This Provisional PDF corresponds to the article as it appeared upon acceptance. Fully formattedPDF and full text (HTML) ve...
http://www.biomedcentral.com/info/authors/BMC Cancer© 2013 Zhou et al.This is an open access article distributed under the...
Polymorphism of A133S and promoterhypermethylation in Ras association domain family1A gene (RASSF1A) is associated with ri...
Li Dong Wang1**Corresponding authorEmail: ldwang2007@126.com1Henan Key Laboratory for Esophageal Cancer Research, The Firs...
promoter methylation of RASSF1A gene was significantly increased the risk to both ESCC(OR=5.90, 95% CI=2.78–12.52) and GCA...
Gastric cardia adenocarcinoma (GCA), with its epicenter located between 1 cm proximal and2 cm distal of the esophago-gastr...
116 GCA and 235 normal controls. Another group of surgically resected esophageal andgastric cardia cancer and matched norm...
genotype was two bands of 136bp and 58bp, Ala/Ser genotype was three bands of 194bp,136bp and 58bp, Ser/Ser genotype was j...
Immunohistochemical analysisPartial of the samples which were inspected by MSP, about 27 ESCC and 24 GCA, as well ascorres...
Table 1 The genotypings of A133S in RASSF1A gene on ESCC and GCA patients andnormal controlsGenotypings* Control ESCC GCAn...
Table 3 Distribution of RASSF1A promoter methylation by clinicopathological classifications and protein expression in ESCC...
N0 91 (64) 48 (53) 43 (47) 44 (48) 28 (64) 16 (36)N1 47 (33) 27 (57) 20 (43) 0.60 1.21 (0.60–2.46) 48 (52) 32 (67) 16 (33)...
Correlations of the RASSF1A protein immunoreactivity andhypermethylation of RASSF1A geneIn ESCC, all the 7 cases with RASS...
Table 5 Correlation between RASSF1A protein expression and clinicopathological parameters of ESCC and GCA patientsClassifi...
However, RASSF1A protein positive immunostaining rate in GCA tissues was much lowerthan in adjacent normal gastric cardia ...
province (58.7%, 54/92)[21], another high incidence area for ESCC and GCA in TaiHangMountain.It is noteworthy that, in the...
Authors’ contributionsLD Wang and SL Zhou conceived of the study, participated in the design of the study anddrafted the m...
9. Li B, Wang B, Niu LJ, Jiang L, Qiu CC: Hypermethylation of multiple tumor-relatedgenes associated with DNMT3b up-regula...
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Polymorphism of A133S and promoter hypermethylation in Ras association domain family 1A gene (RASSF1A) is associated with risk of esophageal and gastric cardia cancers in Chinese population from high incidence area in northern China

The role of tumor suppressor gene RASSF1A in the esophageal and gastric cardia carcinogenesis is still inconclusive. In this study, the polymorphism, promoter methylation and gene expression of RASSF1A were characterized in esophageal squamous cell carcinoma (ESCC) and gastric cardia adenocarcinoma (GCA).
Published on: Mar 4, 2016
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Transcripts - Polymorphism of A133S and promoter hypermethylation in Ras association domain family 1A gene (RASSF1A) is associated with risk of esophageal and gastric cardia cancers in Chinese population from high incidence area in northern China

  • 1. This Provisional PDF corresponds to the article as it appeared upon acceptance. Fully formattedPDF and full text (HTML) versions will be made available soon.Polymorphism of A133S and promoter hypermethylation in Ras associationdomain family 1A gene (RASSF1A) is associated with risk of esophageal andgastric cardia cancers in Chinese population from high incidence area innorthern ChinaBMC Cancer 2013, 13:259 doi:10.1186/1471-2407-13-259Sheng Li Zhou (zhou_shengli@126.com)Juan Cui (cuijuan0807@126.com)Zong Min Fan (zmfan@zzu.edu.cn)Xue Min Li (xmli66@sina.com)Ji Lin Li (jilinli2011@126.com)Bao Chi Liu (liubaochi2002@yahoo.com.cn)Dong Yun Zhang (zhangdy79@126.com)Hong Yan Liu (liuhongyan2009@yahoo.com.cn)Xue Ke Zhao (zhaoxueke2007@163.com)Xin Song (xinsong2012@yahoo.com.cn)Ran Wang (wangran2012@126.com)Ze Chen Yan (yanzechen@qq.com)Hui Xing Yi (yihuixing2012@126.com)Li Dong Wang (ldwang2007@126.com)ISSN 1471-2407Article type Research articleSubmission date 1 January 2013Acceptance date 21 May 2013Publication date 25 May 2013Article URL http://www.biomedcentral.com/1471-2407/13/259Like all articles in BMC journals, this peer-reviewed article can be downloaded, printed anddistributed freely for any purposes (see copyright notice below).Articles in BMC journals are listed in PubMed and archived at PubMed Central.For information about publishing your research in BMC journals or any BioMed Central journal, go toBMC Cancer© 2013 Zhou et al.This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0),which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
  • 2. http://www.biomedcentral.com/info/authors/BMC Cancer© 2013 Zhou et al.This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0),which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
  • 3. Polymorphism of A133S and promoterhypermethylation in Ras association domain family1A gene (RASSF1A) is associated with risk ofesophageal and gastric cardia cancers in Chinesepopulation from high incidence area in northernChinaSheng Li Zhou1Email: zhou_shengli@126.comJuan Cui2Email: cuijuan0807@126.comZong Min Fan1Email: zmfan@zzu.edu.cnXue Min Li3Email: xmli66@sina.comJi Lin Li4Email: jilinli2011@126.comBao Chi Liu5Email: liubaochi2002@yahoo.com.cnDong Yun Zhang1Email: zhangdy79@126.comHong Yan Liu6Email: liuhongyan2009@yahoo.com.cnXue Ke Zhao1Email: zhaoxueke2007@163.comXin Song1Email: xinsong2012@yahoo.com.cnRan Wang1Email: wangran2012@126.comZe Chen Yan7Email: yanzechen@qq.comHui Xing Yi1Email: yihuixing2012@126.com
