INTRODUCTION

Gastric cancer is one of the most common humancancers and the second leading cause of cancer-relatedhuman death worldwide. Several factors have beenknown to contribute to the tumorigenesis of gastriccancer, including H. pylori infection［1］, smoking［2］ andgastric ulcer ［3］, but the exact mechanisms of thetumorogenesis still remain unknown.In 2007, a novel tumor suppressor gene, Wilms'tumor gene on the X chromosome (WTX), was firstidentified in Wilms' tumor ［4］. WTX belongs to theFAM123 gene family. This family includes 3 members,and the other two are FAM123A and FAM123C. Theroles this gene family plays in signal transduction, cellbehaviors and human diseases are poorly understood. Itwas reported that WTX protein could negatively regulateWnt/β-catenin signaling by forming a complex withAXIN1, β-catenin, APC and β-TrCP2 to result in thedegradation of β-catenin［5-6］. WTX can also influenceantioxidant response ［7］ and cellular differentiation ［8］.Studies have shown that aberrant expression of WTX is

implicated not only in Wilms' tumor ［9］ but also inosteopathia striata with cranial sclerosis［10-12］. So far thefunctional roles of WTX in gastric cancer have not beenreported.In general, three reasons account for theinactivation of tumor suppressor genes, namely genemutation［13］, promoter region methylation［14］, and aberrantmicroRNA regulation ［15］. The condition of DNAmethylation is one of the most important aspects inexploring the functions and actions of genes. DNAmethylation is essential for regulating mammalian tissuedevelopment and suppressing gene activity by changingthe structure of the chromatins［16］. Aberrant DNA methy-lation is associated with gene silencing in cancer［17］andplays an important role in tumorigenesis. As animportant mechanism for the inactivation of tumorsuppressor genes and tumor-related genes, promoterCpG island hypermethylation is found in virtually allhuman cancer tissue types［18］. To explore the functions ofWTX, we detected the promoter methylation levels ofWTX in gastric cancer tissues and gastric cancer celllines.MATERIALS AND METHODS

Cell lines and tissue specimens

Gastric cancer cell lines, MGC803, SCG7901, andBGC823, were obtained from the American TypeCulture Collection (ATCC). All the cell lines werecultured in RPMI 1640 (Hyclone, Logan, Utah, USA)

Received: 2012-11-16 Accepted: 2012-12-20Supported by National Natural Science Foundation of China(81071989, 81272760), Natural Science Foundation of GuangdongProvince (S2011010004178), and Science and Technology Programof Guangdong Province (c1221020700008).*Corresponding author: ZHANG Qingling, E-mail: zqllc8@126.com

Promoter methylation of Wilms' tumor gene on the X-chromosome in gastric cancer

LIU Xia1, 2, WANG Qiming1, 2, NIU Huilin1, 2, YANG Xuexi3, SUN Jingzhe3, ZHANG Qingling1, 2*, DING

Yanqing1, 2

Department of Pathology, Nanfang Hospital1, Department of Pathology, College of Basic Medicine2, School of Biotechnology3, SouthernMedical University, Guangzhou 510515, China

Original Article

Abstract: Objective To investigate the changes in methylation levels of the promoters of the tumor suppressor gene Wilms'tumor gene on the X-chromosome (WTX) and its possible role in gastric cancer. Methods WTX promoter methylation levelswere detected in 20 pairs of specimens of gastric cancer and matched normal tissues and in 3 gastric cancer cell lines (MGC803,SCG7901, and BGC823) using the Sequenom MassARRAY quantitative analysis system. The gastric cancer cell line BGC823was treated with 5-aza-2'-deoxycytidine (5-aza-dC) for demethylation and the changes in the level of WTX promotermethylation were investigated. Results WTX promoter methylation levels were very low and showed no significantdifferences among normal gastric tissues, gastric cancer tissues and the 3 gastric cancer cell lines. In BGC823 cells, treatmentwith 5-aza-dC did not obviously affect the promoter methylation levels of WTX. Conclusion High methylation levels of WTXpromoters are rare in gastric cancer.

