Study Of The Mechanisms Of Genesis And Development Of Barrett's Esophagus By Gene Chip Combined With Tissue Microarray

BackgroudsBarrett's esophagus (Barrett's metaplasia, BE) is characterized by specialized metaplastic intestinal epithelium replacing the normal squamous epithelium in the distal esophagus. Barrett's esophagus is thought to be a premalignant transformation and has been identified in 80% to 100% of esophageal adenocarcinoma of the distal esophagus(EAC). A metaplasia-dysplasia-carcinoma sequence links Barrett's esophagus with EAC, which is one of the fastest-increasing cancers in the Western world. Patients with Barrett's oesophagus have a 2-25% risk of developing mild to severe dysplasia and a 2-5% risk of having adenocarcinoma: 30-150 times higher than the risk in the general population. Forty to fifty percent of Barrett's esophagus patients with severe dysplasia would present adenocarcinoma within 5 years[2]. Along with the progression of biotechnology, the study of the mechanisms of BE got great improvement. However, its exact molecular mechanism was still unclear.Recent advances in biotechnology have revolutionized the high-throughput analysis of gene expression. In particular, the development of gene chip technologies has made it possible to analyse the expression of thousands of genes simultaneously. gene chip technology, in combination with bioinformatics analyses, promises more accurate disease classification, earlier detection, and higher efficiency in the field of cancer diagnosis[4].Gene expression profiling studies have been used in the examination of cancer progression, diagnosis, drug target discovery, and gene therapy evaluation. Many studies have applied this technology for Barrett's metaplasia and adenocarcinoma, and identified a number of candidate genes useful as biomarkers in cancer staging, prediction of recurrence,prognosis, and personalized therapy. Some of these target molecules have been used to develop new serum diagnostic markers and therapeutic targets against BE to benefit patients. Functional classification of differentially expressed genes was then performed to discover molecular pathways and subgroupings associated with each carcinogenic transition. Furthermore, we examined global gene signatures as a potential means of risk stratification at precancerous stages[5].On the other hand, the tissue micro array (TMA)technology can simultaneously detect decades even thousands samples on one chip in situ. As an important branch of biochip, TMA has become a key tool for promoting the quickly transformation of the study results of molecular genetics, genomics, proteome to clinical application. Following by the enforcement of postgenome project, TMA provided an unprecedented high performance methods for discovery biological behaviour, molecular diagnosis, prognosis assessment, and individualized treatment related to tumors. It will be an great contribution to realizing the nature of tumor and finding effective therapy.To identify the molecules involved in Barrett's metaplasia and carcinogenesis and for the development of new molecular therapies, we performed gene expression profile analysis using a cDNA microarray, and detected some related proteins by TMA.Materials and Methods1. Clinical samplesEndoscopic tissue biopsy specimens were taken from BE patients at Gastroenterology Research Institute,Southwest Hospital,Third Military Medical University,Chongqing, China. Routine histopathologic analysis was done to confirm the diagnosis by experienced gastrointestinal pathologists. These samples were labeled and snap frozen in liquid nitrogen and stored at–80°C for future RNA extraction. Informed consent was obtained from all patients. Data of clinicopathologic parameters were obtained from patients'clinical records and pathologic reports. Institutional Human Ethics Committee approved the study.2. RNA preparationTissues were ground into powder in -196℃liquid nitrogen and homogenized using Trizol reagent (Invitrogen Life Technologies, CA) for extraction of total RNA following the instruction of the manufacturer. The integrity of total RNA was checked by 1.2% formaldehyde agarose gel electrophoresis (visual presence of 28S and 18S bands). Total RNA with OD260/OD280 > 1.8 was used for microarray experiments.3. Labeling and hybridizationTwentyμg of total RNA from the tumor and matched normal tissue were labeled with cyanine 3-dUTP and cyanine 5-dUTP by direct labeling method (Perkin Elmer Life Sciences, USA: Micromax Direct labeling kit). The labeled probes