A Comparative Analysis by SAGE of Gene Expression Profiles of Barrett’s Esophagus, Normal Squamous Esophagus, and Gastric Cardia
2005; Elsevier BV; Volume: 129; Issue: 4 Linguagem: Inglês
10.1053/j.gastro.2005.07.026
ISSN1528-0012
AutoresJantine W.P.M. van Baal, Francesca Milano, Agnieszka Magdalena Rygiel, Jacques Bergman, Wilda Rosmolen, Sander J. H. van Deventer, Kenneth K. Wang, Maikel P. Peppelenbosch, Kausilia K. Krishnadath,
Tópico(s)Helicobacter pylori-related gastroenterology studies
ResumoBackground & Aims: The metaplastic process in which the normal squamous epithelium of the distal esophagus is replaced by columnar-lined epithelium, known as Barrett’s esophagus (BE), is poorly understood. The aim of this study was to define, analyze, and compare transcription profiles of BE, normal cardia epithelium, and squamous epithelium to gain more insight into the process of metaplasia and to identify uniquely expressed genes in these epithelia. Methods: Serial analysis of gene expression was applied for obtaining transcription libraries of biopsy specimens taken from a BE-affected patient with intestinal type of metaplasia and from normal squamous and gastric cardia epithelia. Validation of results by reverse-transcription polymerase chain reaction and immunoblotting was performed using tissues of 20 patients with BE. Results: More than 120,000 tags were sequenced. Between BE and squamous 776, and between BE and gastric cardia 534 tags were significantly differentially expressed (P < .05, pairwise comparison). In contrast, squamous compared with gastric cardia epithelia showed significant differential expression of 1316 tags. The most up-regulated genes in BE compared with squamous epithelium were trefoil factors, annexin A10, and galectin-4. Each of the epithelia showed a unique cytokeratin expression profile. Conclusions: This study provides a comparison of the transcriptomes of BE, squamous epithelium, and gastric cardia epithelium. BE proves to be an incompletely differentiated type of epithelium that shows similarities to both normal squamous and gastric cardia epithelia. In addition, several uniquely expressed genes are identified. These results are a major advancement in understanding the process of metaplasia that leads to BE. Background & Aims: The metaplastic process in which the normal squamous epithelium of the distal esophagus is replaced by columnar-lined epithelium, known as Barrett’s esophagus (BE), is poorly understood. The aim of this study was to define, analyze, and compare transcription profiles of BE, normal cardia epithelium, and squamous epithelium to gain more insight into the process of metaplasia and to identify uniquely expressed genes in these epithelia. Methods: Serial analysis of gene expression was applied for obtaining transcription libraries of biopsy specimens taken from a BE-affected patient with intestinal type of metaplasia and from normal squamous and gastric cardia epithelia. Validation of results by reverse-transcription polymerase chain reaction and immunoblotting was performed using tissues of 20 patients with BE. Results: More than 120,000 tags were sequenced. Between BE and squamous 776, and between BE and gastric cardia 534 tags were significantly differentially expressed (P < .05, pairwise comparison). In contrast, squamous compared with gastric cardia epithelia showed significant differential expression of 1316 tags. The most up-regulated genes in BE compared with squamous epithelium were trefoil factors, annexin A10, and galectin-4. Each of the epithelia showed a unique cytokeratin expression profile. Conclusions: This study provides a comparison of the transcriptomes of BE, squamous epithelium, and gastric cardia epithelium. BE proves to be an