Key words: Wilms' tumor gene; X-chromosome; gastric cancer; promoter methylation.

doi 10.3969/j.issn.1673-4254.2013.03.02 J South Med Univ, 2013, 33(3): 318-321··318

supplemented with 5% fetal bovine serum (FBS)(Gibco-BRL, Invitrogen, Paisley, UK) in a humidifiedincubator in 5% CO2 at 37 ℃ . Twenty pairs of surgicalspecimens of human gastric cancer tissues and matchednormal tissues were provided by the Tumor Tissue Bankof Nanfang Hospital. The gastric cancer tissues included3 stages and were not classified in the study. The tissuespecimens were frozen in liquid nitrogen immediatelyafter surgery. All the gastric cancer cases werepathologically confirmed, and the normal tissues weretaken at least 5 cm away from the cancer lesions. TheTumor Tissue Bank of Nanfang Hospital possesses acomprehensive set of clinicopathological data, includingage, gender, size of primary tumor, tumor differentiation,lymph node metastasis and clinical stage. This studywas approved by the ethics committee of SouthernMedical University.WTX promoter methylation detection in tissue samplesand cell lines

DNA was extracted from 20 pairs of gastric cancerand normal tissues and 3 gastric cancer cell lines(MGC803, SCG7901, and BGC823) using a QIAampDNA mini kit (Qiagen, Valencia, CA) according to themanufacturer's instructions. The genomic DNA (300 ng)was treated with sodium bisulfite to convert unme-thylated cytosines to uracil using the EpiTect bisulfitekit (Qiagen, Germany). Sequenom MassARRAYquantitative methylation analysis (Sequenom, CA) wasused to detect WTX gene promoter methylation. Usingthree pairs of primers, PCR was performed to analyzethe three candidate methylation reagions of WTX. Thepromoters include WTX18: 5'-aggaagagagAGGAATTTGGGAAGATTGTAGAAAT-3' (F), 5'-cagtaatacgactcactatagggagaaggctAAACTCTAAATTCCAAATTAACCCTT-3'(R) covering 2044-2340 bp of WTX gene promotersequences; WTX29: 5'-aggaagagagGAGTGGGGAGTTTTAGTTAGTGGTT-3' (F), 5'-cagtaatacgactcactatagggagaaggctCCTAATCCTTAATACCCTCCTCCAA-3' (R) for1352-1834 bp; WTX31:5'-aggaagagagGGTAGGATTGGTTTTTTGAGGTTA-3' (F), 5'-cagtaatacgactcactatagggagaaggctCTTCCCCAAACTTTAAATAACTCCC-3'(R) for1598-1897 bp.PCR was first performed in a thermocycler(Eppendorf Mastercycler gradient, Germany) with 0.2μmol/L of each primer using the following protocol: 2min at 95 ℃ for activation of Platinum® Taq DNApolymerase, and 30 s at 95 ℃, 30 s at 56 ℃, and 30 s at72 ℃ for 45 cycles, followed by 5 min at 72 ℃ . Themethylation status of the PCR product was analyzed withEpi-typer software v1.0 (Sequenom) which generatedquantitative results for each CpG site.Methylation detection in 5-aza-dC-treated BGC-823 celllines

5-aza-2'-deoxycytidine (5-aza-dC) can lower theDNA methylation levels and has been widely used fordetecting the changes in specific gene promotermethylation. Gastric cancer BGC-823 cells were treated

with 5-aza-dC at the dose of 1 or 2.5 μmol/L for 3 days.The culture medium was changed every day to maintaina stable concentration of 5-aza-dC. The genomic DNA oftreated and untreated BGC-823 cells were thenextracted to analyze the methylation levels of WTX genepromoters.RESULTS

WTX promoter methylation in gastric cancer, normaltissues and cell lines

DNA promoter Sequenom MassARRAYquantitative methylation analysis was used for analyzingthe methylation levels in 3 putative DNA-methylationregions of WTX gene. For all of the 3 putativeDNA-methylation regions, 9 segments were amplified by3 methylation promoters, and the quantitative resultswere reported. The data revealed very low methylationlevels of WTX promoters (no more than 30% ) in the 20pairs of cancer and matched normal tissues. Nosignificant differences were found in the methylationlevels of WTX promoters between normal and cancertissues and the cell lines (Fig.1 and Fig.2).Effect of 5-aza-dC on WTX methylation in gastric cancercell lines

To verify WTX methylation in gastric cancer, wecompared the differences of DNA promoter methylationlevels between 5-aza-dC-treated anduntreated gastriccancer BGC-823 cells. The result did not show anyobvious changes of the methylation levels of WTX genepromoters in BGC-823 cells in response to 5-aza-dCtreatment (Fig.2).DISCUSSION

