Identification of Functional Genetic Variants in Cyclooxygenase-2 and Their Association With Risk of Esophageal Cancerстатья из журнала
Аннотация: Background & Aims: Overexpression of cyclooxygenase-2 (COX-2) is implicated in many steps of cancer development. Single nucleotide polymorphisms (SNPs) in the COX-2 promoter might contribute to differential COX-2 expression and subsequent interindividual variability in susceptibility to cancer. This study sought to identify functional SNPs in the COX-2 promoter and evaluated their effects on the risk of developing esophageal squamous cell carcinoma (ESCC). Methods: Thirty individual DNA samples were sequenced to search for SNPs, and the function of the SNPs was examined by a set of biochemical assays. Genotypes and haplotypes were analyzed in 1026 patients and 1270 controls, and odds ratios and 95% confidence intervals (CIs) were estimated by logistic regression. Results: Three SNPs, −1290A→G, −1195G→A, and −765G→C, were identified; the frequencies of variant alleles were 0.04, 0.51, and 0.02, respectively. The −1195G→A change creates a c-MYB binding site and displays a higher promoter activity. The −1195A-containing haplotypes had significantly increased luciferase expression and COX-2 messenger RNA levels in esophageal tissues compared with the −1195G-containing counterparts. A case-control analysis showed a 1.72-fold (95% CI, 1.35–2.20) and 2.24-fold (95% CI, 1.59–3.16) excess risk of developing ESCC for the −1195AA or −765CC genotype carriers compared with noncarriers. A greater risk of developing ESCC was observed for A−1195-C−765-containing haplotypes compared with G−1195-G−765-containing haplotypes, suggesting an interaction between the −1195G→A and −765G→C polymorphisms in the context of haplotype. Conclusions: These findings indicate that genetic variants in COX-2 may play a role in mediating susceptibility to esophageal cancer. Background & Aims: Overexpression of cyclooxygenase-2 (COX-2) is implicated in many steps of cancer development. Single nucleotide polymorphisms (SNPs) in the COX-2 promoter might contribute to differential COX-2 expression and subsequent interindividual variability in susceptibility to cancer. This study sought to identify functional SNPs in the COX-2 promoter and evaluated their effects on the risk of developing esophageal squamous cell carcinoma (ESCC). Methods: Thirty individual DNA samples were sequenced to search for SNPs, and the function of the SNPs was examined by a set of biochemical assays. Genotypes and haplotypes were analyzed in 1026 patients and 1270 controls, and odds ratios and 95% confidence intervals (CIs) were estimated by logistic regression. Results: Three SNPs, −1290A→G, −1195G→A, and −765G→C, were identified; the frequencies of variant alleles were 0.04, 0.51, and 0.02, respectively. The −1195G→A change creates a c-MYB binding site and displays a higher promoter activity. The −1195A-containing haplotypes had significantly increased luciferase expression and COX-2 messenger RNA levels in esophageal tissues compared with the −1195G-containing counterparts. A case-control analysis showed a 1.72-fold (95% CI, 1.35–2.20) and 2.24-fold (95% CI, 1.59–3.16) excess risk of developing ESCC for the −1195AA or −765CC genotype carriers compared with noncarriers. A greater risk of developing ESCC was observed for A−1195-C−765-containing haplotypes compared with G−1195-G−765-containing haplotypes, suggesting an interaction between the −1195G→A and −765G→C polymorphisms in the context of haplotype. Conclusions: These findings indicate that genetic variants in COX-2 may play a role in mediating susceptibility to esophageal cancer. Cyclooxygenases (COXs, also known as prostaglandin endoperoxide H synthases or prostaglandin G/H synthases) are key enzymes in mediating the conversion of free arachidonic acid into prostaglandin H2, the precursor of molecules such as prostaglandins, prostacyclin, and thromboxanes.1Needleman P. Turk J. Jakschik B.A. Morrison A.R. Lefkowith J.B. Arachidonic acid metabolism.Annu Rev Biochem. 