Proteomics Analysis Of Laryngeal Squamous Cell Carcinoma And Clinical Significance Of Candidate Protein MRP8

Objective:To study the protein preparation methods for human laryngeal squamous carcinoma and paired normal tumor-adjacent mucosa epithelia tissue, to establish two-dimensional gelelectrophoresis profiles, to provide a base for identifying different proteins of laryngeal squamous carcinoma and paired normal tumor-adjacent mucosa epithelia tissue.Methods :1. After obtaining 10 samples of the normal laryngeal mucosa tissues , squamous cell carcinoma (SCC). Analyzining Patient information and histological data for the tissue samples, Immobilized pH gradient two-dimensional polyacrylamide gel electrophoresis (2-DE) was used to separate the total proteins of the samples. After Coomassie staining, ImageMaster 2-DE image analysis of software was applied to analyze 2-DE image.2. The differential expression proteins were analyzed by using ImageMaster 2D analysis software,then identified by peptide mass fingerprint(PMF)based on matrix-assisted laser desorption / ionization time of flight mass spectrometry (MALDI-TOF-MS)and database searching. PMF identified program (Mascot:Peptide Mass Fingerprint)of Matrix Science company in net was used to search。3. Immunohistochemical method(SP) and western blotting method were used to examine the expression of MRP8 and CypA in Laryngeal squamous cell carcimoma and nomal Laryngeal tissue, to analyse the clinical significance in clinicopathologic factors.Results:1. We obtained well- resolved , reproducible 2-DE patterns of laryngeal squamous carcinoma and adjacent normal mucosa epithelia1.The differential expression proteins were analyzed by using ImageMaster 2D analysis software. 2-DE profile showed that the proteins in the two samples were located mostly in the range of mass 20.1-116kd and isoelectric points (pI) 4-8.The average protein spots in 3 2-DE profile of laryngeal squamous carcinoma and adjacent normal mucosa epithelia1,one was the standard profile ,then protein spots in the 3 profile were matched.The average matched spots were 1 236±18和943±16,matching rate was 91.5% and 91.2%。Well reproducible 2-DE patterns of protein spots were diplayed in 3 profiles of laryngeal squamous carcinoma ,the deviation in IEF direction was (0.973±0.125) mm, in SDS-PAGE direction was (1.025±0.213)mm. Immobilized pH gradient two-dimensional polyacrylamide gel electrophoresis (2-DE) was used to separate the total proteins of the 10 pairs of samples simultaneously. The ten 2DE profiles of laryngeal squamous carcinoma are similar. Analyze them by using ImageMaster 2D Elite 4.01 software and abtained the average image of the ten images. There are 1236 protein spots are decteded. And there are 943 protein spots are decteded in normal tumor-adjacent mucosa epithelia tissue 2DE average image.2. Differential protein spots were defined as spots in 2-DE gels.13 proteins were preliminarily identified,among which 10 proteins were upregulated in laryngeal cancer tissue.Such as Cofilin-1,Nuclear body protein SP140,GRP94, HSP 90, GSTP1-1, Superoxide dismutase [Mn], Cyclophilin A, Proteasome activator complex subunit 2,Apolipoprotein A-I precursor , CaM-like protein and so on.There are 3 proteins downregulated in laryngeal cancer tissue, which are Fatty acid-binding protein (E-FABP), Calgranulin A and Calgranulin B .3. The result of western blotting method showed that MRP8 and CypA were positive expressed in Laryngeal squamous cell carcimoma and nomal Laryngeal tissue; That expression of CypA in Laryngeal squamous cell carcimoma was higher than nomal Laryngeal tissue; And the expression of MRP8 in Laryngeal squamous cell carcimoma was lower than nomal Laryngeal tissue.4. The result of Immunohistochemical method(SP) showed that MRP8 and CypA were both high expressed in Laryngeal squamous cell carcimoma and nomal Laryngeal tissue;The positive cases of CypA in tumor tissue were more than nomal tissue, MRP8 showed an inverse result ; For MRP8, The number of positive cases declined when differential degree of laryngeal squamous tissue increased. These twe proteins had no relationship with tumor patients' age and gender.Conclusions:1. The well-resolved, reproducible 2-DE profiles from human laryngeal tissues have been established with the optimized protein preparation method.2. In this study, We first characterized 13 proteins which are associated with the tumorigenesis of the laryngeal squamous cell carcinoma.Some proteins were the products of oncogenes,apoptosis and others were involved in signal transduction and self defence. These extensive protein variations indicate that multiple protein molecules should be simultaneously targeted as an effective strategy to counter the disease. It is better for understanding of the oncogenesis and pathogenesis in a global way, which in turn is a basis for the rational designs of diagnostic and therapeutic methods.3. Expression of MRP8 was closely related to differential degree of LSCC,decreased in poorly differential tissue ,increased in well differential tissue. Expression of MRP8 decreased as malignant extent of LSCC deepened.Detection of MRP8 was helpful for judging malignant extent , prognosis and carcinogenesis of LSCC tissues.