WTX is a novel tumor suppressor gene in Wilms'tumor［19］, but its functions in gastric cancer still remainsunknown. Abnormal promoter CpG island methylation isoften associated with a transcriptional block and loss ofthe relevant protein, which is one of the main reasons forgene silencing［20］. Sequenom MassARRAY quantitativemethylation analysis allows the comparison ofmethylation profiles between different samples, and iscurrently the most accurate method for analyzing genemethylation conditions. To explore the potential changesof WTX gene in gastric cancers, we analyzed thepromoter methylation conditions of WTX. Our datashowed that WTX promoter methylation levels were verylow and comparable among normal gastric tissues,gastric cancer tissues and the 3 gastric cancer cell lines.To further verify the results, we treated gastric cancerBGC823 cells with two doses of 5-aza-dC for DNAdemethylation and analyzed the changes in WTXpromoter methylation, and the results showed that5-aza-dC treatment did not obviously reduce themethylation levels of WTX promoters. These datasuggest that high methylation levels of WTX promotersare rare in gastric cancer, and WTX gene promoter

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methylation levels do not show significant changes ingastric cancer.REFERENCES

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［2］ Nomura AM, Wilkens LR, Henderson BE, et al. The association ofcigarette smoking with gastric cancer: the multiethnic cohort study［J］. Cancer causes control, 2012, 23 (1): 51-8.

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［4］ Rivera MN, Kim WJ, Wells J, et al. An X chromosome gene, WTX, iscommonly inactivated in Wilms' tumor［J］. Science, 2007, 315(5812):642-5.

［5］ Major MB, Camp ND, Berndt JD, et al. Wilms' tumor suppressorWTX negatively regulates Wnt/β-catenin signaling［J］. Science,2007, 316(5827): 1043-6.

［6］ Tanneberger K, Pfister AS, Kriz V, et al. Structural andfunctionalcharacterization of the wnt inhibitor APC membranerecruitment 1(Amer1)［J］. J Biol Chem, 2011, 286(22): 19204-14.

［7］ Camp ND, James RG, Dawson DW, et al. Wilms tumor gene on Xchromosome (WTX) inhibits degradation of NRF2 protein throughcompetitive binding to KKEAP1 protein［J］. J Boil Chem, 2012, 287(9): 6539-50.

［8］ Rivera MN, Kim WJ, Wells J, et al. The tumor suppressor WTXshuttles to the nucleus and modulates WT1 activity［J］. Proc NatlAcad Sci USA, 2009, 106(20): 8338-43.

［9］Fukuzawa R, Holman SK, Chow CW, et al. WTX mutations can occurboth early and late in the pathogenesis of Wilms tumour［J］. J MedGenet. 2010, 47(11): 791-4.

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sclerosis and developmental delay in a male with a mosaic deletionin chromosome region Xq11.2［J］. Am J Med Genet A, 2012, 158A(11): 2946-52.

［11］Zicari AM, Tarani L, Perotti D, et al. WTX R353X mutation in afamily with osteopathia striata and cranial sclerosis (OS-CS): casereport and literature review of the disease clinical, genetic andradiological features［J］. Ital J Pediatr, 2012, 38: 27.

［12］Perdu B, de Freitas F, Frints SG, et al. Osteopathia striata withcranial sclerosis owing to WTX gene defect［J］. J Bone Miner Res,2010, 25(1): 82-90.

［13］Win AK, Young JP, Lindor NM, et al. Colorectal and other cancerrisks for carriers and noncarriers from families with a DNA mismatchrepair gene mutation: a prospective cohort study［J］. J Clin Oncol,2012, 30(9): 958-64.

［14］Ko HJ, Kim BY, Jung CH, et al. DNA methylation of RUNX3 inpapillary thyroid cancer［J］. Korean J Intern Med, 2012, 27(4):407-10.

［15］Yamanaka S, Olaru AV, An F, et al. MicroRNA-21 inhibits Serpini1,a gene with novel tumor suppressive effects in gastric cancer［J］. DigLiver Dis, 2012, 44(7): 589-96.

