| IntroductionEsophageal carcinoma is one of the most fatal malignancies world wide and the dominant subtype is esophageal squamous cell carcinoma (ESCC) in China. Though great improving have achieved in diagnosis and treatment, the morbility and mortality of ESCC still remains high. About 150,000 patients died of it every year in China. One of the main reasons might be that the tumor typically remains asymptomatic until have reached a serious stage and the early diagnosis was very inconvenient or difficult. Thus ESCC was often diagnosed at an advanced stage and the prognosis remains poor inevitably. The invasion and metastasis is the main reason for the death of ESCC patients.β-catenin is one of the key mediators of Wnt/β-catenin pathway, and plays a pivotal role in Wnt signal transduction in addition to its function as a cell-adhesion component.β-catenin may guide the normal development of the embryo, promote the proliferation and inhibit the apoptosis of the tumor cells.β-catenin might also play an important role in the invasion and metastasis procession of ESCC. The object of proteomics is to analyze the dynamic alteration of the proteome in a integrity cell in its different state. Because the functional molecules in cells are proteins, proteome analysis is believed to have an advantage over other means. Proteomics has introduced a new approach for cancer research which aims at identifying differential expression proteins associated with the development and progression of cancer, providing new opportunities to uncover biomarkers and therapeutic targets related toβ-catenin in ESCC.ObjectiveOur research attempts to study the effect of transfectedβ-catenin siRNA gene on invasion and metastasis of human esophageal carcinoma Eca109 cells and the differential proteomics between pGen-3-CTNNB1 (Eca109 cells transfected byβ-catenin siRNA) and pGen-3-con (Eca109 cells transfected by control vector) cell lines. The relation between the differential proteins andβ-catenin would be studied, at the same time, the link between the expression of the differential proteins andβ-catenin with the clinical characters of the ESCC patients should be also observed.MethodsThe adherent ability of the three groups cells were detected by MTT method. The ability of migration and invasion of transfected cells was examined by Transwell chamber method. Nude mice metastasis models were established by abdominal cavity transfer method.Two-dimensional gel electrophoresis (2-DE) technology was performed to separate the proteins of pGen-3-con and pGen-3-CTNNB1 cell lines, respectively. The gel spots of differential expression protein were found by software analysis, subjected to in-gel digestion, and identified by Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS). Consequently,13 differentially expressed proteins between the two cell lines were identified, of which 14-3-3σ, prohibitin and nm23-H1 were further verified by Western blotting and quantitative real-time reverse transcriptase polymerase chain reaction (QRT-PCR). The up-regulation of 14-3-3σexpression by transfection with the 14-3-3σexpression plasmid was also researched.Tissue microarray (TMA) and immunohistochemistry (IHC) were used to evaluate the expression of 14-3-3σ, prohibitin and nm23-H1 andβ-catenin in tissues of 195 pairs of ESCC and their normal esophageal epithelium, and then correlate them with clinicopathological and prognosis of ESCC patients.ResultsCompared with Eca109 and pGen-3-con cells, the cell adherence, migration, invasion and metastasis ability of pGen-3-CTNNB1 cells were decreased obviously (P＜0.05). Consequently,13 differentially expressed proteins between the two cell lines were identified, of which 14-3-3σ, prohibitin and nm23-H1 were further verified by Western blotting and QRT-PCR analysis. As a functional identification, the up-regulation of 14-3-3σexpression by transfection with the 14-3-3σexpression plasmid could markedly decrease theβ-catenin protein level in Eca109 cells. The regulation ofβ-catenin related proteins might be correlated with tumor infiltration depth and the lymph node metastasis rate of ESCC. Furthermore, the alteration of their expression was associated with survival after curative surgery in ESCC patients. On the basis of univariate and multivariate Cox-regression analysis, we conclude that 14-3-3σ, prohibitin andβ-catenin could be independent prognostic factors for ESCC patients.Conclusions1. For the first time, we demonstrated that the silence ofβ-catenin could inhibit the cell adherence, migration and metastasis ability of Eca109 cells.2. With the analysis of the proteomics, a total of 13 differential proteins were successfully identified between the cell lines of pGen-3-con and pGen-3-CTNNB1. Of the identified proteins,14-3-3σ, prohibitin and nm23-H1 were selected for further verified with Western blotting and QRT-PCR means.14-3-3σwere also founded had important function in Eca109 cells.3.14-3-3σ, prohibitin, nm23-H1 andβ-catenin might take part in the invasion and metastasis of the ESCC, and 14-3-3σ, prohibitin, nm23-H1 might have closely relation withβ-catenin in ESCC.4.14-3-3σ, prohibitin andβ-catenin could be independent prognostic factors for ESCC patients.5. The expression ofβ-catenin had positive correlation with prohibitin and negative with 14-3-3σand nm23-H1 in the progression of ESCC. |
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