were denatured at 95℃for 5 min and hybridized with a human Agilent oligomicroarray(30,968probes) in a hybridization chamber (Corning Life Sciences, USA) at 65℃water bath for 18 h. Before hybridization, slides were pre-hybridized in 5XSSC, 0.1% SDS and 1% BSA solution at 65℃for 45 min to prevent nonspecific hybridization. After hybridization, the slides were washed in 2XSSC with 0.1% SDS, 0.1X SSC with 0.05% SDS and 0.1XSSC sequentially for 20 min each and then spin-dried.4. Microarray image analysisHybridized arrays were scanned at 5 mm resolution on a Gene Pix 4200A scanner (Axon Instruments Inc. Foster City, CA) at various PMT voltage settings to obtain maximal signal intensities with < 0.1% probe saturation. The Cy5-labelled cRNAs were scanned at 635 nm and the Cy3-labeled cRNA samples were scanned at 532 nm. The resulting TIFF images were analyzed by Gene Pix Pro 6.0.1.27 software (Axon Instrument). Both digital images were overlaid to form a pseudo colored image and a detection method was then used to determine the actual target region based on the information from both red (Cy5) and green (Cy3) pixel values. The ratios of the sample intensity to the reference intensity (green: red) for all of the targets were determined and ratio normalization was performed to normalize the center of the ratio distribution to 1.0. Image processing analysis was used for estimation of spot quality by assigning a quality score to each ratio measurement[8]. Data were acquired using MAS 5.0 software (Agilent) and exported to MS Excel.5. Bioinformatics analysisHierarchical clustering: Average linkage hierarchical clustering was done using the Cluster Software version 3.0 written by Michael Eisen[11]. The Euclidean distance metric was used as a measure of similarity between the gene expression patterns for each pair of samples based on log-transformed ratios across all genes. The results were analyzed and visualized with the Tree View Program Version 1.50 also written by Michael Eisen. Those genes showing progressive fold increases or decreases in gene expression relative to normal mucosa were shown proportionally in red and green, respectively.Pathway prediction analysis: We obtained annotations of the bioprocesses, molecular function and cellular localization using the freely available Gene Ontology and Source database[12].The significant gene clusters were queried with known components of the biological pathways on the freely available KEGG database[13]. We also used the Biointerpreter software (http://www.genotypic.co.in/biointerpreter) for gene ontology.6. Real-time RT-PCRThe expressions of 5 up-regulated genes(FLJ45831,MUC1,LYN,ITGβ1,RNF121) and 2 down-regulated genes (CFL1,RPS6)were analyzed by TaqMan SYBR GreenⅠ(Roche Laboratories) fluorescence and LC quantitative PCR(Roche Diagnostics),using GAPDH as a house-keeping gene. The target genes were amplified according to the instruction of Invitrogen RT-PCR kits. After 45 cycles, the cycle threshold(Ct) of each tube and the relative original concentration C were first obtained,then these original data was analyzed by ABI Prism7000 SDS software. Through the relative original concentration ratio, the average value and standard deviation(SD) were calculated.7. TMAThe construction of TMA was performed according by the manuscript of TMA. The samples were dehydrolysised and died by HE, then different tissue samples were arrayed and fixed on the chip. The expression of different proteins on BE, EAC and ESC were detected by immunohistochemistry (IHC) using specific antibody. Three interested proteins Cofilin, Inergrin beta 1 and RNF121 were detected by TMA.8. Western-BlotThe tissue protein was extracted by BCA protein assay kit according to the manuscript. 50μg proteins were denatured in 2×loading buffer at 100°C for 5min, separated on SDS-PAGE gel, and transferred onto nitrocellulose membrane. The proteins were then detected using specific antibodies and appropriate secondary antibodies and visualized by radioautography using ECL. The interested proteins Cofilin and RNF121 were detected by Western-Blot.9. Statistical analysisData were expressed as mean±SD of three or more independent measurements. Paired data were subjected to two-tailed Students t test. A P value less than 0. 