Methylation of ZNF331 Is an Independent Prognostic Marker of Colorectal Cancer and Promotes Colorectal Cancer Growth Yuzhu Wang1,2, Tao He1, James G
Total Page：16
File Type：pdf, Size：1020Kb
Wang et al. Clinical Epigenetics (2017) 9:115 DOI 10.1186/s13148-017-0417-4 RESEARCH Open Access Methylation of ZNF331 is an independent prognostic marker of colorectal cancer and promotes colorectal cancer growth Yuzhu Wang1,2, Tao He1, James G. Herman3, Enqiang Linghu1, Yunsheng Yang1, François Fuks4, Fuyou Zhou5, Linjie Song6,7 and Mingzhou Guo1* Abstract Background: ZNF331 was reported to be a transcriptional repressor. Methylation of the promoter region of ZNF331 has been found frequently in human esophageal and gastric cancers. The function and methylation status of ZNF331 remain to be elucidated in human colorectal cancer (CRC). Methods: Six colorectal cancer cell lines, 146 cases of primary colorectal cancer samples, and 10 cases of noncancerous colorectal mucosa were analyzed in this study using the following techniques: methylation specific PCR (MSP), qRT-PCR, siRNA, flow cytometry, xenograft mice, MTT, colony formation, and transfection assays. Results: Loss of ZNF331 expression was found in DLD1 and SW48 cells, reduced expression was found in SW480, SW620, and HCT116 cells, and high level expression was detected in DKO cells. Complete methylation of the ZNF331 in the promoter region was found in DLD1 and SW48 cells, partial methylation was found in SW480, SW620, and HCT116 cells, and unmethylation was detected in DKO cells. Loss of/reduced expression of ZNF331 is correlated with promoter region methylation. Restoration of ZNF331 expression was induced by 5-aza-2′-deoxycytidine (DAC) in DLD1 and SW48 cells. These results suggest that ZNF331 expression is regulated by promoter region methylation in CRC cells. ZNF331 was methylated in 67.1% (98/146) of human primary colorectal cancer samples. Methylation of ZNF331 was significantly associated with tumor size, overall survival (OS), and disease-free survival (DFS) (p < 0.01, p < 0.01, p < 0.05). Methylation of ZNF331 was an independent poor prognostic marker for 5-year OS and 5-year DFS (both p < 0.05). ZNF331 suppressed cell proliferation and colony formation in CRC cells and suppressed human CRC cell xenograft growth in mice. Conclusions: ZNF331 is frequently methylated in human colorectal cancer, and the expression of ZNF331 is regulated by promoter region methylation. Methylation of ZNF331 is a poor prognostic marker of CRC. Keywords: ZNF331, Epigenetics, DNA methylation, Colorectal cancer Background Numerous prospective cohort epidemiology studies have Colorectal cancer (CRC) is the third most commonly di- identified specific dietary and lifestyle factors that either agnosed cancer in males and the second in females world- promote or protect against CRC [5–7]. Consumption of wide [1]. The incidence has sharply increased in the past red meat and animal fats increases CRC risk, whereas two decades in Eastern countries with changing environ- dietary fiber decreases risk [8, 9]. Other studies suggest mental factors, such as lifestyle and diet [2, 3]. Accumula- that the human gut microbiome is relatively stable in indi- tion of aberrant genetic and epigenetic changes plays an viduals over time except in the case of certain events such important role in CRC initiation and progression [4]. as food poisoning/infection or international travel. This likely reflects the hegemony of long-term dietary patterns on our gut microbiome [10, 11]. All these factors may * Correspondence: [email protected] 1Department of Gastroenterology & Hepatology, Chinese PLA General cause colorectal epithelial cell epigenetic changes and fur- Hospital, 28 Fuxing Road, Beijing 100853, China ther induces tumorigenesis. Thus, identification of new Full list of author information is available at the end of the article © The Author(s). 2017 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. 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. Wang et al. Clinical Epigenetics (2017) 9:115 Page 2 of 12 epigenetic biomarkers for diagnosis, prognosis, prediction, spectrophotometric analysis were used to check RNA and targeting therapy for CRC is necessary. quality and quantity. Total RNA (5 μg) was used to Zinc finger protein 331 (ZNF331) was first identified synthesize first-strand complementary DNA (cDNA) ac- from thyroid tumors [12]. It is also known as RITA cording to the manufacturer’s instructions (Invitrogen, (rearranged in thyroid adenoma), ZNF361, and ZNF463 Carlsbad, CA). The reaction mixture was diluted to [13]. The ZNF331 gene is located at chromosome 100 μl with water, and 2.5 μl of diluted cDNA mixture 19q13.42, a region in which loss of heterozygosity was added to each 25 