| Objective:The study aims to screening the differential expression glycoproteins (especially O-glycoproteins) among Tn negative/Tn positive (Tn-/Tn+) colorectal cancer tissues and normal colorectal tissues by using isobaric tags for relative and absolute quantitation (iTRAQ) combined with2D LC-MS/MS technology.We expected to find some potential O-glycoproteins that play a essential role in carcinogenesis and malignant progression of colorectal cancer,and that is also associated with the expression of Tn antigen. These proteins might be useful for colorectal carcinoma diagnosis and monitoring prognosis, may provide new targets for and therapy.Methods:9cases of patients of pathological diagnosis of colorectal cancer.Their fresh cancer tissues and normal mucous tissues that5-10cm far from the abjacent were collected from The Second Xiangya Hospital of Central South University from November2011to April2012. The tissues were classified into three groups according to the Tn staining:Tn+colorectal cancer tissues,Tn-colorectal cancer tissues and normal mucous tissues.The whole protein extracted separately from three groups of tissues.Differential proteomics analysis of these total protein in each group were analysed by iTRAQ combined with2D LC-MS/MS quantitative proteomics technology.After identifying the differential expression proteins,from them glycoproteins and O-glycoproteins were selected by Uniprot and DAVID databases. Interrelationship of glycoproteins, Interrelationship and functional classification of O-glycoproteins were analysed by String, GO, KEGG datebases. According to the analysis results above, Decorin and SORBS1, two of the differently expressed O-glycoprotein were verified by Western Blot in tissues and cells.Results:1. iTRAQ labeling2D LC-MS/MS results:There were1051unused proteins were identificated.750of them had quantitative information and more than2peptides with95%confidence interval were matched to these proteins. We found there were330differential expression proteins between Tn-and Normal group,317between Tn+and Normal group,316between Tn-and Tn+group.Among the differential expression proteins of Tn-and Tn+group, there were55glycoproteins and19O-glycoproteins.12of the19O-glycoproteins also differently expressed between Tn-/Tn+colorectal cancer tissues and normal mucous tissues.2. Interaction analysis of differential expression proteins:Among the55glycoproteins, K8and K18were in a unique interaction network,35proteins were in another interaction network, accounting for63.64%; Among the19O-glycoproteins, K8and K18were still in a unique interaction network,10proteins were in another interaction network, accounting for52.63%.3. GO functional classification analysis and KEGG metobalic pathway classification of differential expression O-glycoproteins:(1) With functional annotation classification of biological process,19O-glycoproteins mainly take part in10biological processes, which mainly include metabolic process, regulation of biological process and cellular metabolic process.63.12%and52.63%of O-glycoproteins take part in metabolic process and regulation of biological process, respectively.(2) These O-glycoproteins involved in binding, structural molecule activity, catalytic activity, enzyme regulator activity, transporter activity, antioxidant activity, transporter activity6molecular function.68.42%of these O-glycoproteins take part in binding molecular activity.(3) These O-glycoproteins mainly involved in cell part, organelle part organelle3cellular component,accounting for57.89%,73.68%,52.63%,respectively.(4)12of these O-glyprotein participated in25KEGG metobalic pathways and4signaling pathyways. Fibronectinand Heat shock protein HSP90-beta take part in the most number of pathyways, Decorin, SORBS1and Heat shock protein HSP90-beta involved in the4signaling pathyways, separately.4. According to the analysis results above, Decorin and SORBS1 were selected to be comfired by Western Blot in Tn-and Tn+colorectal cancer tissues/cells, and normal mucous tissues. The results of western Blot were consistend with outcomes of proteomics.The expression level of Decorin and SORBS1in Tn-/Tn+colorectal cancer tissues were lower than normal mucous tissues, And there had a lower expression in Tn+than Tn-colorectal cancer tissues or colorectal cancer epithelial cells.5. Decorin and SORBS1have a different expression level in different groups, which included different degrees of tumor differentiation, different stages, sex, age. But there is statistical differences only between two groups of different degrees of tumor differentiation.Conclusions:1. In this study,we found those differential expression O-glyproteins between Tn-and Tn+colorectal cancer tissues, and also differently expressed in Tn-/Tn-colorectal cancer and normal colorectal mucous tissues. Among of them, Keratin8, Keratin18, Decorin, sorbin and SH3domin-cotaining protein1(SORBS1), CD44antigen were correlated with carcinogenesis and malignant progression of colorectal cancer, also associated with the expression of Tn antigen. They lay a foundation for screening proteins associated with carcinogenesis and malignant progression of colorectal cancer carcinoma and diagnosis and monitoring prognosis of colorectal cancer. Aslo explain the role of O-glycoprotein play in carcinogenesis and malignant progression of colorectal cancer.2. According to the interaction, GO functional classification and KEGG metobalic pathway classification analysis of differential expression proteins, we found that12of19O-glycoprotein involved in two different interaction network, extra7proteins were not in any network. These19O-glycoproteinmail mainly take part in metabolic process, regulation of biological process and cellular metabolic process, involved in molecular function of binding, structural molecule activity, catalytic activity, enzyme regulator activity, transporter activity, antioxidant activity, transporter activity. But the relationship between these proteins and colorectal cancer is needed to be studied.3. O-glycoproein of Decorin and SORBS1had a lower expression may contribute to the process and development of colorectal cancer, and which may be expected to be a new target of diagnosis, monitoring prognosis, prevention and cure of colorectal carcinoma. |
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