A Potential Urinary Biomaker Identified By Proteomics For Detecting NSCLC

Objective:Urinary exosomes, a low-density solid component secreted by renal epithelial cells, contain proteins, lipids, and micro-RNA. Proteins in urinary exosomes derived from the urinary and circulatory systems. Thus, these exosomes carry a wealth of physiological and pathological biomarker information, and changes in urinary exosome protein composition may reflect the pathological process of systemic and urinary system disease. Because urine can be obtained noninvasively in large quantities, urinary proteomics has received increased attention. In the present research, we aimed to screen for NSCLC-related proteins in urinary exosomes for early clinical diagnosis of NSCLC by comparing urinary exosomes proteome of normal controls and non-small cell lung cancer (NSCLC) patients.Methods:Urinary exosomes were isolated by ultracentrifugation and identified by electron microscopy. Exosomal proteins were separated by 1D SDS-PAGE and the differentially expressed bands between healthy controls and NSCLC patients ranging in size from 35 kD to 45 kD were cut from the gel. After tryptic digestion,exosomal proteins were identified by nano-HPLC-chip-MS/MS.Then, MS/MS data were input into UniProtKB/SWISS-PROT to find the differential expression of exosomal proteins .The differential expression of leucine-richα-2-glycoprotein (LRG1) was further validated in urinary exosomes by western blot and in lung tissue by immunohistochemistry.Results:The vesicles were visualized by qualitative negative staining electron microscopy: exosome is relatively round vesicles ranging from approximately 60nm in diameter. MS/MS data were input into UniProtKB/SWISS-PROT database. Eighteen proteins were identified in the 35 kD-45 kD bands, of which eleven were identified in samples from the NSCLC patients, three were identified in samples from both normal subjects and NSCLC patients, and four were identified in samples from normal patients. The LRG1 was found to be expressed at higher levels in urinary exosomes and lung tissue of NSCLC patients by western blot and in lung tissue by immunohistochemistry.Discussion: exosomes were selectively enriched in specific proteins. Glomeruli mainly filters proteins with molecular weight lower than 60 kD, suggesting that the 35 kD-45 kD protein bands observed upon electrophoresis of urinary exosomes may be derived from the urinary system or the circulatory system. We thus focused our study on these bands. In this study, 1D SDS-PAGE combined with HPLC-chip-MS/MS analysis were used to analyze differentially expressed proteins in the 35 kD-45 kD bands. In these bands, eighteen proteins were identified in the 35 kD-45 kD bands, of which eleven were identified in samples from the NSCLC patients, three were identified in samples from both normal subjects and NSCLC patients, and four were identified in samples from normal patients. In this study, we focused on one LRG family member in urinary exosomes from NSCLC patients: leucine-richα-2-glycoprotein (LRG1). Past research has shown that LRG1 is involved in important biological and pathological processes such as protein-protein interaction, signal transduction, and cell adhesion. LRG1 is also expressed during granulocyte differentiation. Our results not only showed that LRG1 increased in the urinary exosomes of NSCLC patients, but also showed high expression in tumor tissue of NSCLC patients, which suggested that LRG1 in urinary exosomes in NSCLC patients may be derived from tumor tissues, and LRG1 may be a candidate marker for NSCLC-related tumors in urine.