  • 4. Li Dong Wang1**Corresponding authorEmail: ldwang2007@126.com1Henan Key Laboratory for Esophageal Cancer Research, The First AffiliatedHospital of Zhengzhou University, Zhengzhou, Henan 450052, China2Cancer Research Center, Xinxiang Medical University, Xinxiang, Henan453003, China3Department of Pathology, Cixian Hospital, Cixian, Hebei 056500, China4Department of Pathology, Linzhou Esophageal Cancer Hospital, Linzhou,Henan 456500, China5Department of Surgery, Shanghai Public Health Clinical Center Affiliated toFudan University, Shanghai 201508, China6Henan Medical Genetics Institute, Henan People’s Hospital, ZhengzhouUniversity, Zhengzhou, Henan 450003, China7Department of Urology, The First Affiliated Hospital, Zhengzhou University,Zhengzhou, Henan 450052, ChinaAbstractBackgroundThe role of tumor suppressor gene RASSF1A in the esophageal and gastric cardiacarcinogenesis is still inconclusive. In this study, the polymorphism, promoter methylationand gene expression of RASSF1A were characterized in esophageal squamous cell carcinoma(ESCC) and gastric cardia adenocarcinoma (GCA).MethodsWe firstly analyzed the prevalence of RASSF1A A133S in a total of 228 cancer patients withESCC (n=112) and GCA (n=116) and 235 normal controls by polymerase chain reaction(PCR) and restriction enzyme-digestion assay. Then, the promoter methylation status of theRASSF1A in ESCC (n=143), GCA (n=92) and corresponding adjacent normal tissues werefurther investigated using methylation-specific PCR (MSP) approach. Finally, the RASSF1Aprotein expression were determined in ESCC (n=27), GCA (n=24) and the matched adjacentnormal tissues by immunohistochemical method.ResultsThe frequency of 133Ala/Se and Ser/Ser genotype was significantly higher in GCA patientsthan in normal controls (19.0% vs. 10.2%, P=0.02). Compared with Ala/Ala genotype,Ala/Se and Ser/Ser genotype significantly increased susceptibility to GCA (OR=2.06, 95%CI=1.09–3.97). However, this polymorphism had no association with ESCC (P=0.69). The
  • 5. promoter methylation of RASSF1A gene was significantly increased the risk to both ESCC(OR=5.90, 95% CI=2.78–12.52) and GCA (OR=7.50, 95% CI= 2.78–20.23). Promotermethylation of RASSF1A gene in ESCC was also associated with age and cancer celldifferentiation (for age: OR=3.11, 95% CI=1.10–8.73; for differentiation: OR=0.29, 95%CI=0.12–0.69). RASSF1A positive expression was significantly decreased the risk of GCA(OR=0.16, 95% CI=0.03–0.83). In contrast, there was no statistical significance betweenRASSF1A positive expression and ESCC. The expression of RASSF1A protein trend to bepositively related with older GCA patients (OR=16.20, 95% CI=1.57–167.74).ConclusionsThe present findings suggest that alterations of RASSF1A may play an important role ingastric cardia carcinogenesis in terms of polymorphism, promoter hypermethylation andprotein expression. Whereas, RASSF1A hypermethylation may probably also be involved inesophageal squamous cell carcinogenesis.KeywordsEsophageal squamous cell carcinoma, Gastric cardia adenocarcinoma, A133S in RASSF1A,Polymorphism, Methylation, Protein expressionBackgroundEsophageal squamous cell carcinoma (ESCC) remains the main predominant histologicaltype of esophageal cancer and the leading cause of cancer-related deaths in China[1,2]. ESCChas a striking geographic distribution in China, with higher prevalence in some areas ofChina, especially in Taihang Mountain areas of Henan, Hebei and Shanxi provinces[3],where nutritional deficiencies, intake of pickled vegetables, nitrosamine-rich or mycotoxin-contaminated foods and low socioeconomic status are likely to contribute to ESCC[4]. Also,in these high-risk areas there is a strong tendency toward familial aggregation of ESCC[5],suggesting that genetic susceptibility, in combination with exposure to environmental riskfactors, contributes to the high rates of ESCC in these areas[6]. ESCC has been generallyrecognized as a multi-stage progression process, in which multiple genetic and epigeneticalterations may be involved. Recent genome-wide association study (GWAS) for ESCC hasindicated that the tumor suppressor gene of Ras association domain family 1A gene(RASSF1A) may be associated with high risk to ESCC[6]. RASSF1A locates at 3p21.3 andparticipates in regulating cell cycle, apoptosis, microtubule stability and other physiologicalactivities[7,8]. Accumulated evidences have indicated the possible crucial role of RASSF1Amethylation on esophageal carcinogenesis in Chinese population, the methylation rates variedfrom 14.9%[9] in Beijing with a low incidence for ESCC, to 48.5%[10] in Hangzhou with ahigher incidence for ESCC, indicating the disparity of RASSF1A methylation with differentenvironment background. The single nucleotide polymorphism (SNP) Ala133Ser (A133S) inRASSF1A has been reported to be involved in the lung and breast cancer[11,12]. Linzhou city(formerly Linxian) in Henan province has been well documented as the highest incidencearea for ESCC in China[2,3]. However, the effects of RASSF1A polymorphism andmethylation on esophageal carcinogenesis have not been well characterized in the populationfrom this highest incidence area in China.