incompletely differentiated type of epithelium that shows similarities to both normal squamous and gastric cardia epithelia. In addition, several uniquely expressed genes are identified. These results are a major advancement in understanding the process of metaplasia that leads to BE. Barrett’s esophagus (BE) is a precancerous condition in which the normal squamous epithelium of the esophagus is replaced by metaplastic, columnar-lined epithelium.1Spechler S.J. Zeroogian J.M. Antonioli D.A. Wang H.H. Goyal R.K. Prevalence of metaplasia at the gastro-oesophageal junction.Lancet. 1994; 344: 1533-1536Abstract PubMed Scopus (550) Google Scholar, 2Ransford R.A. Jankowski J.A. Genetic versus environmental interactions in the oesophagitis-metaplasia-dysplasia-adenocarcinoma sequence (MCS) of Barrett’s oesophagus.Acta Gastroenterol Belg. 2000; 63: 18-21PubMed Google Scholar In Western countries, the prevalence of BE and the incidence of esophageal adenocarcinoma have been increasing rapidly.3Devesa S.S. Blot W.J. Fraumeni Jr, J.F. Changing patterns in the incidence of esophageal and gastric carcinoma in the United States.Cancer. 1998; 83: 2049-2053Crossref PubMed Scopus (1951) Google Scholar The increasing prevalence of BE is alarming and calls for screening programs of high-risk populations and development of preventive therapies.4Sharma P. McQuaid K. Dent J. Fennerty M.B. Sampliner R. Spechler S. Cameron A. Corley D. Falk G. Goldblum J. Hunter J. Jankowski J. Lundell L. Reid B. Shaheen N.J. Sonnenberg A. Wang K. Weinstein W. A critical review of the diagnosis and management of Barrett’s esophagus the AGA Chicago Workshop.Gastroenterology. 2004; 127: 310-330Abstract Full Text Full Text PDF PubMed Scopus (469) Google Scholar The transition of BE into cancer is a process known to go along with the accumulation of several genetic events such as aneuploidy, expression of oncogenes, and losses of cell surface receptors and tumor suppressor genes.5Iravani S. Zhang H.Q. Yuan Z.Q. Cheng J.Q. Karl R.C. Jove R. Coppola D. Modification of insulin-like growth factor 1 receptor, c-Src, and Bcl-XL protein expression during the progression of Barrett’s neoplasia.Hum Pathol. 2003; 34: 975-982Abstract Full Text Full Text PDF PubMed Scopus (57) Google Scholar, 6Krishnadath K.K. Reid B.J. Wang K.K. Biomarkers in Barrett esophagus.Mayo Clin Proc. 2001; 76: 438-446PubMed Scopus (66) Google Scholar, 7Sarbia M. Geddert H. Klump B. Kiel S. Iskender E. Gabbert H.E. Hypermethylation of tumor suppressor genes (p16INK4A, p14ARF and APC) in adenocarcinomas of the upper gastrointestinal tract.Int J Cancer. 2004; 111: 224-228Crossref PubMed Scopus (78) Google Scholar, 8Maley C.C. Galipeau P.C. Li X. Sanchez C.A. Paulson T.G. Blount P.L. Reid B.J. The combination of genetic instability and clonal expansion predicts progression to esophageal adenocarcinoma.Cancer Res. 2004; 64: 7629-7633Crossref PubMed Scopus (158) Google Scholar To understand the histologic concept of BE, several gene expression profile and gene chip analysis studies have been performed, for instance through comparing BE and esophageal carcinomas or through comparison of the gene expression profiles of BE and intestinal metaplasia of the cardia.9Selaru F.M. Zou T. Xu Y. Shustova V. Yin J. Mori Y. Sato F. Wang S. Olaru A. Shibata D. Greenwald B.D. Krasna M.J. Abraham J.M. Meltzer S.J. Global gene expression profiling in Barrett’s esophagus and esophageal cancer a comparative analysis using cDNA microarrays.Oncogene. 2002; 21: 475-478Crossref PubMed Scopus (101) Google Scholar, 10Chang Y. Gong J. Liu B. Zhang J. Dai F. Gene expression profiling in Barrett’s esophagus and cardia intestinal metaplasia a comparative analysis using cDNA microarray.World J Gastroenterol. 