肝细胞癌中差异表达N-连接糖蛋白的分析鉴定  

Proteomic fingerprinting of N-linked glycoproteins involved in hepatocellular carcinoma

作　　者：马瑾[1] 齐义军[1] 刘瑞敏[1] 王明[1] 张天[1] 朱晗[1] 马远方[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

出　　处：《解剖学报》2014年第4期493-499,共7页Acta Anatomica Sinica

基　　金：国家自然科学基金资助项目(81072039);河南省高等学校青年骨干教师资助项目(2011)

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。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 carboxylesterase 1 （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.

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 

分 类 号：R735.7[医药卫生—肿瘤]