Identification Of Metastasis Associated Proteins In Human Lung Squamous Carcinoma By Quatitative Proteomic Technique

Objective The study aims to establish two-dimensional gel electrophoresis(2-DE)map among lung squamous carcinoma(with metastasis/without metastasis)using differential in-gel electrophoresis technique(DIGE),coupled with laser capture microdissection(LCM). The differentially expressed proteins were detected by DeCyder^TM2D 6.5 software and identified by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry(MALDI-TOF-MS).These data may be useful for better understanding of lung squamous carcinoma metastatic mechanism and uncovering the metastasis-associated biomarkers.Method In sample preparation,the fresh carcinoma tissue was cut off from the specimen immediately after the specimen was resected and then preserverd in the -80℃refrigeratory.The tissues were classified into 2 groups:lung squamous carcinoma with metastasis and lung squamous carcinoma without metastasis.The former has 16 specimens and the latter has 12 specimens.LCM was utilized to harvest cancer cells from these two groups.Proteins collected from LCM were examined by 2-DE after labeled with CyDye DIGE Fluors Cy2,Cy3 and Cy5.Then the labeled proteins in each gel were visualized using a Typhoon 9000 fluorescence scanner for Cy2,Cy3 and Cy5 dyes.Images were analyzed with the DeCyder^TM2D 6.5 software.Protein spots with intensity changes detected by statistical significance were identified by MALDI-TOF-MS.The differentially expressed protein-spots were incised from the gels and digested by trypsin.We acquired the peptide mass fingerprintings(PMF) by MALDI-TOF-MS and identified the proteins by data searching in the Mascot-database.Part of differentially expressed proteins including AnnexinⅡand Cathepsin D were validated by Western blot and immunohistochemistry.Results Comparative fluorescence differential in-gel electrophoresis map was well established with pure cancer cells of lung squamous carcinoma with metastasis and without metastasis collected by LCM. There were 24 proteins spots differentially expressed between metastasis group and no-metastasis group.All of these proteins spots were selected for mass spectrum and bioinformation analysis.14 proteins were identified.10 of them were upregulated in the group with metastasis,and 4 of them were upregulated in the group without metastasis.AnnexinⅡand Cathepsin D expression were confirmed by Western blot and immunohistochemical analysis.The result showed that these two proteins expression were stronger in lung squmaous tissues with metastasis than lung squmaous tissues without metastasis.The result were in accordance with 2-DE data.Conclusions We successfully established fluorescence differential in-gel electrophoresis map of lung squamous carcinoma with metastasis and without metastasis by LCM and DIGE technique.14 proteins associated with lung squamous carcinoma metastasis were identified by MALDI-TOF-MS.2 of these proteins were selected to verify the liability of differential proteins.These proteins may accelerate or restrain the progression of lung squamous carcinoma.The data paves the way for better understanding the lung squamous carcinoma metastatic mechanism and identifying potential metastasis-associated markers for lung squamous carcinoma.