  • 6. Gastric cardia adenocarcinoma (GCA), with its epicenter located between 1 cm proximal and2 cm distal of the esophago-gastric junction[13], is another common cancer in China, whichbears many similarities to ESCC in terms of concurrent geographic distribution andenvironmental risk factors[6,14]. It is reasonable to clarify the molecular profile of ESCC andGCA, which would be helpful to identify molecular biomarkers for high risk subjectscreening and early detection for these two diseases. Thus, the present study was undertakento determine the effect of RASSF1A polymorphism, promoter methylation status and proteinexpression on esophageal and gastric cardia carcinogenesis in patients from the highincidence area for both ESCC and GCA in Linzhou city, Henan province, northern China.MethodsStudy populationA total of 259 patients were recruited in this study, including 143 ESCC (82 males with amean age (average±standard deviation) of 57±10 years and 61 females with a mean age of59±10 years) and 116 GCA (70 males with a mean age of 55±10 years and 46 females with amean age of 60±10 years). All the patients were from Linzhou, the high incidence area forboth ESCC and GCA. All the patients were performed surgical treatment, withoutchemotherapy and/or radiotherapy before the surgery. In addition, 235 normal controlsubjects were enrolled in this study, including 115 males with a mean age of 58±9 years and100 females with a mean age of 59±9 years. All the subjects were performed biopsy underendoscopy to exclude upper gastrointestinal tumor and questionnaires to exclude tumorhistory and tumor family history. All these subjects were from endoscopy screening for earlycancer detection on symptom-free subjects in Linzhou city. Informed consents were obtainedfrom all participants according to Zhengzhou University and Linzhou Esophageal CancerHospital Review Boards.Histopathological examinationsHistopathlogical examinations and TNM staging were performed by two pathologists (X. M.Li and J. L. Li) based on the UICC criteria in 2002[15]. In brief, all the esophageal cancerswere confirmed as squamous cell carcinoma and gastric cardia cancers as adenocarcinoma.The gross morphological types for ESCC and GCA were classified as medullary, fungating,ulcerating, constriction and intraluminal in ESCC; and protruding, ulcerating and infiltratingin GCA. The differentiation for both ESCC and GCA was classified as high, middle and lowgrades. Lymph node metastasis was recorded as negative and positive, if any.Blood sample and surgically resected ESCC and GCA tissue collectionFive ml peripheral blood samples for each patient and each normal subject, and surgicallyresected ESCC and GCA tissue samples were collected sequentially at Linzhou EsophagealCancer Hospital and endoscopy screening from 2000 to 2008. The blood samples were storedat −40°C until use. The tumor tissues and matched normal tissues were collected aftersurgery, half of the surgically resected sample was formalin-fixed, paraffin-embedded andanother half stored at −80°C. The criteria for matched samples are defined as that the tumortissues and the normal tissues are from the same patients, and the matched normal tissues aretaken at the surgically resected margin excluding the infiltration of carcinoma cells. Finally, atotal of 463 blood samples were recruited for polymorphism detection, including 112 ESCC,
  • 7. 116 GCA and 235 normal controls. Another group of surgically resected esophageal andgastric cardia cancer and matched normal tissue specimens were recruited for the RASSF1Amethylation detection. A total of 143 ESCC tissue specimens and 92 GCA tissue specimenswere used for methylation detection, including 62 matched esophageal cancer and normaltissues, and 30 matched gastric cardia cancer and normal tissues. For further determination ofRASSF1A protein expression, 27 ESCC tissue specimens with 27 matched normalesophageal tissue specimens and 24 GCA tissue specimens with 24 matched normal gastriccardia tissue specimens were recruited from 143 ESCC and 92 GCA, which had been usedfor RASSF1A methylation detection. This study was reviewed and approved by the InstituteResearch Ethics Committee of the Zhengzhou University and informed consents wereobtained from all participants before their blood and tissue samples were used.DNA extractionFor polymorphism detection, genomic DNA from peripheral leucocytes was extracted byphenol/chloroform extraction method, dissolved in TE balanced solution, and then stored at−80°C for next procedure.For methylation profiles and protein expression examination, the paraffin-embedded tissueblocks of tumor tissue (both ESCC and GCA) and corresponding adjacent normal tissue weresectioned for hematoxylin and eosin staining with the purpose of identifying pathologicaldiagnose reviewed by pathologist. Regions with neoplastic compositions of 80% or greaterwere marked as tumor tissue under optical microscope. Depending on the size of the tissue,15 to 20 consecutive 10 µm sections of each block were manually microdissected using aneedle and collected into 1.5 ml microtubes for DNA extraction. The tissue DNA extractionwas carried out using the Puregen Genomic DNA purification kit (Gentra Systems, MN,USA) according to the manufacturer’s protocols. The DNA from tissue was stored at −80°Cfor next procedure.GenotypingThe nest PCR was performed in a 25µl reaction containing 1µl for genome DNA, 10mmol/LdNTP, 10X buffer solution, 5pmol up and down primer respectively and 1U Ex Taq DNApolymerase. All the reagents involved were purchased from TaKaRa, Beijing city, China. Thesequences of both outside and inside primers were designed based on the reference sequence(chr3-50342222-50353371) using Primer 3.0 as follows: outside primer: forward 5′ATG ATTCTG TCT TTC CCT TAT CCA and reverse 5′ACC AAA CCT TGA TAA TAG GTT CCA;inner primer: forward 5′AAG GCA GTC AGT TTC CAA AGA CT and reverse 5′ATG AAGAGG TTG CTG TTG ATC TG. The amplification was conducted on the GennAmp RCRsystem 9700 gene amplifier (ABI, California, USA) under the following thermo-cyclerconditions: pre-degeneration at 94 °C for 5 min; followed by 16 cycles at 94°C for 30 sec, at64°C (−0.3°C/circulation) 30 sec and at 72°Cfor 30 sec; 20X (94°C for 30 sec, 56°C for 40sec and 72°Cfor 30 sec); then, extension at 72°C for 10 min, and finally stored at 4°C. Thefinal product of nest PCR was 194bp. 10µl PCR product was digested with restrictionenzymes AluI (TaKaRa, Beijing city, China.) incubated at 37°C overnight. The digestionproduct was detected on a 3% agarose gel stained with ethidium bromide under UVillumination.The 133rd amino acid which was encoded by the exon 3 of RASSF1A is GCT (Ala) or TCT(Ser), if the locus was allele G, this read was restriction site of AluI. Because of that, Ala/Ala
