二维电泳和质谱技术鉴定肝细胞癌相关N-连接糖蛋白  

Identification of hepatocellular carcinoma associated N-linked glycoproteins by two dimensional electrophoresis and mass spectrometry

作　　者：王明丽[1] 韩大正[2] 晁玮霞[1] 刘瑞敏[1] 赵云岗[1] 张天[1] 齐义军[1] 

机构地区：[1]河南大学医学院细胞与分子免疫学重点实验室,河南开封475004 [2]河南大学第一附属医院消化内科,河南开封475000

出　　处：《解剖学报》2015年第4期495-502,共8页Acta Anatomica Sinica

基　　金：国家自然科学基金资助项目(81072039)

摘　　要：目的鉴定与肝细胞癌(HCC)发生发展相关的差异表达N-连接糖蛋白。方法采用刀豆凝集素A(Con A)、晶状体凝集素(LCH)、雪花凝集素(GNA)等3种植物凝集素组成的亲和层析柱,分别从肝永生化细胞系L02和HCC细胞系Huh7、PLC5、SNU449中纯化N-连接糖蛋白、二维电泳分析差异表达的蛋白质斑点,质谱、生物信息学等技术鉴定差异表达蛋白,Western blotting验证了转录调控肿瘤蛋白(TCTP)、上皮细胞黏附分子(Ep CAM)和膜联蛋白A2(annexin A2)在肝永生化及HCC细胞和HCC及癌旁组织中的表达规律,体外侵袭实验检测TCTP沉默后对SNU449的侵袭力影响。结果质谱、生物信息学等技术共鉴定出42个差异表达的蛋白质分子/异质体(HCC细胞中高、低表达的蛋白/异质体分别为14个、28个),代表32个蛋白质分子,其中的22个蛋白含有至少1个N糖基化位点(Net NGlyc 1.0预测)。这些差异表达的蛋白主要参与氧化还原内稳态维系、碳水化合物/能量代谢、糖酵解、抗凋亡等生物学过程。Western blotting检测表明,TCTP、Ep CAM和annexin A2在6个肝癌细胞系PLC5、Hep G2、SNU449、Huh7、HCC7721和SNU473及肝癌组织中表达显著升高,siRNA介导的TCTP沉默明显抑制了HCC细胞系SNU449的体外侵袭能力。结论 TCTP、Ep CAM和annexin可能参与了HCC发生发展过程,TCTP可能成为HCC靶向治疗的分子靶点之一。Objective To identify differentially expressed N-linked glycoproteins associated with hepatocellular carcinoma（ HCC）. Methods Multi-lectin affinity chromatography comprising concanavalin A（ Con A）,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（ Ep CAM） and annexin A2 between two immortalized cell lines vs six HCC cell lines,HCC vs adjacent non-tumor 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 Net NGlyc 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 ofTCTP,Ep CAM 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 down-regulation of TCTP remarkably inhibited the invasion potential of in SUN449. Conclusion TCTP,Ep CAM and annexin A2 may participate the pathogenesis of HCC and TCTP may become one of molecular targets for the targeted therapy of HCC.

关 键 词：肝细胞癌 N-连接糖蛋白 植物凝集素 质谱 二维电泳 

分 类 号：R735.7[医药卫生—肿瘤]