Proteome And Rnai Analysis Of The Mechanism Of SURVIVIN In Colon Cancer

Colorectal cancer is the second leading cause of cancer related mortality in the Western world. It is important to find a new way to effectively inhibit cancer cell growth and metastasis. Studies suggest that survivin is one of the most tumor-specific genes. Survivin is a member of the inhibitory apoptosis protein (IAP) family. It deserves attention as a selective target gene for cancer therapy due to the fact that it is widely expressed in most malignancies.Some studies have shown that a sustained over-expression of survivin is a characteristic feature of colon cancer. Survivin has attracted abundant interest from several viewpoints of biomedical sciences for dual involvement in apoptosis regulation and mitotic progression. But several studies indicated that the relationship between survivin expression and tumor behavior is still not fully understood due to contradictory results. Some studies showed a positive relationship but others showed negative outcomes or even a lack of correlation. For example, cytoplasmic survivin expression is an unfavorable prognostic indicator. But in contrast, a favorable outcome associated has been reported. For this reason, this study was designed to globally identify the mechanism of survivin.Proteomics is an effective platform to globally detect and characterize proteins. A comparative proteomic approach is the main strategy of proteomics used to analyze and compare the differentially expressed proteins. On the other hand, RNA interference (RNAi), as a sequence-specific and posttranscriptional gene silencing method, is increasingly being used to determine the functions of specific genes. Thus, we silenced the expression of Survivin by the recombinant adenovirus. We detected the survivin gene expression, apoptosis, cell cycle, etc. after shRNA interference. Then, a comparative proteomic approach was performed to identify the differential proteins between SW480/Survivin(-) and SW480/Survivin(+) cells. Our proteomic findings may provide clues to further study the mechanism of the function of survivin in tumors.PARTⅠThe Expression and Localization of Survivin in Colon CanerObjective: To observe the expression and localization of Survivin in colon cancer.Methods: We screened the expression and localization of Survivin by immunohistochemistry and immunocytochemistry in colon cancer cells (including colon cancer tissues and cell lines).Results: Survivin protein expression was 57.6% in colon cancer tissue samples and cell lines (LoVo, HCT116, SW480). Survivin protein expressed mainly in the cytoplasm. Spearman correlation analysis showed that there are positive correlation between Survivin expression and Ducks stage.Conclusion: In the colon carcinoma tissue and cell lines, Survivin expressed at a high level and the localization was in cytoplasm. Survivin plays an important role in control tumor growth.PARTⅡConstruct of Recombinant Adenovirus Vector and its Effect in Colon Cancer CellsObjective: To construct the adenovirus vector which contains the shRNA of Survivin and investigate its effect in colon cancer cellsMethods: We constructed the recombinant adenovirus, which contains the shRNA of Survivin and transfected it into colon cancer cells. Then, we detected Survivin gene expression after shRNA interference. We detected the influence of Ad-Survivin/shRNA on the proliferation, apoptosis and invasion in vitro and vivo by MTT, colony-formation, flow cytometry, transewell and tumorigenic formation.Results: Ad-Survivin/shRNA displayed high transfection efficiency and suppressed Survivin expression efficiency. Survivin slience could induce apoptosis, effect the mitotic cycle and decrease invasion. Cell proliferation was significantly inhibited by Ad-Survivin/shRNA in vitro and vivo.Conclusion: RNAi of Survivin would be a potential therapeutic approach for colon cancer. Survivin plays an important role in control tumor growth by a variety of molecular regulatory mechanisms.PARTⅢProtein Sample Preparation, 2-DE and Image Analysis for ProteomeObjective: To study the protein sample preparation methods and to estabilish 2-DE profiles for SW480 cells under Survivin intervention.Methods: We used liquid nitrogen, RNase-DNase digestion and ultrasonication method to get the protein samples. Protein was focused at different voltagy intensities for two-dimensional electrophoresis.The PDquest sofeware was used to analysis the image.Results: Liquid nitrogen freeze thawing, RNase-DNase digestion and ultrasonication can significantly reduce the impact of nucleic acid and promote the dissolution of protein. The 2-D gels of SW480 displayed about 1,400 protein spots which distributed of pI=4-8 and Mr (15-120)×103. We found more than 80 protein spots were apparently different between before and after survivin gene silencing. Among these protein spots, 36 species were identified.Conclusion: The attained 2-D gels patterns of each cell line were highly reproducible and well-resolved. Optimized protein and 2-DE preparation was established.PARTⅣAnalysis on Differences Proteins by Mass Spectrometry and the Regulation Mechanisms of SurvivinObjective: To identify proteins and analysis their characteristics by MALDI-TOF-MS and HPLC-CHIP-MS/MS.Methods: The differential protein spots were analyzed by MALDI-TOF-MS and HPLC-CHIP-MS/MS and database searching. Gene ontology (GO) lists were downloaded for the functional analysis of proteins. Western-blot method was used to check the different proteins.Results: The differentially expressed proteins identified by 2-D proteome analysis were related to various cellular programs involving cell proliferation, cell cycle, apoptosis, expression of nucleic acid metabolic gene, and regulation of signal transduction.Colon cancer cells effected apoptosis by the regulation of Caspase-10 and cell cycle by the regulation by CDK9.Conclusion: Survivin plays an important role in control tumor growth by a variety of molecular regulatory mechanisms.