2004; 10: 3194-3196PubMed Google Scholar Recently, gene chip analysis has been performed in which BE was compared with duodenal, gastric, and normal squamous epithelium.11Barrett M.T. Yeung K.Y. Ruzzo W.L. Hsu L. Blount P.L. Sullivan R. Zarbl H. Delrow J. Rabinovitch P.S. Reid B.J. Transcriptional analyses of Barrett’s metaplasia and normal upper GI mucosae.Neoplasia. 2002; 4: 121-128Abstract Full Text PDF PubMed Google Scholar Indeed, to develop novel screening and preventive strategies, it is of major importance to understand the biologic pathways involved in the metaplastic transition of normal squamous epithelium into columnar epithelium. We hypothesized that objective, quantitative analyses of large molecular genetic data sets of BE and the surrounding normal epithelia such as normal squamous esophageal and normal gastric cardia mucosa will accurately classify the different phenotypes of these epithelia and improve our insight into the biologic mechanisms involved in the process of metaplasia.In the present study, gene expression profiles were obtained by using serial analysis of gene expression (SAGE). The procedure, as first described by Velculescu et al, allows rapid, quantitative, and simultaneous analysis of thousands of genetic transcripts from tissue samples.12Velculescu V.E. Zhang L. Vogelstein B. Kinzler K.W. Serial analysis of gene expression.Science. 1995; 270: 484-487Crossref PubMed Scopus (3568) Google Scholar SAGE is based on 2 principles. First, a short nucleotide sequence, a tag, is generated. Because the location of the tags within the transcripts is exactly defined, these tags contain sufficient information to identify transcripts through public databases (SAGEgenie; http://cgap.nci.nih.gov). Second, by cloning these tags serially, along with a restriction enzyme recognition sequence that serves as an anchor, a large amount of transcripts can be identified efficiently by sequencing. This reveals the identity of thousands of tags and at the same time quantifies their level of expression.For the present study, SAGE profiles were made of RNA isolated from BE, normal squamous epithelium, and normal gastric cardia mucosa of patients with BE. A panel of another 20 patients with BE was used to validate the profiles by reverse-transcription polymerase chain reaction (RT-PCR) and immunoblotting. SAGE was performed, resulting in 3 unique SAGE libraries with more than 120,000 identified tags. In particular, the whole spectrum of cytokeratins (CK1–CK20) as found in the 3 epithelia is described, and expression of the most informative CKs is validated at protein level.The results indicate that BE is not a fully differentiated phenotype, but rather an incompletely transdifferentiated lesion that has strong similarities to both normal squamous esophageal epithelium and the columnar cardia mucosa. This study provides an important step toward a transcriptome of Barrett’s metaplasia as a comparison to its surrounding epithelia in which Barrett’s metaplasia develops. The unique profiles harbor a wealth of information and provide us the identity of several genes involved in several cell signaling pathways, which will contribute to understanding and elucidating important biologic processes involved in metaplasia. In addition, several unique genes that can be used as novel markers for distinguishing the different type of epithelia are identified.Materials and MethodsPatients and Biopsy SpecimensTissue samples were obtained during routine surveillance endoscopy of 21 patients with known BE but without dysplasia. Eighteen were men, and mean age was 62 years (range, 33–83 years). The average length of the BE segment measured endoscopically was 3.8 cm (range, 2–9 cm). All patients were on long-term proton pump