 95% CI: 1.34–7.80, P = 0.01). CA10 hypermethylation was associated with an increased risk of luminal B subtype (OR = 1.80, 95% CI: 1.09–2.98, P = 0.02). Conclusion: The results of the present study suggest that the hypermethylation of WT1 methylation in leukocytes is significantly associated with an increased risk of breast cancer. The hypermethylation of WT1 is associated with an increased risk of luminal subtypes of breast cancer, and the hypermethylation of CA10 is associated with an increased risk of luminal B subtype of breast cancer. Keywords: Breast cancer, CA10, WT1, DNA methylation, Leukocytes Background including chromosomal instability [3] and gene expres- Breast cancer is one of the most common malignancies sion. The hypermethylation of CpG regions in specific in women worldwide [1] and presents with different mo- genes contribute to neoplastic formation through the lecular subtypes, including luminal A, luminal B, HER2- transcriptional silencing of tumor suppressor genes. Ab- enriched, and basal-like that also called triple negative errant patterns of specific gene methylation can help [2]. As a major type of epigenetic modification, DNA identifying differences in breast cancer subtypes [2], and methylation is involved in regulating cellular processes, showing promise for utilizing in large-scale epidemio- logical studies. It has been suggested that leukocyte * Correspondence: [email protected]; [email protected] DNA methylation, as a simple non-invasive blood 2Department of Breast Surgery, the Tumor Hospital of Harbin Medical marker [4, 5], could serve as a surrogate for systematic University, 150 Haping Street, Nangang District, Harbin 150081, Heilongjiang methylation activity and offers great potential for pre- Province, People’s Republic of China 1Department of Epidemiology, School of Public Health, Harbin Medical dicting the increased risk of breast cancer [6]. University, 57 Baojian Street, Nangang District, Harbin 150081, Heilongjiang Province, People’s Republic of China © The Author(s). 2020 Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data. Ge et al. BMC Cancer (2020) 20:713 Page 2 of 9 Wilm’s Tumor gene (WT1) is a tumor suppressor gene female. In addition, all control participants were asked which involved in human cell growth and differentiation. about their disease history in a questionnaire, and indi- WT1 has been reported to be significantly different viduals who reported a history of any cancer were ex- methylated in the tissues of hepatocellular carcinoma cluded from our final subjects. Finally, 402 female breast [7], lung cancer [8] and breast cancer [9]. WT1 aberrant cancer cases and 557 female controls were included in methylation may lead to a reduction or absence of WT1 our study. Blood sample (5 mL) was collected from each expression, which results in the overexpression of the participant and then stored at − 80 °C. insulin-like growth factor I receptor (IGF 1R) and insulin-like growth factor II (IGF II), thereby promoting Data collection breast cancer process [10–12]. CA10 is a member of the All subjects were interviewed face-to-face by trained in- carbonic anhydrase family, which is a large family of vestigators with normalized questioning methods. The zinc-containing metalloenzymes that catalyze the revers- questionnaire was adopted from the study by Shu et al. ible hydration of carbon dioxide and the dehydration of [17], and included information on demographic informa- carbonic acid [13]. Ivanov et al. suggested that the in- tion (age, ethnicity, and others); daily dietary intake (veg- duction or enhancement of carbonic anhydrase expres- etables, fruits, beverages, and snacks); behaviors sion may contribute to the tumor microenvironment by (smoking, drinking, physical activity and work activity); maintaining an extracellular acidic pH and helping the female-specific questions involving menstruation status, growth and metastasis of cancer cells [14]. Studies have breast disease history (lobular hyperplasia, cyst, and demonstrated that the abnormal expression of carbonic others); gynecologic surgery history (hysterectomy, anhydrase family by aberrant methylation is related with ovariotomy) and family history of cancer and breast can- gastric cancer and the metastasis of ovary tumors [13, cer. The questions involved in dietary and behavioral 15]. Furthermore, Wojdacz et al. reported that both were about the participants’ daily routine of 1 year prior WT1 and CA10 hypermethylation were significantly dif- to the interview. The basic demographic characteristics ferent between breast cancer tumor tissues and non- and environmental factors of the study subjects are pre- malignant tissues [16]. However, how the methylation of sented in Table S1. these two genes in leukocyte DNA affects breast cancer The study was validated with the GEO-GSE51032 susceptibility remains unclear. (IPEC-Italy cohort) dataset with a nested case control In this study, we investigated the associations between study design to analyze the association between the the methylation of WT1, CA10 in peripheral blood methylation of CA10 and WT1 and breast cancer risk. leukocyte DNA and breast cancer risk. We subsequently The blood samples were also collected and other an- used an external dataset of a nested case-control cohort thropometric measurements were taken. The sample se- within the EPIC-Italy cohort study as external data to lection criteria and the methods were reported by Riboli validate the association between gene methylation and et al. [18]. We extracted all 232 female breast cancer breast cancer risk. We also investigated the associations cases and all 340 female controls from this nested case- between the methylation of these two genes and the risk control study and located the methylation probes from of different molecular types of breast cancer. the Illumina 450 K array. The annotated CG sites cov- ered by our MS-HRM sequence are illustrated in Fig. 1. Methods Study subjects DNA extraction and bisulfite conversion We investigated the relationship between WT1 and DNA was extracted from peripheral blood samples using CA10 methylation and breast cancer risk using a case- a commercial DNA extraction kit (QIAamp DNA Blood control study. All the included breast cancer patients Mini Kit, Hilden, Germany). The concentration and the were newly diagnosed females and were recruited from purity of DNA were assessed using a Nanodrop 2000 the Tumor Hospital of Harbin Medical University from Spectrophotometer (Thermo Scientific, USA). Genomic 2010 to 2014. Female breast cancer subjects were in- DNA was bisulfite-modified with an EpiTect Bisulfite kit cluded if they diagnosed with invasive ductal carcinoma (Qiagen, Hilden, Germany). Bisulfite DNA was normal- (IDC) or ductal carcinoma in situ (DCIS), other types of ized to a concentration of 20 ng/mL and was stored at − breast cancer (such as lipoma of the breast, metastatic 20 °C for the following experiment. DNA extraction and breast cancer, etc.) were excluded from our study. Con- DNA sodium bisulfite modification were performed ac- trols were recruited from patients admitted to the cording to the manufacturers’ instructions. Orthopedic and Ophthalmology Department of the Sec- ond Affiliated Hospital of Harbin Medical University Gene methylation status analysis and volunteers from the Xiangfang community of Har- We performed methylation-sensitive high-resolution bin within the same period. All controls were also melting analysis (MS-HRM) to evaluate the methylation Ge et al. BMC Cancer (2020) 20:713 Page 3 of 9 Fig. 1 MS-HRM amplified sequence of WT1 and CA10 and the validated Cg sites in GSE51032 Fig. 2 The MS-HRM based method for WT1 and CA10 methylation detection. The figures showed normalized melting curves and melting peaks for standards methylation level and of WT1(A)(B) and CA10(C)(D).The methylation status of the standards were 0, 0.5, 1, 2, 5, 100%, respectively Ge et al.
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