CAJ | 학술논문

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。
목적：분석감정여간세포암（ HCC）발생발전상관적차이표체적N-련접당단백。방법응용도두단백응집소（ConA）、정상체응집소（LCH）화설화응집소（GNA）조성적친합층석주부집10대HCC화암방비암조직중N-련접당단백、이유전영（2DE）비교분석차이표체적단백질점、천련질보감정차이표체단백，Western blotting 험증인최산지매1（hCE1）、촉주단백（HP）급조직단백매D（CD）적차이표체；체외침습실험검측CD표체침묵후대HCC적침습력영향。결과질보、생물신식학등기술감정료28개여HCC상관적차이표체단백（ HCC중고표체화저표체적단백균위14개），Western blotting험증hCE1、HP재HCC중명현저표체，조직단백매D전체（ pCD）재HCC중고표체；이ConA친화층석주부집적ConA-CD재HCC중현저고표체。 CD-siRNA개도적CD표체침묵능구현저강저간암세포계SNU449、SNU473적체외침습능력。결론 HP、hCE1단백표체변화화CD N-당련변이가능삼여료HCC발생발전과정。
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.