1986; 55: 69-102Crossref PubMed Google Scholar These active products are important regulators of many biologic processes such as inflammation, immune function, cell proliferation, and angiogenesis, which are all relevant to cancer development and progression.2Williams C.S. Mann M. DuBois R.N. The role of cyclooxygenases in inflammation, cancer, and development.Oncogene. 1999; 18: 7908-7916Crossref PubMed Scopus (1293) Google Scholar, 3Fosslien E. Molecular pathology of cyclooxygenase-2 in neoplasias.Ann Clin Lab Sci. 2000; 30: 3-21PubMed Google Scholar, 4Trifan O.C. Hla T. Cyclooxygenase-2 modulates cellular growth and promotes tumorigenesis.J Cell Mol Med. 2003; 7: 207-222Crossref PubMed Scopus (171) Google Scholar, 5Gately S. The contributions of cyclooxygenase-2 to tumor angiogenesis.Cancer Metastasis Rev. 2000; 19: 19-27Crossref PubMed Scopus (348) Google Scholar It has been well known that the COX family consists of 2 isozymes; COX-1 is constitutively expressed in most cell types and involved in the homeostasis of various physiologic functions, whereas COX-2 is an inducible form and its expression can be induced by proinflammatory and mitogenic stimuli such as cytokines and growth factors.6Gasparini G. Longo R. Sarminento R. Morabito A. Inhibitors of cyclooxygenase 2 a new class of anticancer agents?.Lancet Oncol. 2003; 4: 605-615Abstract Full Text Full Text PDF PubMed Scopus (321) Google Scholar Increased expression of COX-2 is observed in many types of cancers, and accumulating evidence indicates that overexpression of COX-2 is associated with many steps of cancer development, including hyperproliferation, transformation, tumor growth, invasion, and metastasis.7Zimmermann K.C. Sarbia M. Weber A.A. Borchard F. Gabbert H.E. Schror K. Cyclooxygenase-2 expression in human esophageal carcinoma.Cancer Res. 1999; 59: 198-201PubMed Google Scholar, 8Shamma A. Yamamoto H. Doki Y. Okami J. Kondo M. Fujiwara Y. Yano M. Inoue M. Matsuura N. Shiozaki H. Monden M. Up-regulation of cyclooxygenase-2 in squamous carcinogenesis of the esophagus.Clin Cancer Res. 2000; 6: 1229-1238PubMed Google Scholar, 9Zhi H. Zhang J. Hu G. Lu J. Wang X. Zhou C. Wu M. Liu Z. The deregulation of arachidonic acid metabolism-related genes in human esophageal squamous cell carcinoma.Int J Cancer. 2003; 106: 327-333Crossref PubMed Scopus (93) Google Scholar, 10Romano M. Claria J. Cyclooxygenase-2 and 5-lipoxygenase converting functions on cell proliferation and tumor angiogenesis implications for cancer therapy.FASEB J. 2003; 17: 1986-1995Crossref PubMed Scopus (222) Google Scholar, 11Buskens C.J. van Rees B.P. Sivula A. Reitsma J.B. Haglund C. Bosma P.J. Offerhaus G.J. van Lanschot J.J. Ristimaki A. Prognostic significance of elevated cyclooxygenase 2 expression in patients with adenocarcinoma of the esophagus.Gastroenterology. 2002; 122: 1800-1807Abstract Full Text Full Text PDF PubMed Scopus (218) Google Scholar, 12Murata H. Kawano S. Tsuji S. Tsuji M. Sawaoka H. Kimura Y. Shiozaki H. Hori M. Cyclooxygenase-2 overexpression enhances lymphatic invasion and metastasis in human gastric carcinoma.Am J Gastroenterol. 1999; 94: 451-455Crossref PubMed Scopus (295) Google Scholar Transcriptional regulation has been shown to be the major mechanism in regulating the expression of COX-2, although posttranscriptional mechanisms such as increased stability of COX-2 mRNA also seem important.6Gasparini G. Longo R. Sarminento R. Morabito A. Inhibitors of cyclooxygenase 2 a new class of anticancer agents?.Lancet Oncol. 