Comparative Proteome Analysis Of Human Esophageal Carcinoma

Esophageal carcinoma is one of the popular malignant tumors in our country. A large number of statistical data indicates that the rates of incidence and mortality of the disease in our country have been going up in recent years. Early forecast and diagnosis of carcinoma is the key to improving survival rate for esophageal carcinoma patients. Now protome study methods and techniques, especially two-dimensional electrophoresis (2-DE), provide useful and important support for the research of esophageal carcinoma.On the basis of these considerations, proteomic strategy, combined with two-dimensional electrophoresis (2-DE) separation and mass spectrometry (MS) identification with advantage of high resolution, high reproducibility was used to separate and identify differentially expressed proteins between normal esophageal and esophageal carcinoma tissue. In this study we hoped to find out a series of protein cluster deeply involved in tumorigenesis and addressed the question whether there were new proteins to be associated with tumorigenesis, moreover, to clarify the tumorigenesis-associated function mediated by new candidate proteins. In the present study, 6 tumorigenesis-associated proteins were separated and identified by comparative proteome technique using established metastatic model, more importantly, the tumorigenesis-associated clinical characterization mediated by HSP27 and ANX1was further characterized.1. Establishment and optimization of two-dimensional polyacrylamide gel electrophoresis for the proteome analysis of the esophageal tissueTo optimize the sample preparation methods for the proteome analysis of the esophageal tissue, to establish a high resolution and reproducible two-dimensional polyacrylamide gel electrophoresis (2-DE) image, and to provide a base for identifying disease-associated proteins. The high-quality separation of 2-DE gel was obtained. The well-resolved, reproducible 2-DE profiles of esophageal tissue have been primarily established.2. Comparative proteomic analysis between normal esophageal tissue and esophageal carcinoma tissueTo better understand the mechanism underlying esophageal tumorigenesis and to search potential markers for esophageal carcinoma, differential proteome analysis on normal esophageal tissue and esophageal carcinoma tissue, was conducted using various proteomics approaches. By two-dimensional gel electrophoresis (2-DE), image analysis and mass spectrometry, the expression levels of 48 protein spots were found quantitatively changed between normal esophageal tissue and esophageal carcinoma, among them the expression levels of 20 protein spots were increased in esophageal carcinoma tissue and the expression levels of 28 protein spots were decreased in normal esophageal tissue. Qualitative analysis between normal esophageal tissue and esophageal carcinoma tissue found that 14 protein spots were only detected in esophageal carcinoma tissue and 18 protein spots in normal esophageal tissue. Combinated with software and manual screening, 6 differentially expressed protein spots between normal esophageal tissue and esophageal carcinoma tissue were further identified by MALDI-TOF MS analysis.3. Further identification and verification of candidate proteinsTwo-dimensional (2-D) gel electrophoresis followed by matrix-assisted laser desorption/time of flight mass spectrometry was utilized to compare the protein expression profiles between normal esophageal tissue and esophageal carcinoma tissue. 6 tumorigenesis-associated proteins were identified successfully. Of the identified proteins, the expressions of squamous cell carcinoma antigen 1, keratin 4, typeâ…¡, cytoskeletal and Annexin A1 were down-regulated in esophageal carcinoma tissue. However, heat shock protein 27, triosephosphate isomerase 1 and manganese superoxide dismutase were elevated in normal esophageal tissue. Most of the candidate proteins have been evidenced to be somehow associated with various aspects of tumorigenesis such as cell growth, invasion, apoptosis and tumor immunity, etc. These results provide the basis for searching for potential markers for esophageal carcinoma and give some clues to elucidate the mechanism of esophageal tumorigenesis.Semi-quantitative RT-PCR analysis were used to further verify candidate proteins in order to ensure the reliabilty of the proteome results. Semi-quantitative RT-PCR analysis indicated that the mRNAs coding SCCA1,KRT4 and ANX1 were up-regulated, but the mRNAs coding TIM1,MnSOD and HSP27 were down-regulated in esophageal carcinoma tissue compared with normal esophageal tissue. The verification results indicated that most of the differences of protein expression displayed by 2-DE between normal esophageal tissue and esophageal carcinoma tissue was convincing.4. HSP27 and ANXI correlates with esophageal tumorigenesisProteomic findings of esophageal carcinoma provide the basis for searching potential markers for esophageal carcinoma and give some clues to elucidate the mechanism of esophageal tumorigenesis. To further explore the clinical significance of the proteomic finding, differential expression and location of HSP27 and ANX1 in esophageal carcinoma were further validated by immunohistochemistry technology. 40 specimens of esophageal carcinoma tissue and paired normal esophageal tissue were examinated by immunohistochemistry staining. A statistically significant difference in HSP27 and ANX1 expression was found between normal esophageal tissue and esophageal carcinoma tissue. Our results indicate that HSP27 expression and ANX1 deletion relates to esophageal tumorigenesis and probably have important role in malignant transformation of esophageal carcinoma.In a word, the present study made a systematic research on tumorigenesis by comparative proteome technique using normal esophageal tissue and esophageal carcinoma tissue. 6 tumorigenesis-associated proteins were identified and characterized, more importantly, there were no clear evidence about the association of candidates HSP27 and ANX1 with esophageal tumorigenesis to the best of our knowledge. Function clinical significance confirmed that HSP27 expression and ANX1 deletion relates to esophageal tumorigenesis and probably have important role in malignant transformation of esophageal carcinoma. HSP27 and ANX1 may be key factors for preventing tumorigenesis and a therapeutic target for the treatment of patients with esophageal carcinoma.