| Liver cancer is one of the most common malignant tumor in digestive system,most(70% to 90%) primary liver cancers occurring worldwide are hepatocellular carcinomaresponsible(HCC). An estimated 782,500 new liver cancer cases and 745,500 deaths occurred worldwide during 2012, with China alone accounting for about 50% of the total number of cases and deaths. Majority cases of initial diagnosis with metastases is the main reason of clinical treatment failure and high mortality, more than 80% of the patients ultimately died of invasion and metastasis. However, the specific molecular mechanism of metastasis is still unclear, biomarkers of metastasis and prognosis have yet to be found, so discover related proteins of liver cancer metastasis is important.Tumor metastasis is a complex dynamic process with interaction between signal molecules and environment. Because the expression of gene function depends on protein molecules, so malignancy is a polygenic and proteomics disease. This study aimed to investigate proteomics of HCC cells with different metastatic by iTRAQ and 2D-LC-MS/MS to screen and identify proteins related to metastasis, which may provide new molecular targets for metastasis and treatment.In this study, there are three HCC cell lines(MHCC97-L, MHCC97-H and HCCLM3) with similar genetic background and stepwise increasing metastatic potential. The cell lines from a freshly isolated tumor tissue with one patient of hepatocellular carcinoma, and further established in nude mice by orthotopic liver transplantation and screening in vitro. Cytoplasmic proteins, extracted from three cell lines were desalted, concentrated, digested and labeled with i TRAQ?. And differentially expressed proteins were identified by 2D-LC –MS/MS, then the proteins were annotated by comparing with known protein database. Metastasis-related proteins were singled out by analyzing the Gene Ontology and related pathways using Panther, KEGG and Webgestalt databases. These proteins were then validated by RT-PCR and western blot. Finally, the correlations between target protein and prognosis were studied by immunohistochemical staining.Using iTRAQ labeling for quantification and detection by LC-MS/MS, 1170 proteins were identified. 47 proteins were detected to be differential expressed in L/H(MHCC97-L / MHCC9-H)groups, and 65 proteins in M/H(HCCLM3 / MHCC97-H)groups. Bioinformatics analysis revealed that differential expression proteins in L/H and M/H groups were mainly related to the catalytic activity of some enzymes and cells adhesion, and play important role in the cellular metabolic process. At transcriptional and translational level, PLEC was up-regulation in MHCC97-H and HCCLM-3, SHMT2 was up-regulation in HCCLM3, LDHA was up-regulation in HCCLM97-H, thus results of RT-PCR and Western blot were accordant with LC-MS/MS. And results of immunohistochemistry showed that the cytomembrane expression of PLEC in HCC tissue was higher than pericarcinous tissues, and PLEC expression in the cancer tissue was associated with TNM stage and tumor size(P=0.019,P=0.044), And the Kaplan-Meier survival curve analysis revealed that the PLEC membrane expression was negatively correlated with survival rate of patients(P =0.016); Cox regression analysis showed that expression of PLEC in the membrane is an independent risk factor for liver cancer poor prognosis, all of which revealed that cytomembrane expression of PLEC may correlated with metastasis and prognosis of HCC.In this study we used proteomics technology basing on high-throughput iTRAQ labeling to analyse differential proteins in three cell lines with different metastatic potentials. Plectin was validated by immunohistochemistry to be a novel protein associated with HCC metastasis and prognosis. |
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