inhibition (40–80 mg daily) to prevent reflux esophagitis. BE was defined as histologically recognized incompletely differentiated intestinal type of metaplasia in the distal esophagus. Paired biopsy specimens, taken next to each other, were obtained of the Barrett’s segment, normal squamous esophagus, and gastric cardia. Biopsy of the Barrett’s segment was performed at least 2 cm above the gastroesophageal junction yet within the Barrett’s segment, recognized endoscopically as typically pink-colored columnar type of metaplasia. Normal squamous epithelium was taken at least 2 cm above the Barrett’s segment, and gastric cardia was taken within 5 cm below the gastroesophageal junction. Endoscopically, none of the patients had reflux esophagitis. All patients had proven incompletely differentiated intestinal type of columnar epithelium without dysplasia in the histologic control biopsy specimens with no signs of active or acute inflammation. Normal gastric cardia and normal squamous esophagus were also confirmed histologically in all the pairwise-taken control biopsy specimens. All patients signed informed consent for the use of their biopsy material.RNA IsolationTotal RNA was isolated from biopsy specimens using TRIzol reagent (Life Technologies Inc, Invitrogen, Breda, The Netherlands) according to the manufacturer’s instructions. Spectrophotometry was performed with 1 μL of total RNA to quantitate on the Nanodrop (type ND-1000; Nanodrop Technologies, Wilmington, DE).SAGE ProcedureThe SAGE libraries were obtained essentially following the SAGE protocol as described by Velculescu et al using the I-SAGE Kit (Life Technologies) and following the manufacturer’s instructions.12Velculescu V.E. Zhang L. Vogelstein B. Kinzler K.W. Serial analysis of gene expression.Science. 1995; 270: 484-487Crossref PubMed Scopus (3568) Google Scholar Electroporate transformation was performed following the manufacturer’s protocol (Bio-Rad, Hercules, CA). Colony PCR was performed with specific primers Sp6-F and T7A-R. DNA sequencing was performed using the Big Dye Terminator Kit (Applied Biosystems, Foster City, CA) and the T7A-R primers. Samples were run on an ABI3730 DNA Analyzer (Applied Biosystems) and analyzed with Sequence Analysis 5.1 software.SAGE and Statistical AnalysisFor analysis of the SAGE data, the program USAGE V2 (Bioinformatics Department, Academic Medical Center) and the public databases of the National Center for Biotechnology Information Web site and SAGE Genie (http://cgap.nci.nih.gov) were used.13van Kampen A.H. van Schaik B.D. Pauws E. Michiels E.M. Ruijter J.M. Caron H.N. Versteeg R. Heisterkamp S.H. Leunissen J.A. Baas F. van der Mee M. USAGE: a web-based approach towards the analysis of SAGE data. Serial Analysis of Gene Expression.Bioinformatics. 2000; 16: 899-905Crossref PubMed Scopus (36) Google Scholar, 14Lash A.E. Tolstoshev C.M. Wagner L. Schuler G.D. Strausberg R.L. Riggins G.J. Altschul S.F. SAGEmap a public gene expression resource.Genome Res. 2000; 10: 1051-1060Crossref PubMed Scopus (352) Google Scholar Statistical analyses and comparison of the SAGE libraries was performed using a comparative Z-test (pairwise comparison, binominal approach) of the USAGE V2 program.15Kal A.J. van Zonneveld A.J. Benes V. van den Berg M. Koerkamp M.G. Albermann K. Strack N. Ruijter J.M. Richter A. Dujon B. Ansorge W. Tabak H.F. Dynamics of gene expression revealed by comparison of serial analysis of gene expression transcript profiles from yeast grown on two different carbon sources.Mol Biol Cell. 1999; 10: 1859-1872Crossref PubMed Scopus (305) Google Scholar, 16Ruijter J.M. Van Kampen A.H. Baas F. Statistical evaluation of SAGE libraries consequences for experimental design.Physiol Genomics. 