2003; 4: 605-615Abstract Full Text Full Text PDF PubMed Scopus (321) Google Scholar Examination of the COX-2 promoter region has identified several key cis-acting regulatory elements, including TATA, PEA3, AP1, nuclear factor interleukin-6, nuclear factor κB, Sp1, adenosine 3′,5′-cyclic monophosphate, c-MYB, and transforming growth factor β response elements, which may play a decisive role in the regulation of COX-2 transcription.13Dixon D.A. Regulation of COX-2 expression in human cancers.Prog Exp Tumor Res. 2003; 37: 52-71Crossref PubMed Scopus (72) Google Scholar Some in vitro experiments have shown that deletion or forced mutations within the consensus sequences of certain cis-acting elements significantly altered gene expression.14Schmedtje Jr, J.F. Ji Y.S. Liu W.L. Dubois R.N. Runge M.S. Hypoxia induces cyclooxygenase-2 via the NF-kappaB p65 transcription factor in human vascular endothelial cells.J Biol Chem. 1997; 272: 601-608Crossref PubMed Scopus (642) Google Scholar, 15Nie M. Pang L. Inoue H. Knox A.J. Transcriptional regulation of cyclooxygenase 2 by bradykinin and interleukin-1beta in human airway smooth muscle cells involvement of different promoter elements, transcription factors, and histone h4 acetylation.Mol Cell Biol. 2003; 23: 9233-9244Crossref PubMed Scopus (107) Google Scholar, 16Xu Q. Ji Y.S. Smedtje J.F. Sp1 increases expression of cyclooxygenase-2 in hypoxic vascular endothelium implication for the mechanisms of aortic aneurysm and heart failure.J Biol Chem. 2000; 275: 24583-24589Crossref PubMed Scopus (105) Google Scholar These findings suggest that naturally occurring sequence variations in the gene might contribute at least in part to differential COX-2 expression and a substantial degree of interindividual variability in susceptibility to cancer and in response to the treatment of patients with COX-2 inhibitors.17Lukiw W.J. Bazan N.G. Cyclooxygenase 2 RNA message abundance, stability, and hypervariability in sporadic Alzheimer neocortex.J Neurosci Res. 1997; 50: 937-945Crossref PubMed Scopus (142) Google Scholar, 18FitzGerald G.A. Patrono C. The coxibs, selective inhibitors of cyclooxygenase-2.N Engl J Med. 2001; 345: 433-442Crossref PubMed Scopus (1444) Google Scholar Several single nucleotide polymorphisms (SNPs) in COX-2 have been reported previously, but most of these polymorphisms seem to be functionally insignificant and not associated with susceptibility to cancer,19Humar B. Giovanoli O. Wolf A. Attenhofer M. Bendik I. Meier R. Muller H. Dobbie Z. Germline alterations in the cyclooxygenase-2 gene are not associated with the development of extracolonic manifestations in a large Swiss familial adenomatous polyposis kindred.Int J Cancer. 2000; 87: 812-817Crossref PubMed Scopus (26) Google Scholar, 20Fritsche E. Baek S.J. King L.M. Zeldin D.C. Eling T.E. Bell D.A. Functional characterization of cyclooxygenase-2 polymorphisms.J Pharmacol Exp Ther. 2001; 299: 468-476PubMed Google Scholar, 21Spirio L.N. Dixon D.A. Robertson J. Robertson M. Barrows J. Traer F. Burt R.W. Leppert M.F. White R. Prescott S.M. The inducible prostaglandin biosynthetic enzyme, cyclooxygenase 2, is not mutated in patients with attenuated adenomatous polyposis coli.Cancer Res. 1998; 58: 4909-4912PubMed Google Scholar whereas a coding polymorphism (Val511Ala) has been linked to reduced risk of colorectal neoplasia.22Lin H.J. Lakkides K.M. Keku T.O. Reddy S.T. Louie A.D. Kau I.H. Zhou H. Gim J.S.Y. Ma H.L. Matthies C.F. Dai A. Huang H.