  • 8. genotype was two bands of 136bp and 58bp, Ala/Ser genotype was three bands of 194bp,136bp and 58bp, Ser/Ser genotype was just one band of 194bp (Figure 1). For qualityassurance, both Ala/Ser and Ser/Ser genotypes were genotyped more than twice, moreover,all of the Ser/Ser, portion of Ala/Ser and some Ala/Ala genotypes which randomly selectedwere sequenced to testify the genotyping results. The sequencing analysis was conductedusing ABI3730XL Sequencers (ABI, California, USA) at Beijing Sunbiotech Co., Ltd.(Beijing, China).Figure 1 The genotying results of the RASSF1A SNP at codon 133. PCR analysis of theRASSF1A SNP at codon 133. Land 1: Marker; Land 2: Ser/Sergenotype; Land 3 and 4:Ala/Ala genotypes; Land 5:Ala/Ser genotype.Bisulfite treatment and methylation specific PCR (MSP)Genomic DNA (1µg) was treated with sodium bisulfite modification in order to convertingunmethylated cytosines to uracils using the CpGenomeTMDNA Modification Kit (S7820)(Chemicon, California, USA) according to manufacturer’s protocol. The modified DNA waspurified by using a Wizard DNA Clean-Up System (Promega Corporation, Madison, USA)following manufacturer’s protocol.The bisulfite-modified DNA was subjected to methylation specific polymerase chain reaction(MSP) as described previously[16]. Primers targeting promoter region of RASSF1A were asfollows: The specific primers for methylated sequences (forward 5′- GTG TTA ACG CGTTGC GTA TC and reverse 5′- AAC CCC GCG AAC TAA AAA CGA) and for unmethylatedsequences (forward 5′- TTT GGT TGG AGT GTG TTA ATG TG and reverse 5′- CAA ACCCCA CAA ACT AAA AAC AA), which generates PCR products of 93 and 105 bp,respectively. The total 25 ml of PCR mix contained 50-100ng bisulfite-modified DNA, 10XPCR buffer (Mg2+Plus), 3.0µl; 2.5 mM dNTPs, 3.0µl; 10µM of each primer, 2.0µl; and 0.5 UTaqHSDNA polymerase, 2.5µl (TaKaRa, Dalian city, China). The amplification wasconducted on the Tgradient RCR system (Biometra, Goettingen, German) under thefollowing thermo-cycler conditions: pre-degeneration at 95°C for 15 min; followed by 40cycles at 95°C for 55 sec, at 65°C for 55 sec and at 72°Cfor 1 min; 20X (94°C for 30 sec,56°C for 40 sec and 72°C for 30 sec; then, extension at 72°C for 10 min. The placenta tissueDNA which was treated with Sss I methyltransferase (TaKaRa, Dalian city, China) was usedas positive control for methylation, while taking the placenta tissue DNA which was notdigested by Sss I methyltransferase as positive control for unmethylation; Distilled water wasused as negative control for PCR. Both of the positive and negative controls were deal with inthe same procedures. Six µl of PCR products were separated on 10% polyacrylamide gel. Thegel was then stained with ethidium bromide, and visualized under UV illumination.Methylated samples were defined as the presence of methylated PCR products in thosesamples (Figure 2).Figure 2 MSP analysis of RASSF1A gene in ESCC and GCA tissue. Representative MSPresults of three ESCC tissues (A, T1, T2, and T3) and (B) was the results of three GCAtissues (T4, T5, and T6). Lane M: indicates the presence of methylated genes; Lane U:indicates the presence of unmethylated genes. T1 and T4 were fully hypermethylation whichrevealed 93bp band (M) with hypermethylated primers; T3 and T6 were unmethylation,having only unmethylated band of 105bp; T2 and T5 were Hemi-methylation with bothhypermethylated band and unmethylated band.
  • 9. Immunohistochemical analysisPartial of the samples which were inspected by MSP, about 27 ESCC and 24 GCA, as well ascorresponding adjacent normal epithelial tissues were subjected to investigate RASSF1Aprotein expression by immunohislochemical staining. Immunohislochemical analysis wasperformed using the avidin-biotin-peroxidase complex method as previously described[17].The rat anti-human monoclonal RASSF1A antibody (eBioscience corporation, San Diego,USA) was used at 1:500 dilution. Intense nuclear or cytoplasm staining was the criterion for a“positive” reaction. We applied the criteria established by our laboratory previously[17] todescribe the types of positive result as follows: “scattered”, in which only some isolatedpositive cells were identified; “papillary”, where immunostain-positive cells were identifiedonly in the papillary area; “focal”, where wide clusters of positive cells were seen in someareas of the epithelia; and “diffuse”, in which the sheets of positive cells were foundthroughout most areas of the lesions. Immunohistochemical labeling was estimated in anoutcome-blinded model by two pathologists on a compound microscope.Statistical analysisConsistency with Hardy–Weinberg equilibrium (HWE) of the genotypes of the ESCC/GCAand control groups was established by Chi-squared tests. The association of Genotype anddiseases were assessed using logistic regression and expressed as odds ratios with 95%confidence intervals. For RASSF1A gene methylation and protein expression, the correlationswith clinic characteristics in ESCC and GCA tissues were evaluated using multiple univariatelogistic-regressions. The statistical analyzes were carried out with SPSS 17.0 softwarepackage. All tests were two tailed. P < 0.05 was considered statistically significant.ResultsFrequency of RASSF1A A133S in ESCC and GCA patientsAll the 235 control, 112 ESCC and 116 GCA samples were analyzed for the presence ofA133S (Table 1). There were no departures from the HWE in the genotyping results ofESCC, GCA or control samples (P=0.83). The homozygous or heterozygous for A133S wereaccounted for 11.6% (13/112) in the ESCC and 10.2% (24/235) in control subjects. Thoughthe frequencies of A133S in ESCC was a little higher than in controls, Ala/Se and Ser/Sergenotype did not increase the risk of ESCC compared with Ala/Ala genotype (11.2% vs.10.2%, P= 0.69; OR=1.15; 95% CI=0.51–2.44).