2002; 11: 37-44Crossref PubMed Scopus (86) Google ScholarRT-PCRComplementary DNAs from biopsy specimens of 20 patients were synthesized from 1 μg of total RNA using an oligo dT primer and Superscript II MMLV reverse transcriptase according to the manufacturer’s instructions (Life Technologies). Primers for selected genes (Table 1) were derived from messenger RNA (mRNA) sequences as deposited in GenBank (National Center for Biotechnology Information Web site). PCR analyses were performed using Reddy Mix PCR Master Mix (Applied Biosystems). The mRNA expression level was determined by the ratio of signal intensity of the mRNA to that of the β-actin. Data are expressed as means ± SEM. Comparison between 2 groups was analyzed using 2-tailed paired t tests.Table 1Primer SequencesGeneForward primerReverse primerAnnealing temperature (°C)Fragment length (base pairs)TFF1TTTGGAGCAGAGAGGAGGTTGAGTAGTCAAAGTCAGAGCAG60438TFF2ATGGATGCTGTTTCGACTCCGGCACTTCAAAGATGAAGTTG55247TFF3GTGCCAGCCAAGGACAGCGTTAAGACATCAGGCTCCAG58303CK7TGAATTAACCGCCGCACAGTGCATTTGGCCATCTCCTCA65277CK20GGGACCTGTTTGTTGGCAATGATTTGCAGGACACACCGAGCAT55247Annexin A10TTGTTCTCTGTGTTCGAGACAAACCGTAGGCAAATTCAGGATAGTAGGC52609Galectin-4GCTCAACGTGGGAATGTCTGTGAGCCCACCTTGAAGTTGATA60461FABP1TCATGAAGGCAATCGGTCTGGTGATTATGTCGTCGCCGTTGAGT55277β-actinGTCAGAAGGATTCCTATGTGGGCTCATTGCCAATGGTGATG52628β2-microglobulinCTCGCGCTACTCTCTCTTTCTTGCTCCACTTTTTCAATTCTCT60185NOTE. Primer sequences used for RT-PCR with corresponding used annealing temperatures and PCR fragment lengths. Open table in a new tab ImmunoblottingImmunoblotting was performed as described by Hardwick et al.17Hardwick J.C. Van Den Brink G.R. Bleuming S.A. Ballester I. Van Den Brande J.M. Keller J.J. Offerhaus G.J. Van Deventer S.J. Peppelenbosch M.P. Bone morphogenetic protein 2 is expressed by, and acts upon, mature epithelial cells in the colon.Gastroenterology. 2004; 126: 111-121Abstract Full Text Full Text PDF PubMed Scopus (209) Google Scholar Biopsy specimens were lysed with 200 μL lysis buffer. Twenty milligrams of protein per lane was loaded onto sodium dodecyl sulfate/polyacrylamide gel electrophoresis. The blots were blocked with 2% bovine serum albumin in Tris-buffered saline supplemented with 0.1% Tween 20. The antibodies used and dilutions are summarized in Table 2.Table 2Antibodies as Used for Immunoblot AnalysisAntibodySpeciesCompanyCountryDilutionCK5/6Mouse monoclonalChemiconUnited States1:500CK7Mouse monoclonalChemiconUnited States1:500CK8Mouse monoclonalChemiconUnited States1:500CK10/13Mouse monoclonalDakoDenmark1:500CK18Mouse monoclonalSigmaUnited States1:500CK20Mouse monoclonalProgenGermany1:500Actin (I-19)Goat polyclonalSanta CruzUnited States1:2000 Open table in a new tab ResultsThree unique SAGE libraries were obtained, in total consisting of more than 120,000 tags. The SAGE library characteristics are described in Table 3. The complete SAGE libraries can be found on the Gene Expression Omnibus Web site (http://www.ncbi.nlm.nih.gov/geo/) (Table 3). A minority of the identified tags correspond with different genes due to the presence of conserved sequences and common repeats in the 3′ untranslated mRNA transcript. An example is the tag TTTTCTGAAA, which matches with several genes, namely thioredoxin and surfeit 5. Various different tags can represent the same gene; for instance, the expressed sequence tags of GATACTGCCT, AAAGCACAAG, and ATGTAATCAC correspond to the gene cluster of keratin 6A. This variation may be the result of alternative splicing, alternative polyadenylation, or polymorphisms in the mRNA from which these tags are derived.Table 3SAGE Library CharacteristicsSquamous esophagusBEGastric cardiaTotal tags50,50846,26925,797Unique tags14,83516,0588810Singletons (%)4168 (21)4430 (25)6485 (25)Tags 5 times present12011202612Tags 10 times present538545262Accession codeGSM52501GSM52502GSM52500NOTE. Number of total tags in the squamous esophagus, BE, and gastric cardia libraries, together with the corresponding accession code in the Gene Expression Omnibus Web site (http://www.ncbi.nlm.nih.gov/geo/), the number of unique tags, the number of singletons, and the number of tags at least 5 times and 10 times present in each of the libraries. Calculation of the percentages of singleton tags was based on the total number of tags present in the libraries. Open table in a new tab Comparison of the Expression Profiles of BE With Normal Squamous Epithelium and Normal Gastric CardiaBetween the BE and squamous esophagus SAGE library, 776 tags were significantly differentially expressed (P < .05), 72 tags were more than 10-fold up-regulated, and 26 tags were more than 10-fold down-regulated (Supplementary Figure 1A, see supplemental material online at www.gastrojournal.org). The BE SAGE library as compared with the gastric cardia library showed 534 tags significantly differentially expressed (P < .05). Thirty-one tags were more than 10-fold up-regulated and 76 tags were more than 10-fold down-regulated in BE compared with gastric cardia (Supplementary Figure 1B, see supplemental material online at www.gastrojournal.org). Between the squamous esophagus and gastric cardia SAGE libraries, 1316 tags were significantly differently expressed (P < .05). From these, 108 tags were more than 10-fold up-regulated and 140 tags were more than 10-fold down-regulated in the squamous epithelium. Genes were also clustered in groups of biologic processes (see supplemental material online at www.gastrojournal.org).Validation of SAGE ResultsExpression levels of trefoil factor (TFF) 1, TFF2, TFF3, galectin-4, annexin A10, and fatty acid binding protein 1 (FABP1) were verified by RT-PCR. In all cases examined, the expression of genes represented by tags in either SAGE library was confirmed. TFF1, TFF2, TFF3, annexin A10, galectin-4, and FABP1 were expressed significantly higher in all BE samples compared with all control squamous esophagus samples (Figure 1).CK Expression PatternsSpecific CK expression patterns were found for the 3 epithelia by SAGE and verified by RT-PCR and immunoblotting. Tags corresponding to CKs 7, 8, 18, 19, and 20 were expressed significantly higher in BE compared with squamous esophagus (P < .05), whereas tags corresponding to CKs 1, 4, 5, 6A, 6B, 13, 14, 15, and 17 were expressed significantly higher in squamous esophagus (P < .05; Figure 2). Similar expression in BE and squamous esophagus was found for CKs 10 and 16. Compared with the gastric cardia SAGE library, CKs 4, 5, 6A, 7, 8, 13, 15, and 19 were expressed significantly higher in BE (P < .05; Figure 2).Figure 2Keratin expression patterns in the SAGE libraries of BE, normal squamous esophagus, and gastric cardia. CKs 4, 6A, 6B, and 13 were expressed higher in squamous esophagus. CKs 7, 8, 18, 19, and 20 were expressed higher in BE compared with squamous esophagus. CK18 and CK20 were highly expressed in both the BE and the gastric cardia SAGE libraries.View Large Image Figure ViewerDownload (PPT)The CK expression profiles were validated by RT-PCR. CK7 and CK20 were both significantly higher expressed in BE compared with normal squamous epithelium (Figure 3). Validation at protein level confirmed the high expression of CKs 7, 8, 18, and 20 in BE epithelium as compared with normal squamous tissue (Figure 4). Immunoblot analysis also showed high expression of CKs 5/6 and 10/13 in normal squamous esophagus compared with BE (Figure 4). CKs 8, 18, and 20 were highly expressed and CK7 was less expressed in gastric cardia (Figure 4).Figure 3(A) Validation