［16］Akahira J, Sugihashi Y, Suzuki T, et al. Decreased expression of14-3-3δIs associated with advanced disease in human epithelialovarivian cancer: its correlation with aberrant DNA methylation［J］.Clin Cancer Res, 2004, 10(8): 2687-93.

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［19］Guertl B, Leuschner I, Guelly C, et al. Is predisposition for nep-hroblastoma linked to polymorphisms of the WTX gene［J］? PatholOncol Res, 2010, 16(2): 189-91.

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Fig.1 Representative results of quantitative analyses of WTX promoter methylation in normal and gastric cancer tissues bySequenom MassARRAY methylation technique. The data show that the methylation levels of WTX promoters including thesegments of promoter WTX31(A), WTX18(B), and WTX29(C). The sets of tissue samples included change 17 (No. 9 normal gastrictissue) and change 18 (No. 9 gastric cancer tissue). The colored circles indicate the degree of methylation with red representing 0%and yellow representing 100% methylation. Although two yellow points are present in WTX29, gene sequence analysis show thatthey are not CpG islands and therefore can not represent the actual methylation levels.

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胃癌中新抑癌基因胃癌中新抑癌基因WTXWTX启动子区域甲基化水平的检测启动子区域甲基化水平的检测刘 霞 1，2，王启明 1，2，牛会林 1，2，杨学习 3，孙静哲 3，张庆玲 1，2，丁彦青 1，2

南方医科大学 1南方医院病理科，2基础医学院病理系，3生物技术学院，广东 广州 510515

摘要：目的 探讨抑癌基因WTX启动子区域甲基化水平及其在胃癌中的作用。方法 运用MassARRAY定量分析系统分析20例

胃癌及配对正常组织和3种胃癌细胞株（MGC803、SCG7901和BGC823）中WTX基因启动子区域甲基化水平。应用5-杂氮-2'-

脱氧胞苷(5-aza-dC）对胃癌细胞BGC823进行去甲基化处理，分析其对WTX基因启动子区域甲基化水平的影响。结果 在胃癌

及其配对正常组织和胃癌细胞株中，WTX基因启动子区域甲基化水平普遍较低，并且3组之间无统计学差异。用5-aza-dC处理后

并不能改变胃癌细胞株中WTX基因启动子区域的甲基化水平。结论 胃癌中基本不存在WTX基因启动子区域高甲基化状态。

关键词：抑癌基因；WTX；胃癌；甲基化

收稿日期：2012-11-16

基金项目：国家自然科学基金(81071989，81272760)；广东省自然科学基金 (S2011010004178)；广东省科技计划项目(c1221020700008)

作者简介：刘 霞，硕士，E-mail: a200550346@yahoo.com.cn

通信作者：张庆玲，博士，副教授，硕士生导师，E-mail: zqllc8@126.com

WTX18-CpG-1WTX18-CpG-2WTX18-CpG-3WTX18-CpG-4, 5,6WTX18-CpG-7, 8WTX18-CpG-9, 10WTX18-CpG-11WTX18-CpG-13WTX18-CpG-14, 15

WTX29-CpG-1WTX29-CpG-3, 4, 5WTX29-CpG-6WTX29-CpG-7, 8WTX29-CpG-9, 10WTX29-CpG-11WTX29-CpG-12WTX29-CpG-13, 14WTX29-CpG-15

WTX31-CpG-1, 2WTX31-CpG-3WTX31-CpG-4, 5WTX31-CpG-6, 7WTX31-CpG-8WTX31-CpG-9, 10WTX31-CpG-11WTX31-CpG-12, 13WTX31-CpG-15

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Fig.2 Quantitative analysis of WTX gene promoter methylation in normal and gastric cancer tissues and gastric cancer cell lines.The data show the methylation levels of WTX gene promoters including the segments of promoters WTX18 (A), WTX29 (B), andWTX31 (C). In normal and gastric cancer tissues and gastric cancer cell lines, the prevalence of methylation were very lowranging from 3% to 12%, 2% to 8%, and 0% to 25%, respectively, showing no significant differences among them. Treatment with1 or 2.5 μmol/L 5-aza-dC did not cause any obvious changes in the methylation levels in BGC-823 cells. BGC823-1 andBGC823-2.5: BGC823 cells treated with in 1 and 2.5 μmol/L 5-aza-dC, respectively; NT: Mean value of 20 normal tissues; CT:Mean value of 20 gastric cancer tissues.

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