  • 10. Table 1 The genotypings of A133S in RASSF1A gene on ESCC and GCA patients andnormal controlsGenotypings* Control ESCC GCAn (%) n (%) P OR (95%CI) n (%) P OR (95%CI)Ala/Ala 211 (89.8) 99 (88.4) 94 (81.0)Ala/Ser 23 (9.7) 11 (9.8) 0.96 1.02 (0.48–2.17) 18 (15.5) 0.09 1.76 (0.91–3.41)Ser/Ser 1 (0.4) 2 (1.8) 0.24 4.26 (0.38–47.02) 4 (3.5) 0.04 9.01 (0.99–83.31)Ala/Se and Ser/Ser 24 (10.2) 13 (11.6) 0.69 1.15 (0.51–2.44) 22 (19.0) 0.02 2.06 (1.09–3.97)*: Ala/Ala: homozygous for wild-type codon 133, Ala/Ser: heterozygote for codon 133,Ser/Ser: homozygous for codon A133S.Intriguingly, in GCA, there was a significant difference of the frequency of the RASSF1AA133S T allele compared with the controls (19.0% vs. 10.2%). The individuals carryingA133S (Ser/Ser) genotype had a much higher susceptibility to GCA compared with peoplecarrying Ala/Ala genotype (P=0.04, OR=9.01, 95% CI= 0.99–83.31). In addition, comparedwith Ala/Ala genotype, Ala/Se and Ser/Ser genotype also significantly increasedsusceptibility to GCA (P=0.02, OR=2.06, 95% CI=1.09–3.971).The promoter methylation of RASSF1A in ESCC and GCA patientsThe promoter methylation of RASSF1A in ESCC tissue was 3.4-fold higher than in adjacentnormal mucosa (76/143, 53% vs. 10/62, 16%, P<0.001). The promoter hypermethylation ofRASSF1A gene significantly increased almost 6-fold higher the risk to ESCC development(OR=5.90, 95% CI=2.78–12.52). Interestingly, the similar results were observed in GCA, thepromoter hypermethylation of RASSF1A gene significantly increased almost 7.5-fold higherthe risk to GCA development (65% in GCA vs. 20% in adjacent normal mucosa, OR=7.50,95% CI=2.78–20.23) (Table 2 and Figure 2).Table 2 The profiles of RASSF1A promoter hypermethylation in ESCC and GCAtissues and in normal tissues adjacent to the corresponding ESCC and GCATissues#N Methylated Unmethylated P OR (95%CI)n (%) n (%)ESCC 143 76 (53) 67 (47) <0.001 5.90 (2.78–12.52)ENOR 62 10 (16) 52 (84)GCA 92 60 (65) 32 (35) <0.001 7.50 (2.78–20.23)GNOR 30 6 (20) 24 (80)#: ESCC: esophageal squamous cell carcinoma, ENOR: esophageal normal epithelial tissue,GCA: gastric cardia adenocarcinoma, GNOR: gastric cardia normal epithelial tissue.Furthermore, the promoter methylation of RASSF1A in ESCC patients with the age groupfrom 50–60 years old had a significantly higher risk to ESCC than in those with the age lessthan 50 years old (OR=3.11, 95% CI=1.10–8.73) (Table 3). The ESCC with moderatedifferentiation had a lower frequency of RASSF1A methylation than in those with higherdifferentiation (OR=0.28, 95% CI=0.12–0.69) (Table 3). However, multivariate analysis didnot show did not show any significant association for RASSF1A promoter methylation andgender, gross pathologic classification, infiltration degree, lymph node metastasis and clinicalstages both in ESCC and GCA.
  • 11. Table 3 Distribution of RASSF1A promoter methylation by clinicopathological classifications and protein expression in ESCC and GCAtissuesClassification ESCC(n=143) GCA(n=92)N MethylatedUnmethylatedP OR (95%CI) N Methylated UnmethylatedP OR (95%CI)n (%) n (%) n (%) n (%) n (%) n (%)GenderMale 82 (57) 43 (52) 39 (48) 58 (63) 37 (64) 21 (36)Female 61 (43) 33 (54) 28 (46) 0.84 0.94 (0.48–1.82) 34 (37 23 (68) 11 (32) 0.71 0.84 (0.34–2.07)Age(y)≤50 25 (17) 13 (52) 12 (48) 21 (23) 16 (76) 5 (24)50–60 48 (34) 37 (77) 11 (23) 0.03 3.11 (1.10–8.73) 32 (35) 19 (59) 13 (41) 0.21 0.46 (0.13–1.56)>60 70 (49) 26 (37) 44 (63) 0.20 0.55 (0.22–1.37) 39 (42) 25 (64) 14 (36) 0.34 0.56 (0.17–1.85)Gross pathologic classificationΦ1 38 (27) 18 (47) 20 (53) 17 (18) 10 (59) 7 (41)2 25 (17) 16 (64) 9 (36) 0.58 0.74 (0.25–2.20) 32 (34) 24 (67) 8 (33) 0.25 2.10 (0.60–7.36)3 29 (20) 19 (66) 10 (34) 0.91 0.92 (0.23–3.77) 33 (36) 21 (64) 12 (36) 0.74 1.23 (0.37–4.06)4 19 (13) 15 (79) 4 (31) 0.06 0.39 (0.14–1.05) ND ND ND ND ND NDUnclear 32 (22) 8 (25) 24 (75) 10 (11) 5 (50) 5 (50)Differentiation classificationHigh 32 (22) 22 (69) 10 (31) 20 (22) 14 (70) 6 (30)Middle 70 (49) 27 (39) 43 (61) 0.0060.28 (0.12–0.69) 31 (34) 22 (71) 9 (29) 0.94 1.05 (0.31–3.59)Low 34 (24) 26 (76) 8 (24) 0.48 1.48 (0.50–4.39) 41 (45) 24 (59) 17 (41) 0.39 0.61 (0.19–1.89)Unclear 7 (5) 1 (14) 6 (86) ND ND ND ND ND NDTumor stage (T) classificationT1+T2 25 (17) 13 (52) 12 (48) 14 (15) 9 (64) 5 (36)T3+T4 113 (79) 62 (55) 51 (45) 0.38 1.38 (0.68–2.82) 78 (85) 51 (65) 27 (35) 0.77 1.20 (0.36–4.04)Unclear 5 (3) 2 (40) 3 (60) ND▲ND ND ND ND NDTumor stage (N) classification
  • 12. N0 91 (64) 48 (53) 43 (47) 44 (48) 28 (64) 16 (36)N1 47 (33) 27 (57) 20 (43) 0.60 1.21 (0.60–2.46) 48 (52) 32 (67) 16 (33) 0.76 1.143 (0.48–2.70)Unclear 5 (3) 1 (20) 4 (80) ND ND ND ND ND NDTumor stage (TNM) classificationI+II 91 (64) 47 (52) 44 (48) 47 (51) 30 (64) 17 (36)III+IV 47 (33) 28 (60) 19 (40) 0.38 1.38 (0.68–2.82) 45 (49) 30 (67) 15 (33) 0.78 1.13 (0.48–2.68)Unclear 5 (3) 1 (20) 4 (80) ND ND ND ND ND NDRASSF1A proteinpositive 17 (63) 6 (35) 11 (65) 14 (58) 5 (36) 9 (14)negative 10 (37) 7 (70) 3 (30) 0.09 0.23 (0.04–1.25) 10 (42) 10 (100) 0 (0) 0.001Φ: Histopathologically, the gross pathologic types were different for ESCC and GCA, in ESCC, 1: medullary; 2: fungating; 3: ulcerating; 4:constriction & intraluminal; in GCA, 1: protruding; 2: ulcerating; 3: infiltrating.▲: Not detected.