-F. Materi A.M. Lin J.H. Frankl H.D. Lee E.R. Hardy S.I. Herschman H.R. Henderson B.E. Kolonel L.N. Le Marchand L. Garavito R.M. Sandler R.S. Haile R.W. Smith W.L. Prostaglandin H synthase 2 variant (Val511Ala) in African Americans may reduce the risk for colorectal neoplasia.Cancer Epidemiol Biomarkers Prev. 2002; 11: 1305-1315PubMed Google Scholar Recently, 2 SNPs (−490C→G and −765G→C) in the COX-2 promoter region (from −1122 to 54 base pairs) have been described in a UK population.23Papafili A. Hill M.R. Brull D.J. McAnulty R.J. Marshall R.P. Humphries S.E. Laurent G.J. Common promoter variant in cyclooxygenase-2 represses gene expression evidence of role in acute-phase inflammatory response.Arterioscler Thromb Vasc Biol. 2002; 22: 1631-1636Crossref PubMed Scopus (324) Google Scholar It has been shown that the −765G→C polymorphism, which appears to disrupt an Sp1-binding site and thus displays a lower promoter activity, is associated with the phenotype of lower level of C-reactive protein in acute inflammatory response to coronary artery bypass graft surgery. In another study,24Panguluri R.C. Long L.O. Chen W. Wang S. Coulibaly A. Ukoli F. Jackson A. Weinrich S. Ahaghotu C. Isaacs W. Kittles R.A. COX-2 gene promoter haplotypes and prostate cancer risk.Carcinogenesis. 2004; 25: 961-966Crossref PubMed Scopus (101) Google Scholar a 1400-base pair DNA upstream of the ATG start site of COX-2 was resequenced and 4 SNPs (−1285A→G [rs3918304], −1265G→A [rs20415], −899G→C [rs20417], and −297C→G [rs5270]) were detected in black and Bini Nigerian patients. However, only 2 of them (−1265G→A and −297C→G) were presented in European Americans and the reported allelic frequencies of these polymorphisms vary greatly among the 3 populations. All of these findings suggest that the genetic variation in COX-2 displays a striking ethnic difference. The diseases in which a role for COX-2 has been shown are usually characterized by varying individual and even ethnic susceptibility, implying the role of genetic factors. The specific function of COX-2 in the formation of prostaglandins and cancer development (vide supra) makes it a strong candidate for several heritable traits such as common cancers. Genetic polymorphisms that alter the level of protein expressed would be anticipated to have a substantial influence on disease activity. In this study, we sought to identify new and functional polymorphisms in the promoter (∼2 kilobases) of the human COX-2 gene and conducted a large case-control study to evaluate the contribution of detected polymorphisms to the risk of developing esophageal squamous cell carcinoma (ESCC), a common cancer in which COX-2 has been shown to play an important role.7Zimmermann K.C. Sarbia M. Weber A.A. Borchard F. Gabbert H.E. Schror K. Cyclooxygenase-2 expression in human esophageal carcinoma.Cancer Res. 1999; 59: 198-201PubMed Google Scholar, 8Shamma A. Yamamoto H. Doki Y. Okami J. Kondo M. Fujiwara Y. Yano M. Inoue M. Matsuura N. Shiozaki H. Monden M. Up-regulation of cyclooxygenase-2 in squamous carcinogenesis of the esophagus.Clin Cancer Res. 2000; 6: 1229-1238PubMed Google Scholar, 9Zhi H. Zhang J. Hu G. Lu J. Wang X. Zhou C. Wu M. Liu Z. The deregulation of arachidonic acid metabolism-related genes in human esophageal squamous cell carcinoma.Int J Cancer. 2003; 106: 327-333Crossref PubMed Scopus (93) Google Scholar, 11Buskens C.J. van Rees B.P. Sivula A. Reitsma J.B. Haglund C. Bosma P.J. Offerhaus G.J. van Lanschot J.J. Ristimaki A. Prognostic significance of elevated cyclooxygenase 2 expression in patients with adenocarcinoma of the esophagus.Gastroenterology. 