| [Background]Gastric cancer is one of the most common cancers worldwide. It’s the second leading cause of cancer death. Most patients are diagnosed as advanced gastric cancer when they first visit. Local recurrence and metastasis is the key factor of treatment failure and mortality. With the development of molecular biology, more and more studies aimed at finding crucial factors of the process of invasion and metastasis.DIXDC1was a new member of Wnt signaling pathway, which was isolated as an Axin2C-terminus binding protein. It’s the human homolog of Ccdl (Coiled-coil-DIX1) gene, which was certified as a positive regulator in the Wnt signaling pathway during zebrafish neural development. Meanwhile, DIXDC1was seemed as an actin-binding protein participating in modulating the cellular morphology and movement. There were few reports about the role of DIXDC1in tumor carcinogenosis and development. Our group found that over-expression of DIXDC1might target p21and CyclinD1to promote colon cancer cell proliferation and tumorigenesis at least partially through activation of PI3K/Akt pathway. Then few researches are managed to study the biological role of DIXDC1working on cancer.[Objectives]To investigate the expression of DIXDC1in different types of gastric cancer (GC) and and analyse its relationship with clinicopathological features in GC patients.To explore the role of DIXDC1in the metastasis of gastric cancer and the relating mechanism.[Methods]The expression of DIXDC1in paraffin-embedded GC tissues was determined by Immunohistochemistry (IHC) assay. The expression of DIXDC1in gastric cancer cells were determined by western blotting. Recombinant lentiviral vectors infected with over-expression plasmids were designed to up-regulate DIXDC1expression in gastric cancer cell lines. Wound healing assay, invasion assay were used to investigate the impact of DIXDC1on the migration, invasion of GC cells respectively. Proteins relating GC metastasis were also detected by western blotting. Dual-luciferase assay was performed to detect the effect of DIXDC1on the transcriptional activity of β-catenin/TCF. Co-immunoprecipitation confirmed the interaction between DIXDC1and β-catenin in GC cells. Laser scanning confocal microscope was used to observe the subcellular location of DIXDC1and β-catenin. Finally, the effects of DIXDC1on the β-catenin protein and its phosphorylated level in GC cells were detected by western blotting assay.[Results]1. DIXDC1was overexpressed in gastric cancer tissue and correlated with the TNM stage and lymphatic metastasis.Using the IHC assay, DIXDC1expression was compared among the gastric cancer, precancerous lesion and tissue adjacent to cancer. DIXDC1proteins were expressed in56.0%(37/66) of GC,28.6%(8/28) in precancerous lesions and no DIXDC1were detected in tissue adjacent to cancer. Chi-square test showed that the expression of DIXDC1was highly related to the clinical stage, the lymphatic metastasis and histological types of GC. In29.8%of the94cases, β-catenin were abnormally expressed and located in cell nucleus. Remarkable correlation beween DIXDC1expression and nuclear β-catenin location was suggested through the Spearman correlation analysis.2. DIXDC1overexpression significantly promoted the migration and invasion of GC cells. And DIXDC1knockdown inhibited the migration of GC cells.Construct Lentiviral vector overexpressing DIXDC1, and use it to infect GC cells. After the successful establishment of stable cell lines that overexpress DIXDC1and its negative control, these cells were pushed into the cellular function analysis. Wound healing assay and Transwell chamber assay (including with or without Matrigel) suggested that after up-regulating DIXDC1, the BGC-23cells seemed much powerfull in the migration or invasion experiments. Then we design DIXDC1-specific siRNA to knockdown it, and the cell’s migration and invasion ability is largely down-regulated. Taken together, these results suggest that DIXDC1can highly promote gastric cancer metastasis by enhancing the migration and invasion abilities of gastric cancer cells.3. Through modulating the phosphorylation of β-catenin, up-regulating its expression and promoting its nuclear location, DIXDC1finally promote gastric cancer metastasis.Dual-luciferase report showed that DIXDC1overexpression increased the transcriptional activity of β-catenin/TCF. Co-IP confirmed the interaction between DIXDC1and P-catenin. With the overexpression of DIXDC1, the phosphorylation level of P-catenin declined, and the total protein and nucleoprotein of β-catenin all increased. Finally, immunofluorescence also showed the same results. In summary, DIXDC1finally promoted gastric cancer metastasis through modulating the phosphorylation of β-catenin, up-regulating its expression and promoting it to transfer to cell nucleus.[Conclusion]DIXDC1is overexpressed in gastric cancer tissues, and significantly correlated with the histological types, the lymphatic metastasis and the clinical stage of gastric cancer. DIXDC1overexpression can remarkably promote the migration and invasion of gastric cancer cells. However, DIXDC1knockdown inhibits these abilities of GC cells. DIXDC1promoted gastric cancer invasion and metastasis through regulating phosphorylation of β-catenin, insitituting its transfer to nucleus and finally binding to the TCF. |
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