05 was considered statistically significant.Results1. Extraction of Total RNA and Purification of mRNA RNA quality of BE and normal esophageal epithelium was assessed by A260/ A280. These results showed that A260/A280 of total RNA was 1.843 - 1.951 , and that of mRNA was 1.90 - 2.0. RNA purity was examined by electrophoresis. Clear signal appearance of 28S and 18S of rRNA was seen on the electrophorogram of total RNA , suggesting no degradation in RNA.2. Differentially Expressed GenesThe image of gene expression profiles in BE was shown in fig. 2,3. From the original number of 30,968 gene probes, 426 genes had a quality score of P <0 .05 and were subjected to further comparison. Of the 426 genes that had significantly different expression between BE and normal esophagus, 142 were upregulated and 284 were downregulated in BE. On the basis of Gene Ontology and KEGG Pathway, four different molecular functional pathways (cobalamin transport, cobalt ion transport, negative regulation of transcription from RNA polymerase II promoter, transition metal ion transport) were most significantly upregulated and six different molecular functional pathways (MAPK signaling pathway, T cell receptor signaling pathway, Ribosome, Wnt signaling pathway, VEGF signaling pathway, Apoptosis) were most significantly downregulated. The reverse transcription-PCR confirmed the results of microarrays. These genes were listed on table 1. These differentially expressed genes were classified based on Gene Ontology (GO) system and TreeView.Many EAC-assoeiated genes were screened by the high-throughput gene chip method.There were 212 up-regulated genes and 126 down-regulated genes am- ong 2-fold DEGs. including 16 genes related to cytochrome P450 (CYP)3. Confirmation by Real time RT PCRThe arrays screening results of 7 genes were conformed by real-time RT-PCR . We found that there was a good correlation between this two methods. The results of 7 target genes were not only consistent with the results of Agilent gene hybridization, but the gene expression levels were also very similar.4. The expression of Cofilin, Intergrinβ1,RNF121 detected by TMAThe expression of Cofilin,Intergrinβ1,RNF121in BE were all significantly increased compared to normal esophagus mucous, and their expression in EAC and ESC were increased than in latero-cancer tissues. Furthermore, The expression of Cofilin,Intergrinβ1,RNF121in EAC were all significantly increased compared to in BE,but not in EAC. 5. The expression of Cofilin and RNF121 detected by Western-BlotThe expression of Cofilin and RNF121 in BE were both significantly increased compared to normal esophagus mucous, and their expression in EAC and ESC were increased than in latero-cancer tissues. Furthermore, The expression of Cofilin, RNF121in EAC were all significantly increased compared to in BE,but not in EAC. These results were matched to the results of TMA.Conclusion1. Two biopsies by disposable jumbo biopsy forceps provided approximately 5 microg required for microarrays. Microarray-based studies are feasible in endoscopically obtained tissues.2. 426 different expressed genes including 142 up-regulated genes and 284 down-regulated genes were screened by gene chip. These genes involved in cell cycle, signal transduction, mucoprotein, oncogene and anti-oncogene, bone morphous protein, apoptosis inhibiting, and BCL-2 family. These different expressed genes maybe related to the genesis and development of BE, and at the same time these genes maybe related to the transformation of BE to EAC.3. The up-regulated expression of MMP related genes,down-regulated expression of CYP related genes and gene polymorphism of CYP2 subfamily may be involved in the onset and progress of EAC.4. The results of Real-time RT-PCR were matched to that of gene chip and both gene chip and Real-time RT-PCR were credible. Gene chip has the feature of high flux and large scale, but the Real-time RT-PCR was suit for the study of single gene. They are mutual for supplement and verification.5. It was discovered by TMA that the expression of Cofilin ,Intergrinβ1and RNF121 were all increased in BE,EAC and ESC, and The expression of Cofilin,Intergrinβ1,RNF121in EAC were all significantly increased compared to in BE,but not in EAC. It is demonstrated that Cofilin,Intergrinβ1and RNF121 were related to the genesis of BE and played a great role for BE transformed to EAC. This was an original discovery for the mechanism of BE.6. The results of Western-blot were matched to that of TMA. It demonstrated that TMA was a reliable and high flux protein analytical system. Gene chip combined with TMA would provide an integrity analytical system for gene expression, amplification and function detection. The results we got provided vigorous evidence for deeply understanding the genesis and improvement of BE.