2002; 122: 1800-1807Abstract Full Text Full Text PDF PubMed Scopus (218) Google Scholar Thirty DNA samples deriving from blood of randomly selected healthy subjects (all were Han Chinese) were used to search for SNPs within the promoter region of COX-2 (2310 base pairs). These samples included 60 chromosomes, providing at least a 95% confidence level to detect alleles with frequencies >5%. In reference to the human COX-2 gene promoter sequence (GenBank accession no. AY382629), 5 sets of polymerase chain reaction (PCR) primers (provided on request) were designed for SNP screening. The 5 COX-2 promoter fragments from each subject were amplified, and SNPs were identified by directly sequencing the PCR products with ABI PRISM Dye Terminator Sequencing Kits (Applied Biosystems, Foster City, CA) and loading the samples onto an ABI 3700 sequencer. Finally, we used the Mutation Explorer program (Todaysoft Inc, Beijing, China) to identify SNP candidates that were then confirmed by 2 independent observers. We further confirmed these SNP positions and individual genotypes by reamplifying and resequencing the SNP site from the opposite strand. This study included 1026 patients with ESCC and 1270 cancer-free controls. All subjects were unrelated ethnic Han Chinese. Patients were consecutively recruited between July 1999 and July 2003 at the Cancer Hospital of the Chinese Academy of Medical Sciences in Beijing. All patients with histopathologically confirmed ESCC were enrolled, and there was no sex and age restriction. The response rate for patients was 92%. The exclusion criteria included previous cancer and previous chemotherapy or radiotherapy. Among 1026 patients, 588 were participants in a molecular epidemiologic study of esophageal cancer as described previously.25Sun T. Miao X. Zhang X. Tan W. Xing P. Lin D. Polymorphisms of death pathway genes FAS and FASL in esophageal squamous-cell carcinoma.J Natl Cancer Inst. 2004; 96: 1030-1036Crossref PubMed Scopus (181) Google Scholar Controls were cancer-free individuals randomly selected from a community cancer screening program for early detection of cancer conducted in the same regions during the same period the patients were recruited. The response rate for controls was 89%. The characteristics of most controls (n = 648) were described previously.25Sun T. Miao X. Zhang X. Tan W. Xing P. Lin D. Polymorphisms of death pathway genes FAS and FASL in esophageal squamous-cell carcinoma.J Natl Cancer Inst. 2004; 96: 1030-1036Crossref PubMed Scopus (181) Google Scholar The selection criteria for the controls included no individual history of cancer and frequency matched to case patients based on sex and age (±5 years). In this study, we also selected 622 more controls from the same database frequency matched to case patients based on age and sex as described previously, for a total of 1270 controls. At recruitment, informed consent was obtained from each subject and personal data from each participant regarding demographic characteristics such as sex and age and related risk factors including tobacco smoking were collected via questionnaire. This study was approved by the institutional review board of the Chinese Academy of Medical Sciences Cancer Institute. Genomic DNA was extracted from peripheral blood lymphocytes of all controls and most patients. Twenty-eight percent of DNA samples from patients were isolated from surgically removed normal tissues adjacent to the tumors. Genotypes were determined by PCR-based restriction fragment length polymorphism. The PCR primer pairs used to amplify the COX-2 promoter region containing −765G→C, −1195G→A, and −1290A→G sites were 765F5′-tattatgaggagaatttacctttcgc-3′/765R5′-gctaagttgctttcaacagaagaaat-3′, 1195F5′-ccctgagcactacccatgat-3′/1195R5′-gcccttcataggagatactgg-3′, and 1290F5′-caggttttatgctgtcattttcc-3′/1290R5′-tagtgctcagggaggagcat-3′. PCR was performed at a 25-μL reaction mixture containing 100 ng of DNA, 0.1 μmol/L of each primer, 0.2 mmol/L of deoxynucleoside triphosphate, 1.0 U of Taq DNA polymerase (Promega, Madison, WI), and 1× reaction buffer. The concentration of MgCl2 was 3.0 mmol/L for the −765G→C site and 1.5 mmol/L for both the −1195G→A site and the −1290A→G sites. The PCR profile consisted of an initial melting step of 2 minutes at 95°C, followed by 35 cycles of 30 seconds at 94°C, 30 seconds at 