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Identification of hepatocellular carcinoma associated N-linked glycoproteins by two dimensional electrophoresis and mass spectrometry
WANG Ming-li HAN Da-zheng CHAO Wei-xia LIU Rui-min ZHAO Yun-gang ZHANG Tian QI Yi-jun*
Acta Anatomica Sinica ›› 2015, Vol. 46 ›› Issue (4) : 495-502.
Identification of hepatocellular carcinoma associated N-linked glycoproteins by two dimensional electrophoresis and mass spectrometry
Objective To identify differentially expressed N-linked glycoproteins associated with hepatocellular carcinoma (HCC). Methods Multi-lectin affinity chromatography comprising concanavalin A (ConA), lentil lectin (LCH), snowdrop lectin (GNA) were utilized to isolate N-linked glycoproteins from human immortalized liver cell line L02 and HCC cell lines Huh7, PLC5 and SNU449, followed by 2 dimensional electrophoresis-based quantification and MS/MS identification. Western blotting was used to verify different expression of translationally-controlled tumor protein (TCTP), epithelial cell adhesion molecule (EpCAM) and annexin A2 between two immortalized cell lines vs six HCC cell lines, HCC vs adjacent nontumor liver tissue. Invasion potential in vitro was examined after si-RNA mediated TCTP gene scilencing. Results A total of 42 proteins/isoforms including 14 up-regulated and 28 down-regulated proteins/isoforms were identified. These proteins/isoforms represented 32 unique proteins of which 22 had more than one glycosylation site predicted by NetNGlyc 1.0. These differentially expressed proteins plaid biological functions in redox homeostasis, carbohydrate/energy metabolism, glycolysis, anti-apoptosis, etc. Western blotting validated the up-regulated expression of TCTP, EpCAM and Annexin A2 in 6 HCC cell lines and HCC tissue in comparison with two immortalized cell lines L02, chang liver and adjacent non-tumor tissue, respectively. siRNA mediated downregulation of TCTP remarkably inhibited the invasion potential of in SUN449. Conclusion TCTP, EpCAM and annexin A2 may participate the pathogenesis of HCC and TCTP may become one of molecular targets for the targeted therapy of HCC.
Hepatocellular carcinoma / N-lined glycoproteins / Lectin / Mass spectrometry / Two-dimensional electrophoresis
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