Identification Of Stromal Differentially Expressed Proteins In The Colon Carcinoma By Quantitative Proteomics

Background:The role which tumor microenvironment plays in tumor carcinogenesis is more and more important and now is becoming a hotspot. Colon cancer is a common malignancy in the world, the morbidity and mortality is the third in Europe and the United States. In recent years, because of the deterioration of the environment and the adjustment of diet, colon cancer morbidity and mortality increased year by year, and is a great threat to people’s health and lives.Identification of differentially expressed proteins in tumor stromal cells and normal tissues stromal cells will help to identify the unique characteristics of the tumor microenvironment. Looking for early diagnostic marker and lay a solid foundation for targeted stromal therapy.We try to use LCM to purify the cancer stroma (CS) and normal stroma (NS) from colon adenocarcinoma (CAC) and normal colon mucosa (normal colon mucosa, NCM). Isotope labeling relative and absolute quantification (iTRAQ) labeling quantitative proteomics would be performed to identify the differentially expressed proteins between CS and NCS. Exploring the role of the tumor stroma in tumor development.Chapter1Identification of differentially expressed proteins by iTRAQ labeling quantitative proteomics and LCMObjective:To separeate and identify differentially expressed proteins(DEPs) of CAC. And DEPs bioinformatics analysis.Methods:①we performed LCM to purify CS and NS from12cases of CAC and NCM;②The CS and NCS were labeled by iTRAQ-118and iTRAQ-121, and then identification of CS and NCS differentially expressed protein by two-dimensional liquid chromatography tandem mass spectrometry (2D LC-MS/MS);③Cellular components, biological pathways and molecular functional analysis of DEPs was performed by David Functional Annotation;④Biological function analysis, the canonical pathway and networks were performed by Ingenuity Pathway Analysis, in order to clarify the relationship between DEPs and understand the relevant signaling pathways.Results:①Purify successfully colon cancer stromal and normal colon stromal.②A total of70DEPs were identified and quantified. Among these proteins,37protein are up-regulated in CAC and33protein are down-regulated in CS.③DEPs was analyzed by David Functional Annotation software. In accordance with the cellular components, most belong to the extracellular matrix proteins (37.5%), organelles proteins (28.6%) and cytoskeletal proteins (25%). In accordance with the biological function involved in extracellular matrix (37.5%), followed by the endoplasmic reticulum stress and the cytoskeleton, and involved in metabolism, inflammation response protein only small proportion. In accordance with the molecule function, it can be seen that91.8%of DEPs is binding protein. Proteins involved in the enzyme regulator activity accounted for only1%.④Bioinformatics analysis of DEPs were performed by Ingenuity Pathway Analysis. Bioinformatics analysis found51significant canonical pathways,4significant networks and56network eligible proteins."Cancer" was identified as the main disease associated with the stromal DEPs. Some common pathways affected by the stromal DEP expression in the CAC and NCM were related to cancer, including actin cytoskeleton signaling, ILK signaling,14-3-3-mediated signaling, VEGF signaling, Wnt/B-catenin signaling and so on. Four significant networks functioned in inflammatory disease and immunological disease, developmental disorder and hereditary disorder, digestive system development and its function, inflammatory disease and connective tissue disorders.⑤56network eligible proteins was found closely related to the biological function of tumor development:inflammation, extracellular matrix, metabolism, endoplasmic reticulum stress, cytoskeleton and protein aggregates. And plotted in figure,in order to presents clearly the relationship of the DEPs in the biological function of preoteins associated with tumors. Most of the protein is not only involved in one biological function, also participate in the regulation of the biological functions of two or even three. DEPs involved in tumor development from a variety of sources.Conclusions:①For the first time reported that iTRAQ-based quantitative proteomics coupled with LCM could identify and quantify the stromal DEPs of CAC.②DEPs were analysis by GO and IPA shows molecular function, biological function, signaling pathways and networks, it is provide clues to the mechanism for colon cancer. Chapter2Validation of stromal DEPs and screening for colon cancer diagnostic markerObjective:To validate reliability of iTRAQ-based quantitative proteomics, and look for the biomarkers which can be used for early diagnosis of colon cancer.Methods:①Western blotting was used to detect the expression levels of eight stromal DEPs (DCN, FN1, PKM2, HSP90B1, S100A9, MYH9, TUBB, and YWHAZ). Eight stromal DEPs are located in different biological functions. They are located in the core position of siginificant network, and plays a key role;②By reviewing the literature, select four proteins which has an important role in tumor development:DCN, FN1, PKM2and HSP90B1. Immunohistochemical (IHC) were performed to detect the expression of DCN, FN1, PKM2and HSP90B1;③DCN, FN1, PKM2and HSP90B1was evaluated for early detection of CAC by receiver operating characteristic analysis.Results:①The results of western blotting showed that the express levels of eight proteins. DCN, PKM2, HSP90B1were higher in CAC than NCM. FN1, S100A9, Tubb, MYH9, YWHAZ was downexpressed in CAC then NCM.②The results of IHC showed: DCN, PKM2and HSP90B1was downregulated in the CAC, FN1was upregulated in the CAC.③The results of ROC analysis showed that detection alonely of DCN,FN1, PKM2and HSP90B1achieving a sensitivity of70%-78%and a specificity of70%-87%, the combined detection of DCN, FN1, PKM2and HSP90B1could perfectly discriminate CAC from NCM, achieving a sensitivity of82.6%and a specificity of82.6%, the combined detection of three proteins could achieving a sensitivity of65.2%-82.6%and a specificity of82.6%-87%, the combined detection of two proteins could achieving a sensitivity of69.6%-82.6%and a specificity of65.2%-87%. The combined detection of DCN, FN1, PKM2and HSP90B1could perfectly discriminate CAC from NCM which as same as the combined detection of DCN, FN1and HSP90B1. The combined detection three proteins including PKM2have the same sensitivity and specificity as the combined detection two proteins. It showed that PKM2haven’t significance in the combined detection.Conclusion:①The results of western blotting and IHC were well consistent with the iTRAQ-based quantitative proteomics data, which suggested that iTRAQ-based quantitative proteomics are accurate and reliable.②the combined detection of DCN, FN1and HSP90B1improved the sensitivity and specificity. It provide experimental basis for new molecular targets for screening colon cancer diagnosis.