Pro-oncogene MET Mediates Epithelial Mesenchymal Transition And Its Role In The Acquired Radioresistance Of Nasopharyngeal Carcinoma Cell Lines

Part I Irradiation induces epithelial-mesenchymal transition in nasopharyngeal carcinoma cell linesObjective:To investigate the effect of irradiation on epithelial-mesenchymal transition (EMT) and explore the role of EMT in the acquired radioresistance of nasopharyngeal carcinoma (NPC).Methods:Various NPC cell lines were irradiated with X-rays, including HNE1, CNE2, CNE1 and CNE1-LMP1. Invert microscope was adopted to observe and record the cell phenotype conversion. Western blot was used to measure the expression of EMT-related molecular markers and matrix metalloproteinase 2,9 (MMP-2,9). Immunofluorescent assay was employed to detect the position and expression of EMT-related molecular markers. The migration capability of NPC cells was assayed by wound-healing migration assay and the invasion capability was tested by Transwell invasion assay.Results:NPC cells presented with typical morphological changes of EMT after exposed to irradiation. The expression of E-cadherin was decereased in irradiated and radioresistant NPC cells, while Vimentin and N-cadherin were up-regulated in these cells. Immunofluorescent assay indicated that irradiation induced a decreased expression of E-cadherin at the cell-cell contact site and an increased expresssion of Vimentin in cytoplasm. The migration capability of NPC cells increased markedly after exposed to irradiation. However, the invasion capability of NPC cells was not accelerated significantly. Moreove, irradiation did not increase the expression of MMP-2 and MMP-9 in NPC cells.Conclusion:Irradiation induces EMT in nasopharyngeal carcinoma cell lines, while radioresistant NPC cells harbored the phenotype of mesenchymal cells. These results indicated that EMT participated in the acquired radioresistance of NPC.Part ? MET activated by irradiation and its role in irradiation-induced EMT and radioresistance of nasopharyngeal carcinoma cell linesObjective:To investigate the effects of irradiation on the activation of MET and explore the role of MET in irradiation-incuced EMT and acquired radioresistance of nasopharyngeal carcinoma (NPC) cells.Methods:ELISA was applied to quantify the level of hepatocyte growth factor (HGF). Real-time polymerase chain reaction (RT-PCR) was used to detect the expression of MET mRNA. c-Met and its downstream signal molecules were detected by western blot. Lentivirus transfection technique was used to silence MET expression. Cloning formation experiment assay was performed to measure the survival fraction and compare the proliferation ability of NPC cells.Results:After exposed to irradiation, the levels of HGF in NPC cells elevated slightly without statistical significance. However, the mRNA and protein expression of MET increased markedly. MET and its downstream signal molecules, p-c-Met, p-Stat3, p-Akt and p-erk1,2, were up-regulated in irradiated NPC cells. Irradiation-induced EMT and MET activation were inhibited when MET gene was silenced. The colony forming assay demonstrated that the proliferation ability of NPC cells was significantly decreased in MET silencing groups.Conclusion:Irradiation was able to activate MET and its downstream signal molecules. Silencing MET could inhibit irradiation-induced EMT, MET activation and the proliferation ability of NPC cells. These results indicated that MET played an important role in irradiation-induced EMT and the acquired radioresistance of NPC cell lines.Part ? The molecular mechanism of MET-mediated EMT in regulating radioresistance of nasopharyngeal carcinoma cell linesObjective:To clarify the molecular mechanism of MET-mediated EMT in regulating radioresistance of nasopharyngeal carcinoma (NPC) cell lines.Methods:Western blot was adopted to analyze the expression of E-cadherin, ?-catenin, c-Myc and Cyclin D1. Immunofluorescent assay was performed to detect the position and expression of E-cadherin and ?-catenin. Flow cytometry was used to measure the cell cycle and cell apoptosis.Results:After exposed to irradiation, the expression of E-cadherin decreased while P-catenin transportation to the nucleus increased. Silencing MET resulted in decreased P-catenin transportation to the nucleus. Besides, an inhibition of c-Myc and Cyclin D1, the downstream targeted genes of ?-catenin, presented in MET silencing NPC cells. Immunofluorescent assay revealed that irradiation induced a decreased expression of E-cadherin at the cell-cell contact site and an increased ?-catenin expression in the nucleus. However, the ?-catenin transportation to the nucleus decreased while MET was silenced. Flow cytometry showed that cell cycle at G2/M phase as well as the cell apoptosis increased markedly in MET silencing groups.Conclusion:Silencing MET could decrease the ?-catenin transportation to the nucleus and inhibit the c-Myc and Cyclin D1 expression. Besides, silencing MET was able to arrest the cell cycle at G2/M phase and increase the cell apoptosis. These results suggested that MET-mediated EMT could down-regulate the expression of E-cadherin and promoted the P-catenin transportation to the nucleus, which then activated c-Myc and Cyclin D1 and regulated cell cycle and cell apoptosis resulting in the acquired radioresistance of NPC cells.