解剖学报

解剖学报 ›› 2014, Vol. 45 ›› Issue (4): 493-499.doi: 10.3969/j.issn.0529-1356.2014.04.010

• 肿瘤生物学 • 上一篇    下一篇

肝细胞癌中差异表达N-连接糖蛋白的分析鉴定

马瑾 齐义军* 刘瑞敏 王明 张天 朱晗 马远方   

  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
  • 收稿日期:2013-12-09 修回日期:2014-01-18 出版日期:2014-08-06 发布日期:2014-08-06
  • 通讯作者: 齐义军 E-mail:qiqiyijun@gmail.com
  • 基金资助:

    国家自然科学基金;河南省高等学校青年骨干教师资助计划

Proteomic fingerprinting of N-linked glycoproteins involved in hepatocellular carcinoma

MA Jin  QI Yi-jun*  LIU Rui-min  WANG Ming  ZHANG Tian  ZHU Han  MA Yuan-fang   

  1. Key Laboratory of Cellular and Molecular Immunology, Medical School of He’nan University, He’nan Kaifeng 475004, China
  • Received:2013-12-09 Revised:2014-01-18 Online:2014-08-06 Published:2014-08-06
  • Contact: QI Yi-jun E-mail:qiqiyijun@gmail.com
  • Supported by:

    the National Natural Science Foundation of China

摘要：

目的 分析鉴定与肝细胞癌（HCC）发生发展相关的差异表达的N-连接糖蛋白。方法 应用刀豆蛋白凝集素（ConA）、晶状体凝集素（LCH）和雪花凝集素（GNA）组成的亲合层析柱富集10对HCC和癌旁非癌组织中N连接糖蛋白、二维电泳（2DE）比较分析差异表达的蛋白质点、串联质谱鉴定差异表达蛋白，Western blotting验证人羧酸酯酶1 (hCE1)、触珠蛋白（HP）及组织蛋白酶D（CD）的差异表达；体外侵袭实验检测CD表达沉默后对HCC的侵袭力影响。
结果 质谱、生物信息学等技术鉴定了28个与HCC相关的差异表达蛋白（HCC中高表达和低表达的蛋白均为14个），Western blotting验证hCE1、HP在HCC中明显低表达，组织蛋白酶D前体（pCD）在HCC中高表达；而ConA亲和层析柱富集的ConA-CD在HCC中显著高表达。CD-siRNA介导的CD表达沉默能够显著降低肝癌细胞系SNU449、SNU473的体外侵袭能力。 结论 HP、hCE1蛋白表达变化和CD N-糖链变异可能参与了HCC发生发展过程。

关键词: 原发性肝癌, 糖蛋白, 组织蛋白酶D, 触珠蛋白, 人羧酸酯酶1, 二维电泳, 质谱, 人

Abstract:

Objective To identify differentially expressed N-linked glycoproteins between hepatocellular carcinoma (HCC) and adjacent non-tumorous liver tissues.
Methods N-linked glycoproteome was extracted by multi-lectin affinity chromatography comprising concanavalin A (ConA), lentil lectin (LCH), and snowdrop lectin (GNA) and subsequently subjected to two-dimensional electrophoresis (2DE) and mass spectrometry(MS) for identification of differential glycoproteins between 10 pairs of HCC and adjacent non-cancer tissue. Western blotting was used to verify different expression of human liver carboxylesterase1 (hCE1), haptoglobin (HP）and cathepsin D (CD). Invasion potentialin vitro was examined after si-RNA mediated CD gene scilencing. Results LC-ESI-MS/MS identified a total of 28 differentially expressed glycoproteins (14 up-regulation and 14 down-regulated). Western blotting detected consistent down-regulation of hCE1 and HP, and up-regulation of pro-cathepsin D (pCD) in HCC. Up-regulation of ConA-binding CD (ConA-CD), however, was verified in HCC only after ConA-CD enrichment by ConA chromatography. Down-regulation of CD expression mediated by CD-siRNA markedly inhibited the in vitroinvasive potential of SNU449 and SNU473.
Conclusion Dysregulation of HP, hCE1 expression and alteration of glycans linked to CD may play crucial roles in pathogenesis of HCC.

Key words: Hepatocellular carcinoma, Glycoproteins, Cathepsin D, Haptoglobin, Human liver carboxylesterase1, Two-dimensional electrophoresis, Mass spectrometry, Human