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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 associated with the development of hepatocellular carcinoma (HCC). Methods 10 pairs of HCC and non-cancerous non-cancerous tissue N-linked glycoprotein were enriched by affinity chromatography consisting of ConA, LCH and GNA. The differentially expressed proteins were analyzed by 2DE and the differentially expressed proteins were identified by tandem mass spectrometry. The differential expressions of human carboxylesterase 1 (hCE1), HP and cathepsin D (CD) were detected by Western blotting. In vitro invasion assay was used to detect the impact of CD expression on invasiveness of HCC. RESULTS: 28 HCC-related differentially expressed proteins were identified by mass spectrometry and bioinformatics techniques (14 of them were high expression and low expression in HCC). Western blotting showed that hCE1 and HP were significantly lower in HCC and cathepsin D The precursor (pCD) was highly expressed in HCC, while the ConA affinity column enriched ConA-CD was significantly higher in HCC. CD-siRNA-mediated CD expression silencing can significantly reduce the in vitro invasiveness of hepatocellular carcinoma cell lines SNU449 and SNU473. Conclusion The changes of HP and hCE1 protein expression and CD-N mutation may be involved in the development of HCC.