by RT-PCR on RNA from BE (BA) and squamous esophagus (SQ) biopsy specimens from several patients. RT-PCR shows a higher expression of CK7 and CK20 in BE versus squamous esophagus. (B) Quantification of RT-PCR results of gene expression of CK7 and CK20 in BE and squamous esophagus of 20 patients shows that these are significantly higher expressed in the BE biopsy specimens (2-tailed paired t tests; *P < .05, **P < .01). The gene expression levels were determined by the ratio of signal intensity of the mRNA to that of the β-actin. Data are expressed as means ± SEM.View Large Image Figure ViewerDownload (PPT)Figure 4Immunoblot analysis of CK 5/6, 7, 8, 10/13, 18, and 20 expression in squamous esophagus, BE, and gastric cardia shows higher expression levels of CKs 5/6 and 10/13 in squamous epithelium, while CKs 7, 8, and 20 are highly expressed in BE and less in the cardia biopsy specimens. β-actin was used as a control.View Large Image Figure ViewerDownload (PPT)DiscussionIn this study, SAGE technology was applied to identify the entire transcription profile of BE as a comparison to the profiles of normal squamous and gastric cardia epithelia. The specific information gained from this study helps us to identify factors involved in the metaplastic process and to identify uniquely expressed tissue-specific genes.The main advantage of SAGE compared with expression microarrays and other gene chip technologies is that it allows the generation of a library of thousands of expressed genes without any previous knowledge of the cell’s repertoire. The obtained SAGE transcriptome conveys not only the identity of each expressed gene but also quantifies its level of expression.In this study, more than 120,000 tags were analyzed. Comparison of the SAGE-generated tag expression profiles of BE, normal squamous esophagus epithelium, and gastric cardia epithelium identified hundreds of differentially expressed transcripts. Yet, it should be noted that because of sequence artefacts, some tags could incorrectly be assigned to a certain gene cluster. Particularly, singleton tags should be carefully considered; although these generally correspond to mRNAs expressed at very low levels, some may be due to sequencing errors. Singleton tags were found in approximately 25% of each library, which is in accordance with other SAGE libraries previously published.18Ibrahim A.F. Hedley P.E. Cardle L. Kruger W. Marshall D.F. Muehlbauer G.J. Waugh R. A comparative analysis of transcript abundance using SAGE and Affymetrix arrays.Funct Integr Genomics. 2005; 5: 163-174Crossref PubMed Scopus (33) Google ScholarMapping the SAGE tags to known genes and mRNAs in the SAGE Genie database revealed a large number of genes known to be expressed in BE and many genes not previously recognized in BE. For instance, the Barrett SAGE library confirmed high expression of mucin 5 (TGCACAATAT), TFF1 (CTGGCCCTCG), and TFF3 (CTCCACCCGA).19Guillem P. Billeret V. Buisine M.P. Flejou J.F. Lecomte-Houcke M. Degand P. Aubert J.P. Triboulet J.P. Porchet N. Mucin gene expression and cell differentiation in human normal, premalignant and malignant esophagus.Int J Cancer. 2000; 88: 856-861Crossref PubMed Scopus (111) Google Scholar In the gastric cardia SAGE library, a high number of tags was found, for instance, for glutathione peroxidase 2 (GGTGGTGTCT), known to be highly expressed in the stomach.20Lee S. Baek M. Yang H. Bang Y.J. Kim W.H. Ha J.H. Kim D.K. Jeoung D.I. Identification of genes differentially expressed between gastric cancers and normal gastric mucosa with cDNA microarrays.Cancer Lett. 2002; 184: 197-206Abstract Full Text Full Text PDF PubMed Scopus (64) Google Scholar Also, the tag CAGTGCTTCT (mapping to Deleted in Lymphocytic Leukemia 1 or Gastric Lipase) is known to be highly expressed in the stomach (http://cgap.nci.nih.gov/). The squamous SAGE library showed high expression of the tags GGCAGAGAAG and AAAGCGGGGC corresponding to keratin 4 and 13; both keratins are known to be expressed at protein level in squamous esophagus and not in BE.21El-Zimaity H.M. Graham D.Y. Cytokeratin subsets for distinguishing Barrett’s esophagus from intestinal metaplasia in the cardia using endoscopic biopsy specimens.Am J Gastroenterol. 