62°C (for the −765G→C site) or 60°C (for both the −1195G→A and the −1290A→G sites), 45 seconds at 72°C, and a final elongation step of 7 minutes at 72°C. Restriction enzymes HhaI, PvuII, or RsaI (New England Biolabs, Beverly, MA) was used to distinguish the −765G→C, −1195G→A, or −1290A→G genotypes, respectively. Genotyping was performed without knowledge of the case/control status of the subjects. A 10% random sample of cases and controls was tested twice by different persons, and all results were 100% concordant. Unconditional logistic regression was used to assess the association between genotypes and the risk of ESCC using SAS software (version 6.12; SAS Institute, Inc, Cary, NC). Odds ratios (ORs) were all adjusted for age, sex, and smoking where appropriate. A P value of <.05 was used as the criterion of statistical significance, and all statistical tests were 2-sided tests. Linkage disequilibrium was analyzed using LDA software.26Ding K. Zhou K. He F. Shen Y. LDA—a java-based linkage disequilibrium analyzer.Bioinformatics. 2003; 19: 2147-2148Crossref PubMed Scopus (116) Google Scholar Haplo.score approach was used to test the association of statistically inferred haplotypes with ESCC.27Schaid D.J. Rowland C.M. Tines D.E. Jacobson R.M. Poland G.A. Score tests for association between traits and haplotypes when linkage phase is ambiguous.Am J Hum Genet. 2002; 70: 425-434Abstract Full Text Full Text PDF PubMed Scopus (1575) Google Scholar Because haplo.score does not provide the magnitude of the effect of each haplotype, haplo.glm was performed to calculate adjusted ORs and 95% confidence intervals (CIs) for each haplotype.28Lake S.L. Lyon H. Tantisira K. Silverman E.K. Weiss S.T. Laird N.M. Schaid D.J. Estimation and tests of haplotype-environment interaction when linkage phase is ambiguous.Hum Hered. 2003; 55: 56-65Crossref PubMed Scopus (395) Google Scholar Both haplo.score and haplo.glm were implemented in the haplo.stats software developed using the R language. The method assigns the probability for each haplotype pair in each individual and then directly models an individual’s phenotype as a function of each inferred haplotype pair, weighted by their estimated probability, to account for haplotype ambiguity. This program has the advantage that adjustment for covariates and computation of simulation P values for global and each haplotype can be performed. The number of simulations for empirical P values was set as 1000. To verify whether the identified SNPs influence transcriptional activity of COX-2, we constructed a reporter plasmid encompassing −1940 to +204 base pairs of human COX-2 promoter. The primers used for amplifying this DNA fragment were 5′-atacgctagcgccctgtttttccctcactt-3′ and 5′-gataaagcttgcgccaggtactcacctgta-3′, which contain 5′-NheI and 3′-HindIII cloning sites (underlined sequences), respectively. To ensure high-fidelity amplification, LA Taq polymerase (TaKaRa, Dalian, China) was used in the PCR. The PCR product was digested with NheI and HindIII and ligated, respectively, into an appropriately digested pGL3-Basic vector (Promega) containing the firefly luciferase gene as a reporter. The resulting construct was designated as pA-A-C according to sequence analysis. This pA-A-C product was subsequently used as a template to generate 7 other constructs containing all of the possible haplotypes (ie, pA-G-C, pA-A-G, pA-G-G, pG-A-C, pG-G-C, pG-A-G, and pG-G-G; Figure 2A) by using site-specific mutagenesis. All constructs used in this study were restriction mapped and sequenced to confirm their authenticity. HeLa cells were cultured in Dulbecco’s modified Eagle medium supplemented with 10% fetal bovine serum, 2 mmol/L l-glutamine, 100 U/mL penicillin, and 100 U/mL streptomycin (Invitrogen, Carlsbad, CA) in a humidified, 5% co2 incubator