| The gene PTEN (phosphatase with tensin homology deleted on chromosome 10) has been cloned and named at 1997 by three different groups, and its role was anti-cancer. Recently, PTEN has been a hot topic, since some experts believes that the importance of its function is comparable with the famous anti-cancer p53. It has been revealed that in many cancers PTEN has changed. The phenomenon was found by us that gastric carcinoma occurred at 100% when the PTEN gene was knocked out in epithelium of mouse stomach specifically. In addition, the downregulation of PTEN in gastric cancer has been observed in many clinical researches and the expression level of PTEN was related with the migration and prognosis of gastric cancer. Taken together, these suggested that there were links between dysfunctions in PTEN signal pathway and gastric carcinogenesis. The development of gastric cancer induced by knockout of PTEN has not been reported until now.Although genetic alterations in proto-oncogenes, tumor-suppressor genes, cell cycle regulators, and cell growth factors have been implicated in the process of human gastric carcinogensis, the principle carcinogenic mechanisms are not fully understood. To reduce the mortality and improve the effectiveness of therapy, many studies have tried to find key biomarkers, which contribute to early diagnosis, and the target of drugs. In order to make clear the role of PTEN in gastric carcinoma generation, the PTEN specific knockout mouse in stomach epithelium was obtained with the Cre-LoxP system. The phenotype of the mouse was analyzed systematically, and the conclusion could be taken that the mouse gastric carcinoma model which is stable in phenotype, clear in genetic history and similar to human carcinoma development was got.To understand clearly the role of PTEN signal pathway and its related proteins in gastric carcinogenesis and screen for the key biomarkers and target of drugs, we used proteomic approach to establish different expression map and identify the proteins which express differently in knockout and wildtype epithelium of mouse stomach.Proteins from epithelium of mouse stomach were separated by two-dimensional electrophoresis (2-DE) and identified by mass spectrometry (MS). Using 2-DE of the stomach cancer tissue PTEN knockout or wildtype at different weeks, the different expression map was established. And proteins express differently was determined by comparing the level of each dots with gel imaging software and manually. Digest in gel was performed first and proteins were identified by peptide fingerprinting with matrix-assisted laser desorption/ionization-time-of-flight-mass spectrometry (MALDITOF-MS) and peptide sequence tag with tandem MS combining with database respectively. After that, the function of these identified proteins was known and classified by search in the PUBMED and SWISSPROT databases. After verification in transcription and protein level, some key biomarkers and target of drugs should be found. And, importantly the function of some interesting protein related with PTEN pathway should be investigate in cell model.To make clear the proteomic change systematically, 2-DE was used to separated the tissue extract at different weeks age. After comparing the map, normal tissue as control, there were 78 and 83 differential expression proteins in the region of molecular masses of 8-170 kDa and an isoelectric point of 5-8 respectively. Among them, the maximum and minimus change is 8.6 folds and 0.1 folds. And, 57 proteins in one month age , 26 proteins were up-regulated and 31 proteins were down-regulated ,and 53 proteins in two month age , 33 proteins were up-regulated and 20 proteins were down-regulated was identified by MS. The relationship between 24 proteins of identified proteins with gastric cancer has been reported. The identified proteins included cytoskeletal proteins, degradation system related proteins, proteins involved in signal transduction, cell transcription and translation, and metabolism.To sum, proteins involved in signal transduction account for 29% and 32% respectively. Some proteins has related to PTEN signal pathway, such as Annexin A3, Annexin A2, Phosphatidylinositol transfer protein alpha isoform. Annexin A2 can directly bound to PtdIns(4,5)P2, an important component of PTEN signal pathway, and recruit it to definite location. Some proteins are involved in tumor generation and invade , such as Ezrin , prohibitin whose expression is known to be altered in other types of tumors. These proteins take important clues to make clear the PTEN signal pathway and its role in gastric cancer development.Combining well defined PTEN specific knockout animal models with proteome analysis, We identified more than 100 proteins expressed differently, these will improve our understanding of the PTEN signal pathway and its contribution to the process of carcinogenesis and finding key biomarkers in gastric cancer. |
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