| Gastric cancer is one of the most malignancies worldwide which remains a great threaten to the public health. The prognosis of patients with gastric cancer is poor, and the5-year survival rate remains below30%. Gastric cancer at early stage could be asymptomatic, and it often proceeds to advanced stage with lymph node and distant metastases, which is one of the reasons for the treatment failure and death of gastric cancer. For this reason, it would be of great value to explore metastasis-associated molecules for better therapeutic effects in gastric cancer.Series of gastric cancer specific monoclonal antibodies have been generated by hybridoma technique, and of these, the antigen recognized by MGb2was showed to be associated with gastric cancer metastasis. In previous studies, Coronin3was found to interact with MGb2-Ag and promote metastasis in gastric cancer. It has been reported that Coronins play important roles in cytoskeleton reorganization, invadopodia formation and cell migration, and therefore, we speculated that Coronin3might promote metastasis by regulating F-actin nucleation and invadopodia formation.In this study, we identified Arp2as a protein that directly interact with Coronin3. The expression level of Arp2was significantly higher in the metastatic lymph node tissues than that in the primary cancer tissues. Meanwhile, it was up-regulated in MKN-28M cells with increased migratory potential than in its parental cells. By gain-and loss-of-funtion studies, we found that Arp2could promote metastasis of gastric cancer in vitro. This study could help develop novel strategies to treat metastases in gastric cancer.[Objectives]1. To study the expression of Coronin3in gastric cancer tissues and its correlation with prognosis;2. To screen for the candidate F-actin associated molecules interacting with Coronin3;3. To study the expression of target protein in gastric cancer tissues and its correlation withclinicopathological parameter;4. To study the influrences of target protein on metastasis of gastric cancer cells.[Methods]1. Immunohistochemistry was used to detect Coronin3in gastric cancer and the paired para-cancerrous tissues, and the correlation of Coronin3with clinicopathological parameters of gastric cancer is analysed; Log-rank test was performed to analyse the relation between Coronin3and the prognosis of gastric cancer;2. Immunoprecipitation (IP), MALDI-TOF-MS and bioinformatic analysis were used to screen for proteins that interact with Coronin3, and their co-expression was further confirmed by IP;3. Immunohistochemistry was used to detect the expression of Arp2in gastric cancer tissues, and the correlation of Arp2with clinicopathological parameters of gastric cancer is analysed; Log-rank test was performed to analyse the relation between Arp2and the prognosis of gastric cancer;4. Western blot and qPCR were used to detect Arp2in multiple gsatric cancer cell lines. Gastric cancer cells were transfected with Arp2plasmid and siRNAs, after which the migratory ability of cells was examined by wound healing assay and transwell. [Results]1. Coronin3was positive in68%(51/75) of gastric cancer tissues while it was detected in only50.7%(38/75) of all para-cancerous tissues. The intensity of Coronin3was not associated with gender and age, but correlated with differentiation and TNM staging of gastric cancer. The overall survival of patients with stronger expression of Coronin3was relatively poor.2. Actin-related protein2(Arp2) was identified as a protein interacting with Coronin3by means of IP, MALDI-TOF-MS and bioinformatic analysis. The IP confirmed the interaction between Coronin3and Arp2.3. The expression of Arp2was stronger in gastric cancer tissues than that in adjacent normal tissues (47/75,62.7%vs.16/75,21.3%). It was also increased in metastatic lymph nodes that in primary cancer (33/40,82.5%vs.21/40,52.5%). Arp2was not associated with gender and age, but related with differentiation and TNM staging of gastric cancer. The stronger intensity of Arp2could serve as a marker for poor prognosis.4. Compared with MKN-28and MKN-28NM cells, Arp2was up-regulated in MKN-28M cells. Meanwhile, its expression was increased in multiple gastric cancer cell lines than in immortalized gastric epithelial cell line GES. Down-regulation of Arp2was sufficient to suppress the mobility and invasive potential of MKN-28M cells while its up-regulation could lead to increased migration and invasion of MKN28-NM cells.[Conclusion]Overexpression of Coronin3was correlated with poor prognosis of gastric cancer. Arp2was found to directly interact with Coronin3by means of proteomics study, and its expression was associated with differentiation, TNM staging and metastatic status of gastric cancer. In addition, Arp2could also serve as a biomarker for poor prognosis in gastric cancer. Arp2was up-regulated in gastric cancer cell lines with higher migratory and invasive potential and could promote migration, invasion and metastasis of gastric cancer cells in vitro. |
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