  • 13. Correlations of the RASSF1A protein immunoreactivity andhypermethylation of RASSF1A geneIn ESCC, all the 7 cases with RASSF1A promoter methylation-positive tissues (7/10, 70%),showed completely lack of immunoreactivity for RASSF1A, similar results were observedfor the matched adjacent normal tissue. Interestingly, of the 17 cases with RASSF1A promotermethylation-negative tissues, 11 cases showed positive immunoreactivity for RASSF1A(11/17, 65%). RASSF1A positive staining was negatively associated with RASSF1A promotermethylation in the ESCC (P=0.09, Fisher’s exact test) (Table 3).In GCA, all the 10 cases with RASSF1A promoter methylation-positive tissues, RASSF1Aprotein expression was not detected (Table 3). But, of the 14 cases with RASSF1A promotermethylation-negative tissues, 9 cases showed RASSF1A positive expression (64%). Therewas a significant correlation between RASSF1A promoter hypermethylation and loss ofRASSF1A protein expression in GCA (P=0.001, Fisher’s exact test).RASSF1A protein expression in ESCC and GCA patientsThe positive expression of the RASSF1A protein was located in cell nucleus and cytoplasm.The positive immunostaining rate for RASSF1A in ESCC tissue was slightly lower than inadjacent normal tissues (17/27, 63% vs. 21/27, 78%). But, the difference was not significant(P=0.24) (Table 4). No association was found for RASSF1A protein expression and gender,age, cancer cell differentiation, infiltration degree and lymph node metastasis or clinicalstages in ESCC (Table 5).Table 4 RASSF1A protein expression in ESCC and GCA tissues and in normal tissuesadjacent to the corresponding ESCC and GCATissues N Postive Negative P OR (95%CI)n (%) n (%)ESCC 27 17 (63) 10 (37 ) 0.24 0.47 (0.13–1.67)ENOR 23 18 (78) 5 (22)GCA 24 14 (58) 10 (42) 0.03 0.16 (0.03–0.83)GNOR 20 18 (90) 2 (10)#: ESCC: esophageal squamous cell carcinoma, ENOR: esophageal normal epithelial tissue,GCA: gastric cardia adenocarcinoma, GNOR: gastric cardia normal epithelial tissue.
  • 14. Table 5 Correlation between RASSF1A protein expression and clinicopathological parameters of ESCC and GCA patientsClassificationESCC(n=27) GCA(n=24)N Positive NegativeP OR(95%CI)N Positive NegativeP OR(95%CI)n (%) n (%) n (%) n(%) n(%) n(%)GenderMale 18(67) 10(56) 8(44) 14(58) 6(43) 8(57)Female 9(33) 7(78) 2(22) 0.27 0.36(0.06–2.22) 10(42) 8(80) 2(20) 0.08 0.19(0.03–1.23)Age(y)≤40 12 (44) 10(83) 2(17) 10(42) 9(90) 1(10)≥70 15 (56) 7(47) 8(53) 0.06 5.71(0.92–35.48) 14(58) 5(36) 9(64) 0.02 16.20(1.57–167.74)Differentiation classificationHigh+Middle 23 (85) 13(57) 10(43) 14(58) 6(43) 8(57)Low 4(15) 4(100) 0(0) 0.10 10(42) 8(80) 2(20) 0.08 0.19(0.03–1.23)Tumor stage (T) classificationT1+T2 6(22) 4(67) 2(33) 2(8) 1(50) 1(50)T3+T4 21(78) 13(62) 8(38) 0.57 1.88(0.22–15.93) 22(92) 13(59) 9(41) 0.73 0.75(0.15–3.83)Tumor stage (N) classificationN0 20(74) 13(65) 7(35) 15(63) 9(60) 6(40)N1 7(26) 4(57) 3(43) 0.41 2.18(0.35–13.76) 9(38) 5(56) 4(44) 0.83 0.83(0.16–4.44)Tumor stage (TNM) classificationI+II 20(74) 12(60) 8(40) 21(88) 12(57) 9(43)III+IV 7(26) 5(71) 2(29) 0.59 1.67(0.26–10.79) 3(13) 2(67) 1(33) 0.76 1.50(0.12–19.24)
  • 15. However, RASSF1A protein positive immunostaining rate in GCA tissues was much lowerthan in adjacent normal gastric cardia tissue (14/24, 58% vs. 22/24, 92%, P=0.02) (Table 4).Furthermore, RASSF1A expression in GCA patients over 70 years old was significantlyhigher than in those under 40 years old (58% vs. 42%; OR=16.20, 95% CI=1.57–167.74)(Table 5). No association was found for RASSF1A protein expression and gender, cancer celldifferentiation, infiltration degree and lymph node metastasis in GCA (Table 5).DiscussionIn the present study, we firstly investigated the RASSF1A A133S polymorphism and the riskto ESCC and GCA development on the patients from Linzhou, the high incidence area forboth ESCC and GCA. Our results demonstrated that the existence of polymorphic allele ofRASSF1A might participate in gastric cardia carcinogenesis. The carriers with Ser/Sergenotype (the homozygote for codon RASSF1A A133S) and with mutated T allele genotype(Ala/Ser+Ser/Ser genotype) could have 9-fold (OR=9.01, 95% CI=0.99–83.31) and 2-fold(OR=2.06, 95% CI=1.09–3.97) higher risk to GCA, respectively. In contrast, the presence ofSNP A133S in RASSF1A seems to be not involved in esophageal squamous cellcarcinogenesis (P=0.69). Histopathologically, the predominant type for gastric cardia isadenocarcinoma, but the predominant type for esophageal cancer is squmous cell carcinomain Chinese population, which may partially explain the genetic risk difference observed in thepresent study. It has been reported that single nucleotide polymorphism at codon 133 of theRASSF1 gene is preferentially associated