High-throughput proteomics integrated with gene microarray for discovery of colorectal cancer potential biomarkers

// Jiekai Yu 1, * , Xiaofen Li 1, * , Chenhan Zhong 1 , Dan Li 1 , Xiaohui Zhai 1 , Wangxiong Hu 1 , Cheng Guo 1 , Ying Yuan 2 , Shu Zheng 1 1 Cancer Institute (Key Laboratory of Cancer Prevention and Intervention, China National Ministry of Education), the Second Affiliated Hospital, Zhejiang University School of Medicine, China 2 Department of Medical Oncology, The Second Affiliated Hospital, Zhejiang University School of Medicine, Zhejiang, China * These authors have contributed equally to this work Correspondence to: Shu Zheng, email: zhengshu@zju.edu.cn Keywords: iTRAQ-MS, gene microarray, colorectal cancer, biomarkers Received: April 05, 2016 Accepted: August 10, 2016 Published: September 20, 2016 ABSTRACT Proteins, as executives of genes’ instructions, are responsible for cellular phenotypes. Integrating proteomics 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. ELISA results 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.