Identification And Functional Study Of The Molecular Markers In Colorectal Cancer

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.