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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
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 carboxylesterase1 (hCE1), haptoglobin (HP）and cathepsin D (CD). Invasion potentialin 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 vitroinvasive 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.
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
Hepatocellular carcinoma / Glycoproteins / Cathepsin D / Haptoglobin / Human liver carboxylesterase1 / Two-dimensional electrophoresis / Mass spectrometry / Human
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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
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