title: High-throughput proteomics integrated with gene microarray for discovery of colorectal cancer potential biomarkers.
abstract: Proteins, as executives of genes' instructions, are responsible for cellular phenotypes. Integratingproteomics with gene microarray, we conducted this study to identify potential protein biomarkers of colorectal cancer (CRC). Isobaric tags with related and absolute quantitation (iTRAQ) labeling mass spectrometry (MS) was applied to screen and identify differentially expressed proteins between paired CRC and adjacent normal mucosa. Meanwhile, Affymetrix U133plus2.0 microarrays were used to perform gene microarray analysis. Verification experiments included immunohistochemistry (IHC), western blot and enzyme-linked immunosorbent assay (ELISA) of selected proteins. Overall, 5469 differentially expressed proteins were detected with iTRAQ-MS from 24 matched CRC and adjacent normal tissues. And gene microarray identified 39859 differential genes from 52 patients. Of these, 3083 differential proteins had corresponding differentially expressed genes, with 245 proteins and their genes showed >1.5-fold change in expression level. Gene ontology enrichment analysis revealed that up-regulated proteins were more involved in cell adhesion and motion than down-regulated proteins. In addition, up-regulated proteins were more likely to be located in nucleus and vesicles. Further verification experiments with IHC confirmed differential expression levels of 5 proteins (S100 calcium-binding protein A9, annexin A3, nicotinamide phosphoribosyltransferase, carboxylesterase 2 and calcium activated chloride channel A1) between CRC and normal tissues. Besides, western blot showed a stepwise increase of annexin A3 abundance in normal colorectal mucosa, adenoma and CRC tissues. ELISAresults revealed significantly higher serum levels of S100 calcium-binding protein A9 and annexin A3 in CRC patients than healthy controls, validating diagnostic value of these proteins. Cell experiments showed that inhibition of annexin A3 could suppress CRC cell proliferation and aggressiveness. S100 calcium-binding protein A9, annexin A3, nicotinamide phosphoribosyltransferase, carboxylesterase 2 and calcium activated chloride channel A1 were probably potential biomarkers of colorectal cancer. Annexin A3 was a potentially valuable therapeutic target of CRC.
authors.affiliation: Cancer Institute (Key Laboratory of Cancer Prevention and Intervention, China National Ministry of Education), the Second Affiliated Hospital, Zhejiang University School of Medicine, China.
authors.forename: Jiekai
authors.initials: J
authors.lastname: Yu
authors.affiliation: Cancer Institute (Key Laboratory of Cancer Prevention and Intervention, China National Ministry of Education), the Second Affiliated Hospital, Zhejiang University School of Medicine, China.
authors.forename: Xiaofen
authors.initials: X
authors.lastname: Li
authors.affiliation: Cancer Institute (Key Laboratory of Cancer Prevention and Intervention, China National Ministry of Education), the Second Affiliated Hospital, Zhejiang University School of Medicine, China.
authors.forename: Chenhan
authors.initials: C
authors.lastname: Zhong
authors.affiliation: Cancer Institute (Key Laboratory of Cancer Prevention and Intervention, China National Ministry of Education), the Second Affiliated Hospital, Zhejiang University School of Medicine, China.
authors.forename: Dan
authors.initials: D
authors.lastname: Li
authors.affiliation: Cancer Institute (Key Laboratory of Cancer Prevention and Intervention, China National Ministry of Education), the Second Affiliated Hospital, Zhejiang University School of Medicine, China.
authors.forename: Xiaohui
authors.initials: X
authors.lastname: Zhai
authors.affiliation: Cancer Institute (Key Laboratory of Cancer Prevention and Intervention, China National Ministry of Education), the Second Affiliated Hospital, Zhejiang University School of Medicine, China.
authors.forename: Wangxiong
authors.initials: W
authors.lastname: Hu
authors.affiliation: Cancer Institute (Key Laboratory of Cancer Prevention and Intervention, China National Ministry of Education), the Second Affiliated Hospital, Zhejiang University School of Medicine, China.
authors.forename: Cheng
authors.initials: C
authors.lastname: Guo
authors.affiliation: Department of Medical Oncology, The Second Affiliated Hospital, Zhejiang University School of Medicine, Zhejiang, China.
authors.forename: Ying
authors.initials: Y
authors.lastname: Yuan
authors.affiliation: Cancer Institute (Key Laboratory of Cancer Prevention and Intervention, China National Ministry of Education), the Second Affiliated Hospital, Zhejiang University School of Medicine, China.
authors.forename: Shu
authors.initials: S
authors.lastname: Zheng
mesh_terms: D014408:Biomarkers, Tumor; D049109:Cell Proliferation; D015179:Colorectal Neoplasms / Q000235:genetics* / Q000378:metabolism* / Q000473:pathology; D019295:Computational Biology / Q000379:methods; D018450:Disease Progression; D005260:Female; D063990:Gene Ontology; D057166:High-Throughput Screening Assays; D006801:Humans; D007413:Intestinal Mucosa / Q000378:metabolism / Q000473:pathology; D008297:Male; D060787:Neoplasm Grading; D009367:Neoplasm Staging; D020411:Oligonucleotide Array Sequence Analysis; D020543:Proteome; D040901:Proteomics / Q000379:methods
chemical_list: D014408:Biomarkers, Tumor; D020543:Proteome
keywords: biomarkers; colorectal cancer; gene microarray; iTRAQ-MS