at 37°C. For transient transfection experiments, 6 × 104 cells were plated in 10-mm 24-multiwell plates and grown to 60%–70% confluence. Transfection was performed using Lipofectamine reagent (Life Technologies, Inc, Rockville, MD) according to the manufacturer’s protocol. Cells were cotransfected with 50 ng of reporter plasmid and 1.0 ng of pRL-SV40 (Luciferase Assay System; Promega); the latter containing Renilla reniformis luciferase was used to standardize transfection efficiency. Luciferase activity was determined according to the manufacturer’s protocol using a luciferase assay system (Promega). Briefly, cells were scraped into lysis reagent, transferred to microfuge tubes, and centrifuged for 30 seconds at 12,000g. Luciferase activity was measured using a manual luminometer (TD-20/20; Turner Designs, Sunnyvale, CA) by mixing 100 μL of luciferase assay reagent with 20 μL of 1:10-diluted cell lysate, and the value for each sample was recorded 3 times at 10-second intervals. For each plasmid construct, 3 independent transfection experiments were performed, and each was done in triplicate. The empty pGL3 Basic vector cotransfected with pRL-SV40 plasmid served as a control. Fold increase was calculated by defining the activity of empty pGL3 Basic vector as 1. Differences were determined by t test, and P < .01 was considered significant. Synthetic double-stranded oligonucleotides 5′-atgaaattccagctgtcaaaatc-3′ and 5′-atgaaattccaactgtcaaaatc-3′ corresponding to the −1195G or −1195A sequence from the COX-2 promoter region were labeled with biotin according to the Biotin 3′ End DNA Labeling Kit protocol (Pierce, Rockford, IL). The gel shift reaction was accomplished by using the LightShift Chemiluminescent EMSA Kit (Pierce) according to the manufacturer’s instructions. For each gel shift reaction, an aliquot of labeled oligonucleotide (10 fmoles) was incubated with 3 μg of nuclear extract from HeLa cells (Promega) for 20 minutes at room temperature in a 10-μL mixture containing 1× binding buffer, 2.5% glycerol, 10 mmol/L MgCl2, 50 mg/L poly(deoxyinosinic-deoxycytidylic acid), and 0.05% Nonidet P-40. For competition experiments, a 200-fold molar excess of unlabeled −1195G or −1195A probe, a c-MYB recognition element (5′-gcattataacggttttttagcgc-3′), or a mutant c-MYB recognition element (5′-cacattatatgccattttttagc-3′)29Gaillard C. Le Rouzic E. Creminon C. Perbal B. Alteration of C-MYB DNA binding to cognate responsive elements in HL-60 variant cells.J Clin Pathol Mol Pathol. 2002; 55: 325-335Crossref Scopus (1) Google Scholar was preincubated for 2 minutes at room temperature with the nuclear extracts before the addition of the labeled probe. Samples were run on a nondenaturing 6% polyacrylamide gel in 0.5× Tris-borate EDTA at 100 V for 70 minutes at 4°C. The electrophoresed binding reactions were transferred to positively charged nylon membrane (Pierce) in a Mini Trans-Blot Cell (BioRad, Hercules, CA) at 380 mA for 40 minutes, and cross-link was performed for 90 seconds in a GS Gene Linker UV chamber (BioRad). Detection of biotin-labeled DNA was performed using stabilized streptavidin/horseradish peroxidase conjugate (Pierce) according to the manufacturer’s procedures. Chromatin immunoprecipitation assays were performed using the chromatin immunoprecipitation assay kit (Upstate Biotechnology, Lake Placid, NY) according to the manufacturer’s protocol. Briefly, 1 × 106 KYSE30 cells (a cell line of ESCC with the COX-2 −1195AA genotype) were treated with 1% formaldehyde for 10 minutes. After washing with PBS, cells were lysed in detergent lysis buffer. Lysates were washed and sonicated. One microgram of rabbit anti-c-MYB polyclonal antibody (Santa Cruz Biotechnology, Santa Cruz, CA) or rabbit anti-β-actin antibody was added and incubated overnight. Salmon sperm