with human lung adenocarcinoma risk, but not forhuman lung squamous cell carcinoma[11].In this study, we also investigated RASSF1A promoter methylation in ESCC and GCA.Aberrant RASSF1A promoter methylation was found in 53% (76/143) of Chinese ESCC,significantly increased 5.9-fold higher the risk to ESCC (OR=5.90, 95% CI=2.78–12.52). Itwas apparently higher than in Korean ESCC (14%, 7/50)[18] and Japanese ESCC (24%,13/55)[19]. Interestingly, another two reports also show higher frequency of RASSF1Apromoter methylation in Chinese ESCC from Hong Kong (34%, 22/64)[20] and Hangzhou(48.5%, 32/66)[10], the high incidence area for ESCC in China. It is noteworthy that ChineseESCC from low incidence area seems to have low frequency of RASSF1A promotermethylation (14.9% 22/64)[9]. Obviously, the RASSF1A promoter methylation may beinterfered by genetic and environmental factors from different population. In this study, wefound that the prevalence of RASSF1A promoter methylation and the risk to ESCC in ESCCpatients with the age of more than 50 years old were higher than those with less than 50 yearsold (OR=3.11, 95% CI=1.10–8.73), which further indicate the impact of environmental factorin terms of exposure time on RASSF1A promoter methylation.Of particular interest, we also demonstrated the similar hypermethylation of RASSF1A inGCA (65%, 60/92), the promoter hypermethylation of RASSF1A significantly increased 7.5-fold higher the risk to GCA (OR=7.5, 95% CI=2.78–20.23), which is very similar as inESCC. It is noteworthy that these GCA patients enrolled in our study were from the samehigh incidence area as the ESCC patients, suggesting that there may be similar environmentalcarcinogenic factors involved in ESCC and GCA. GCA seems to occur together with ESCCin China and bears many similarities in terms of concurrent geographic distribution andenvironmental risk factors including nutritional deficiencies, low intake of vegetables andfruit, and low socioeconomic status[14]. Recent GWAS studies have demonstrated thesimilar genetic changes for ESCC and GCA in Chinese population[6]. Furthermore, ourresults is close to the findings of RASSF1A gene methylation in GCA patients from Hebei
  • 16. province (58.7%, 54/92)[21], another high incidence area for ESCC and GCA in TaiHangMountain.It is noteworthy that, in the present study, we also observed the RASSF1A promotermethylation in the normal esophageal and gastric cardia epithelial tissue adjacent tocorresponding ESCC and GCA (in ESCC: 13%, 23/143; in GCA: 20%, 19/92). Thesehistological normal tissue has been exposed to same carcinogenic factors with tumor tissues.The present results indicate that RASSF1A promoter methylation may be a promising earlyindicator for esophageal and gastric cardia carcinogenesis. Further studies are required tocharacterize the RASSF1A promoter methylation in esophageal and gastric cardiaprecancerous lesion.Another interesting finding in this study is that RASSF1A promoter hypermethylation couldsignificantly downregulated RASSF1A protein expression both in ESCC and GCA, furtherindicating the possible crucial role of RASSF1A promoter hypermethylation and proteinexpression in esophageal and gastric cardia carcinogenesis.Finally, considering the obvious impact of environmental factors on DNA methylation, theadvantage of our study is the large sample size with similar lifestyle background. All theESCC and GCA patients and control subjects enrolled in this study were from the same highincidence area for both ESCC and GCA.ConclusionsIn conclusion, this is the first study to reveal the interactions of RASSF1A polymorphism,promoter methylation and protein expression on the risk of esophageal and gastric cardiacarcinogenesis. RASSF1A promoter hypermethylation could significantly increase 6-fold and8-fold higher risk to ESCC and GCA development, respectively, and induce the inactivationof RASSF1A protein expression both in ESCC and GCA, which indicate that RASSF1Apromoter hypermethylation may play an important role both in esophageal and gastric cardiacarcinogenesis. The T allele (Ala/Ser + Ser/Ser genotype) of RASSF1A A133S could increasethe risk to GCA development, but not to ESCC. Certainly, further work should be done toillustrate the mechanism of RASSF1A in the development of ESCC and GCA, and ultimatelydevelop RASSF1A gene as one of the molecular biomarkers for high-risk subject screeningand early detection for ESCC and GCA in future.AbbreviationsRASSF1A, Ras association domain family 1A gene; ESCC, Esophageal squamous cellcarcinoma; SNP, Single nucleotide polymorphism; GCA, Gastric cardia adenocarcinoma;PCR, Polymerase chain reactionCompeting interestsAll authors declare no competing financial interests.
