| Objective To observe whether DDR1 affects the invasion and metastasis of gastric cancer cells via regulating the epithelial-mesenchymal transition by using molecular biological methods, and to explore its underlying molecular mechanism.Methods(1) The immunohistochemical analysis was used to select gastric cancer cell lines with either high or low DDR1 expression.(2) DDRl-targeted siRNA was constructed and transiently transfected into DDR1 high-expressing gastric cancer cells, and changes before and after transfection in DDR1 expression were detected by RT-PCR and W-B assays. Changes before and after transfection in the proliferation, the migration and invasion, and the cell cycle and apoptosis in gastric cancer cells were examined by MTT assay, Transwell method, and flow cytometry method (FCM), respectively, and changes in the expression of EMT-related proteins E-cadherin and Vimentin, and of EMT signaling-associated protein Snail, were explored by western blot (W-B) analysis.(3) DDR1 recombinant lentivirus was constructed to stably transfect DDR1 low-expressing gastric cancer cells to overexpress DDR1, and RT-PCR and W-B assays were employed to examine the changes before and after transfection in DDR1 expression. The effects of DDR1 overexpression on the proliferation, the migration, the invasion, and the cell cycle and apoptosis of gastric cancer cells were examined by MTT assay, colony formation assay, Transwell method, and flow cytometry method (FCM), respectively. Changes in the expression of EMT-related proteins E-cadherin and Vimentin, and of EMT signaling-associated protein Snail, were detected by western blot (W-B) analysis.(4) A model of nude mice with a transplantation tumor was constructed by subcutaneously injecting the mice with BGC-823 cells which were stably transfected by DDR1 recombinant lentivirus carrying GFP. The mice were subcutaneously inoculated with 2×106 cells, and the small animal in vivo imaging (SAIVI) was conducted after 7d,21d,35d, and 49d respectively, to measure the size of the transplantation tumor, and to observe the effects of DDR 1 overexpression on the growth of the transplantation tumor in nude mice. The mice were sacrificed after 49d, and the tumor was taken to study its tissue morphology and the expression of angiogenesis-associated protein CD34.Results(1) The immunohistochemical analysis on gastric cancer cells MKN-45, BGC-823, SGC-7901, and on normal gastric mucosal cell GES-1 showed that DDR1 expression was all positive in the above gastric cancer cells while negative in the normal gastric mucosal cell GES-1. DDR1 expression was strongest in MKN-45 while weakest in BGC-823.(2) RT-PCR and Western Blot results showed that DDR1 expression significantly decreased (p<0.05) in the gastric cancer cell MKN-45 after MKN-45 was transfected with DDRl-siRNA, and that DDR1 expression significantly increased (p<0.01) in BGC-823 after BGC-823 was stably transfected with DDR1 recombinant lentivirus.(3) MTT results showed that inhibiting DDR1 expression in MKN-45 remarkably (p<0.05) inhibited the proliferation of MKN-45, whereas DDR1 overexpression in BGC-823 significantly (p<0.01) promoted the proliferation of BGC-823; the colony formation assay confirmed that DDR1 overexpression resulted in a significantly (p<0.05) enhanced monoclonal forming ability of BGC-823 cells.(4) FCM results showed that DDR1 overexpression or inhibition of DDR1 expression in gastric cancer cells had neither effects on the cell cycle (p>0.05) nor effects on apoptosis (p>0.05) of gastric cancer cells.(5) Transwell cell migration and invasion experiments verified that inhibition of DDR1 expression in MKN-45 cells resulted in a significantly reduced (p<0.01) number of cells across the membrane, while DDR1 overexpression in BGC-823 cells significantly increased (p<0.01) the number of cells across the membrane.(6) Western blot results confirmed that inhibition of DDR1 expression in gastric cancer cell MKN-45 resulted in increased expression of the EMT associated protein E-cadherin (p<0.01), and decreased expression of Vimentin (p<0.05) and signaling-associated protein Snail (p<0.05). In contrast, DDR1 overexpression in gastric cancer cells resulted in opposite outcomes in terms of the expression of the above proteins.(7) The animal experiments confirmed that DDR1 overexpression significantly (p<0.05) accelerated the growth of BGC-823 cells in the subcutaneous transplantation tumor in nude mice. And the immunohistochemical staining of tumor tissue showed that CD34 expression significantly (p<0.01) increased in DDR1 overexpressing group.Conclusions(1) DDR1 expression in gastric cancer cells promoted the proliferation, the migration, and the invasion of gastric cancer cells, indicating that DDR1 may be a therapeutic target for gastric cancer.(2) DDR1 expression in gastric cancer cells had no significant effects on the cell cycle and apoptosis of gastric cancer cells.(3) DDR1 may activate Snail signaling pathway to promote the epithelial-mesenchymal transition, thereby affecting the invasion and metastasis of gastric cancer cells.(4) DDR1 can promote the growth of transplantation tumor in gastric cancer cells, and promote the formation of the MVD of transplantation tumor. |
PDF Full Text Request