2001; 96: 1378-1382Crossref PubMed Google Scholar, 22Moll R. Franke W.W. Schiller D.L. Geiger B. Krepler R. The catalog of human cytokeratins patterns of expression in normal epithelia, tumors and cultured cells.Cell. 1982; 31: 11-24Abstract Full Text PDF PubMed Scopus (4495) Google ScholarUnlike the genome, the transcriptome is variable and depends on gene function and the developmental and disease state of the individual. To prevent as much as possible confounding of the results by inflammatory factors, we only included patients with long-term acid suppression and without active reflux esophagitis. We preferred to use tissue samples of one male individual known to have nondysplastic BE to make the SAGE libraries. For confirmation, it is mandatory to verify RNA expression levels on a larger panel of samples. In this study, tissue samples of 20 patients with BE were used to validate expression of several genes by RT-PCR. For instance, the expression of FABP1, galectin-4, annexin A10, TFF1, TFF2, TFF3, CK7, and CK20 (Figure 1, Figure 3) was validated. The high expression of TFFs in BE and in gastric cardia as seen in our profiles are in concordance with several other reports.11Barrett M.T. Yeung K.Y. Ruzzo W.L. Hsu L. Blount P.L. Sullivan R. Zarbl H. Delrow J. Rabinovitch P.S. Reid B.J. Transcriptional analyses of Barrett’s metaplasia and normal upper GI mucosae.Neoplasia. 2002; 4: 121-128Abstract Full Text PDF PubMed Google Scholar, 23Warson C. Van De Bovenkamp J.H. Korteland-Van Male A.M. Buller H.A. Einerhand A.W. Ectors N.L. Dekker J. Barrett’s esophagus is characterized by expression of gastric-type mucins (MUC5AC, MUC6) and TFF peptides (TFF1 and TFF2), but the risk of carcinoma development may be indicated by the intestinal-type mucin, MUC2.Hum Pathol. 2002; 33: 660-668Abstract Full Text Full Text PDF PubMed Scopus (71) Google Scholar, 24Labouvie C. Machado J.C. Carneiro F. Sarbia M. Vieth M. Porschen R. Seitz G. Blin N. Differential expression of mucins and trefoil peptides in native epithelium, Barrett’s metaplasia and squamous cell carcinoma of the oesophagus.J Cancer Res Clin Oncol. 1999; 125: 71-76Crossref PubMed Scopus (43) Google Scholar TFFs are peptides often cosecreted with mucins by goblet cells and play an important role in protecting the epithelium.25Kindon H. Pothoulakis C. Thim L. Lynch-Devaney K. Podolsky D.K. Trefoil peptide protection of intestinal epithelial barrier function cooperative interaction with mucin glycoprotein.Gastroenterology. 1995; 109: 516-523Abstract Full Text PDF PubMed Scopus (306) Google Scholar, 26Hoffmann W. Trefoil factor family (TFF) peptides regulators of mucosal regeneration and repair, and more.Peptides. 2004; 25: 727-730Crossref PubMed Scopus (44) Google Scholar, 27Emami S. Rodrigues S. Rodrigue C.M. Le Floch N. Rivat C. Attoub S. Bruyneel E. Gespach C. Trefoil factor family (TFF) peptides and cancer progression.Peptides. 2004; 25: 885-898Crossref PubMed Scopus (105) Google Scholar Galectin-4 is a member of the galectin family, which are carbohydrate-binding proteins with high affinity for β-galactosides. They are involved in apoptosis and proliferation.28Barondes S.H. Cooper D.N. Gitt M.A. Leffler H. Galectins. St
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