  • 17. Authors’ contributionsLD Wang and SL Zhou conceived of the study, participated in the design of the study anddrafted the manuscript. J Cui performed the statistical analysis and clinical data collection.ZM Fan and BC Liu carried out polymorphism examination. XM Li and J L Li participated inpathological finding analysis. SL Zhou and DY Zhang carried out the methylation detection.HY Liu, XK Zhao and X Song participated in the immunohistochemical analysis. R Wang,ZC Yan and HX Yi helped to worked out the methylation detection. All authors read andapproved the final manuscript.AcknowledgmentsWe thank Dr. Wan Cai Yang of Xinxiang Medical University at Xinxiang, Henan, China forhis carefully discussion of this manuscript. We thank Professor Jun Yan Hong at RutgersUniversity and Professor Liang Wang at Medical College of Wisconsin for their help inEnglish writing and discussion for the manuscript. This project was funded by 863 HighTechKey Projects (2012AA02A503, 2012AA02A209 and 2012AA02A201) and National NaturalScience Foundations (81071783 and 30971133).References1. Jemal A, Bray F, Center MM, Ferlay J, Ward E, Forman D: Global cancer statistics. CACancer J Clin 2011, 61:69–90.2. Ke L: Mortality and incidence trends from esophagus cancer in selected geographicareas of China circa 1970–90. Int J Cancer 2002, 102:271–274.3. Sun XB, Liu ZC, Liu SZ, Li BY, Dai DX, Quan PL, et al: Descriptive analysis ofincidence and time trends of esophageal and gastic cancers in Linzhou city. ZhouhuaZhong Liu Za Zhi 2007, 29:764–767.4. Sun G, Wang S, Hu X, Su J, Huang T, Yu J, et al: Fumonisin B1 contamination ofhome-grown corn in high-risk areas for esophageal and liver cancer in China. FoodAddit Contam 2007, 24:181–185.5. Guohong Z, Min S, Duenmei W, Songnian H, Min L, Jinsong L, et al: Geneticheterogeneity of oesophageal cancer in high-incidence areas of southern and northernChina. PLoS One 2010, 5:e9668.6. Wang LD, Zhou FY, Li XM, Sun LD, Song X, Jin Y, et al: Genome-wide associationstudy of esophageal squamous cell carcinoma in Chinese subjects identifiessusceptibility loci at PLCE1 and C20orf54. Nat Genet 2010, 42:759–763.7. Máthé E: RASSF1A, the new guardian of mitosis. Nat Genet 2004, 36:117–118.8. Agathanggelou A, Cooper WN, Latif F: Role of the Ras-association domain family 1tumor suppressor gene in human cancers. Cancer Res 2005, 65:3497–3508.
  • 18. 9. Li B, Wang B, Niu LJ, Jiang L, Qiu CC: Hypermethylation of multiple tumor-relatedgenes associated with DNMT3b up-regulation served as a biomarker for early diagnosisof esophageal squamous cell carcinoma. Epigenetics 2011, 6:307–316.10. Cong DG, Wang SF: Hypermethylation of promoter region of RAS associationdomain family gene1A in esophageal squamous cell carcinoma and significance thereof.Zhonghua Yi Xue Za Zhi 2007, 87:2932–2934.11. Kanzaki H, Hanafusa H, Yamamoto H, Yasuda Y, Imai K, Yano M, et al: Singlenucleotide polymorphism at codon 133 of the RASSF1 gene is preferentially associatedwith human lung adenocarcinoma risk. Cancer Lett 2006, 238:128–134.12. Schagdarsurengin U, Seidel C, Ulbrich EJ, Kölbl H, Dittmer J, Dammann R: Apolymorphism at codon 133 of the tumor suppressor RASSF1A is associated withtumorous alteration of the breast. Int J Oncol 2005, 27:185–191.13. Siewert JR, Hölscher AH, Becker K, Gössner W: Cardia cancer: attempt at atherapeutically relevant classification. Chirurg 1987, 58:25–32.14. Tran GD, Sun XD, Abnet CC, Fan JH, Dawsey SM, Dong ZW, et al: Prospective studyof risk factors for esophageal and gastric cancers in the Linxian general population trialcohort in China. Int J Cancer 2005, 113:456–463.15. Greene FL: AJCC cancer staging manual. New York: Springer Verlag; 2002.16. Herman JG, Graff JR, Myöhänen S, Nelkin BD, Baylin SB: Methylation-specific PCR:a novel PCR assay for methylation status of CpG islands. Proc Natl Acad Sci U S A 1996,93:9821–9826.17. Wang LD, Hong JY, Qiu SL, Gao H, Yang CS: Accumulation of p53 protein in humanesophageal precancerous lesions: a possible early biomarker for carcinogenesis. CancerRes 1993, 53:1783–1787.18. Kim YT, Park JY, Jeon YK, Park SJ, Song JY, Kang CH, et al: Aberrant promoterCpG island hypermethylation of the adenomatosis polyposis coli gene can serve as a goodprognostic factor by affecting lymph node metastasis in squamous cell carcinoma of theesophagus. Dis Esophagus 2009, 22:143–150.19. Yamaguchi S, Kato H, Miyazaki T, Sohda M, Kimura H, Ide M, et al: RASSF1A genepromoter methylation in esophageal cancer specimens. Dis Esophagus 2005, 18:253–256.20. Wong ML, Tao Q, Fu L, Wong KY, Qiu GH, Law FB, et al: Aberrant promoterhypermethylation and silencing of the critical 3p21 tumour suppressor gene, RASSF1A,in Chinese oesophageal squamous cell carcinoma. Int J Oncol 2006, 28:767–773.21. Guo W, Dong Z, Chen Z, Yang Z, Wen D, Kuang G, et al: Aberrant CpG islandhypermethylation of RASSF1A in gastric cardia adenocarcinoma. Cancer Invest 2009,27:459–465.
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