【作者】 马怡茗；

【导师】 黄常志； 张叔人； 冉宇靓； 马洁； 袁兴华； 肖建平； 孙克林；

【作者基本信息】 中国协和医科大学 ， 生化与分子生物学， 2010， 博士

【摘要】 本课题旨在通过差异蛋白质组学的方法,寻找与大肠癌发生发展及转移相关的蛋白质分子,研究这些蛋白质与大肠癌发生发展的关系。近期的研究表明,肿瘤的进展和淋巴结转移程度都是决定患者预后的重要因素。寻找与大肠癌发生发展及转移相关的分子,对于揭示大肠癌发生发展的分子机理,以及预测患者的预后都具有非常重要的意义。本课题收集了15例临床组织标本,通过双向电泳技术对组织总蛋白进行分离分析,并对差异表达蛋白点进行MALDI-TOF-MS分析,得到了33个相关分子。其中FHC的表达水平随肿瘤转移的发生进展而下降,而UCHL1的表达水平则随转移的发生进展而上升。随后,我们通过Western blot和免疫组化的方法对这一结果进行了研究。免疫组化的实验结果显示,UCHL1高表达与肿瘤T分期及淋巴结转移具有显著相关性。UCHL1是泛素水解酶家族的重要成员,它参与调控细胞内很多重要代谢活动。我们的研究发现,UCHL1高表达能够促进HCT8细胞在体外的增殖和迁移能力。而其突变体C90S则失去了这种能力,这实验结果说明,UCHL1的泛素水解酶功能对于其促进生长、转移的功能是必不可少的。我们的研究还发现,UCHL1可能是通过激活β-catenin/TCF4通路来发挥其功能的。免疫荧光实验的结果显示,在UCHL1高表达的细胞中,β-catenin的表达量也有所提高,两者存在共定位现象,并且主要定位于细胞核。另外,UCHL1的高表达能够使β-catenin的蛋白量呈现高水平,而mRNA的表达量却基本不变。同时,UCHL1能够促进β-catenin/TCF4通路下游调控基因的表达。这说明,UCHL1可能是通过降低β-catenin的泛素化水平,使其在核内堆积,从而激活β-catenin／TCF4通路来发挥功能的。另外,免疫组化结果显示,FHC在大肠癌组织中的表达量有所下降,且其表达下降与肿瘤的转移具有明显的相关性。FHC在铁代谢途径中发挥重要的作用,具有贮存和释放铁离子的功能。我们初步研究了FHC的生物学功能,发现FHC高表达能够降低细胞体外生长和迁移的能力。上述研究结果表明,UCHL1和FHC具有重要的生物学功能,在大肠癌发生及进展中起很重要的作用,可能是重要的标志性分子。研究这些相关的蛋白质分子对揭示大肠癌发生发展及转移的机制有重要意义,也可能为肿瘤诊断和治疗提供有效的标志物和靶点。更多还原

【Abstract】 The focus of this study was to use differential protein expression to investigate operative pathways in development and metastasis of human colorectal cancer (CRC). The presence or absence of lymph node metastases is a strong independent prognostic factor for CRC survival. Investigating proteins associated with tumor process and lymph node metastasis (LNM) process is crucial for understanding the molecular mechanisms underlying the CRC process and predicting the CRC prognosis. In the present study, proteins from CRC tissues and adjacent normal mucosa were examined using two-dimensional gel electrophoresis coupled with MALDI-TOF-MS. Thirty-three differentially expressed proteins were identified. The expression levels of Ferritin Heavy Chain (FHC) were decreased in LNM CRC as compared to those of non-LNM CRC, while the expression of Ubiquitin C-terminal hydrolase-L1 (UCH-L1) were increased. The results were confirmed by western blotting and immunohistochemical staining.For UCHL1, immunohistochemichal staining showed that UCHL1 high expression was significantly related to tumor (T) stage and lymph node metastasis (LNM). UCHL1 is an important deubiquitinating enzyme, it is involved in the regulation of distinct critical cellular processed. We found UCHL1 overexpression increased cell growth and migration activities of HCT8 cells in vitro. We further elucidated that the mutant C90S had no effection on cell growth and migration in vitro, which proved that deubiquitinating activity of UCHL1 was crucial for its functions. We also proved that UCHL1 may promote cellular growth and migration by activatingβ-catenin/TCF4 pathway. Immunofluorescence analysis showed that cells transiently transfected with UCHL1 expressed moreβ-catenin protein consistently. Moreover, UCHL1 overexpression can increaseβ-catenin expression in vitro in protein manner but not mRNA manner, and activateβ-catenin-dependent gene expression.For FHC, immunohistochemical staining showed that FHC expression was decreased in colorectal cancer tissues and was significantly related to lymph node metastasis. FHC plays an important role in the storage and release of iron and is pivotal to coordinating iron metabolism. We found that FHC overexpression decreaed cell growth and migration activities of HCT8 cells in vitro.Our results proved that UCH-L1 and FHC were not only biomarkers for CRC, but functional proteins that played a significant role in cell migration. The proteins we identified will contribute significantly to understanding the tumor process and metastasis process of CRC and may provide useful markers for diagnosis and targets for therapeutic intervention.更多还原