μl PCR reaction. The ZNF331 (LOH) was detected in prostate cancer [14]. In our pre- PCR primer sequences were as follows: 5′-TAGGTCA vious study, we found that the ZNF331 gene is fre- GCTCTAGCCTCTC-3′ (forward) and 5′-AGCGTACC quently methylated in human esophageal squamous cell TTCACATATCCAG-3′ (reverse). Thermal cycling pa- cancer (ESCC) and it serves as a tumor suppressor in rameters were as follows: 95 °C 5 min; (95 °C 30 s, 58 °C ESCC [15]. The function of ZNF331 in human CRC re- 30 s, and 72 °C 30 s) 35 cycles; 72 °C 5 min. The primers mains unclear. In this study, we analyzed the epigenetic for GAPDH were as follows: 5′-GAGTCAACG regulation and the function of ZNF331 in human CRC. GATTTGGTCGT-3′ (forward), and 5′-GACAAGCTT CCCGTTCTCAG-3′ (reverse). Thermal cycling parame- Methods ters were as follows: 95 °C 5 min; (95 °C 30 s, 58 °C 30 s Human tissue samples and cell lines and 72 °C 40 s) 25 cycles; 72 °C 5 min. The amplified A total of 146 cases of primary CRC and 10 cases of non- PCR products were examined by 1.5% agarose gels. Each cancerous colorectal mucosa were collected from the cDNA sample was analyzed in triplicate with the Ap- Chinese PLA General Hospital in Beijing between May plied Biosystems StepOnePlus Real-Time PCR Systems 2009 and November 2013. All cancer samples were classi- using SYBR Green Realtime PCR Master Mix (Toyobo, fied according to WHO Classification of Digestive Tu- Shanghai, China) according to the manufacturer’s in- mors: the 4th Edition. All samples were collected under structions. The relative amount of ZNF331 mRNA was the guidelines approved by the institutional review board normalized to GAPDH using the ΔΔCt method. at the Chinese PLA General Hospital. Among the patient cases, 98 cases were male and 48 cases were female. The Bisulfite modification, methylation-specific PCR, bisulfite median age was 60 years old (range 33–86 years old). All sequencing, and KRAS and BRAF mutation detection cancer samples were classified according to the TNM sta- Genomic DNA was extracted by the proteinase K ging system (AJCC2010), which included tumor stage I (n method. Cultured cells and fresh tissue samples were = 17), stage II (n =58),stageIII(n = 52), and stage IV (n = digested by DNA digestion buffer (pH 8.0, 10 mM Tris- 19). Six CRC cell lines, DLD1, SW48, HCT116, SW480, Cl, 25 mM EDTA, 1% SDS, 100 μg/ml proteinase K) and SW620, and DKO (DNMT1 and DNMT3b double knock- extracted by phenol/chloroform. The bisulfite modifica- out from HCT116 cells, a generous gift from Stephen tion assay was performed as previously described [16]. Baylin), were examined in this study and maintained in MSP primers were designed according to genomic se- 90% Roswell Park Memorial Institute (RPMI) 1640 media quences around the transcription start sites (TSS) and supplemented with 10% fetal bovine serum, 100 U/ml synthesized (BGI, Beijing, China) to detect unmethylated penicillin and 100 mg/ml streptomycin. HEK-293T cells (U) and methylated (M) alleles. The MSP primers were were cultured in Dulbecco’s modified Eagle’smediumsup- as follows: 5′-TAAGGTAGGACGTTTTTAGGGTCGC- plemented with 10% fetal bovine serum. All cell lines were 3′ (MF) and 5′-AACTCTACACGACGCAAATAAAAC cultured in an atmosphere of 5% carbon dioxide at 37 °C. CG-3′ (MR); 5′-TTTTAAGGTAGGATGTTTTTAGGG TTGT-3′ (UF) and 5′-ACAACTCTACACAACACAAA 5-Aza-2′-deoxycytidine treatment TAAAACCA-3′ (UR). The thermal cycling parameters CRC cell lines (DLD1, SW48, HCT116, SW480, SW620, were as follows: 95 °C 5 min; (95 °C 30 s, 60 °C 30 s and and DKO) were split to a low density (30% confluence) 72 °C 40 s) 35 cycles; 72 °C 5 min. The expected sizes of 12 h before treatment with 2 μM 5-aza-2′-deoxycytidine unmethylated and methylated products were 147 and (DAC, Sigma, MO, USA). Growth medium conditioned 142 bp, respectively. Bisulfite-treated DNA was also with DAC at 2 μM was exchanged every 24 h for a total amplified using bisulfite sequencing (BSSQ) primers that of 96 h. At the end of the treatment course, RNA was included the MSP region. The sequencing primers were extracted from the cells as described below. as follows: 5′-GGTTATGAGTTATATTTTTTAGAAG- 3′ (forward) and 5′-CTCRCTCCTCATTAAACTATAC- RNA isolation, semi-quantitative RT-PCR, and real-time 3′ (reverse). The thermal cycling parameters were as quantitative RT-PCR analyses follows: 95 °C 5 min; (95 °C 30 s, 55 °C 30 s and 72 °C Total RNA was isolated by Trizol reagent (Life Tech- 40 s) 35 cycles; 72 °C 5 min. Methylation status was de- nologies, MD, USA). Agarose gel electrophoresis and tected by MSP in four genes (RUNX3, CACNA1G, IGF2, Wang et al. Clinical Epigenetics (2017) 9:115 Page 3 of 12 and MLH1) to represent CpG island methylator pheno- supernatant was collected and filtered after 48 h.