DNA/protein A agarose were used; after extensive washing, cross-links were removed at 65°C for 4 hours in an elution buffer. DNA was isolated using phenol/chloroform extraction and ethanol precipitation. Five percent of purified DNA was analyzed by PCR with the primers 5′-ccctgagcactacccatgat-3′ and 5′-gcccttcataggagatactgg-3′, which produced a 212-base pair fragment of COX-2 promoter containing the −1195 polymorphic site. Forty normal esophageal tissues adjacent to the tumors were obtained from surgically removed specimens of individual patients (all nonsmokers).30Yu C. Zhou Y. Miao X. Xiong P. Tan W. Lin D. Functional haplotype in the promoter of matrix metalloproteinase-2 predict risk of the occurrence and metastasis of esophageal cancer.Cancer Res. 2004; 64: 7622-7628Crossref PubMed Scopus (186) Google Scholar The normal tissues sampled at least 5 cm away from the margin of the tumor were immediately placed in liquid nitrogen and then stored at −80°C before analysis. Total RNA was isolated from tissues using TRIzol reagent (Molecular Research Center Inc, Cincinnati, OH). An aliquot of total RNA (2 μg) from each specimen was reverse transcribed into single-strand complementary DNA using oligo (dT)15 primer and Superscript II (Invitrogen). Relative gene expression quantitation for COX-2, with β-actin as an internal reference gene, was performed using the ABI Prism 7000 sequence detection system (Applied Biosystems) based on the SYBR-Green method. The primers used for COX-2 were 5′-gctcaaacatgatgtttgcattc-3′ and 5′-gctggccctcgcttatga-3′ and for β-actin were 5′-ggcggcaccaccatgtaccct-3′ and 5′-aggggccggactcgtcatact-3′. The PCR reaction mixture consisted of 0.1 μmol/L of each primer, 1× SYBR Premix EX Taq (Perfect Real Time) premix reagent (TaKaRa), and 50 ng complementary DNA to a final volume of 20 μL. Cycling conditions were 95°C for 5 minutes, followed by 40 cycles at 95°C for 15 seconds and 62°C for 1 minute. The expression of individual COX-2 measurements was calculated relative to expression of β-actin using a modification of the method described by Lehmann and Kreipe.31Lehmann U. Kreipe H. Real-time PCR analysis of DNA and RNA extracted from formalin-fixed and paraffin-embedded biopsies.Methods. 2001; 25: 409-418Crossref PubMed Scopus (320) Google Scholar All analyses were performed in a blinded fashion with the laboratory persons unaware of genotyping data. The SNPs identified were compared with the National Center for Biotechnology Information (NCBI) database (http://www.ncbi.nlm.nih.gov/dbSNP). A search for differences in potential transcription factor binding capability between 2 different alleles at the site of each polymorphism was performed using the programs TRANSFAC and the accompanying Match at http://www.gene-regulation.com.32Matys V. Fricke E. Geffers R. Gossling E. Haubrock M. Hehl R. Hornischer K. Karas D. Kel A.E. Kel-Margoulis O.V. Kloos D.U. Land S. Lewicki-Potapov B. Michael H. Munch R. Reuter I. Rotert S. Saxel H. Scheer M. Thiele S. Wingender E. TRANSFAC transcriptional regulation, from patterns to profiles.Nucleic Acids Res. 2003; 31: 374-378Crossref PubMed Scopus (1680) Google Scholar By sequencing the full-length promoter region of COX-2 from 30 healthy subjects, we identified 3 SNPs: −1290A→G, −1195G→A, and −765G→C (Figure 1). Among them, the −1290A→G variant has never been
Год издания: 2005
Авторы: Xuemei Zhang, Xiaoping Miao, Wen Tan, Baitang Ning, Zhihua Liu, Hong Yuan, Wenguang Song, Yongli Guo, Xinyu Zhang, Yan Shen, Boqin Qiang, Fred F. Kadlubar, Dongxin Lin
Издательство: Elsevier BV
Источник: Gastroenterology
Ключевые слова: Inflammatory mediators and NSAID effects, Estrogen and related hormone effects, Helicobacter pylori-related gastroenterology studies
Открытый доступ: hybrid
Том: 129
Выпуск: 2
Страницы: 565–576