Effect Of VEGF On Radiosensitivity And Metastasis Of Nasopharyngeal Carcinoma

Background Nasopharyngeal carcinoma(NPC)is a malignant tumor originated from the overlying epithelium of the nasopharynx,which occurs most frequently in Southeast Asia,especially in southern China.Nasopharyngeal carcinoma is characterized by poorly differentiated tumor and complex anatomical location.Radiotherapy is the main treatment.Approximately 20% of patients with nasopharyngeal carcinoma have poor treatment response due to radiation resistance,and the majority of patients(approximately 75-90%)have cervical lymph node metastasis at the time of presentation.Therefore,it is of great significance to explore the factors causing radiation resistance and metastasis of nasopharyngeal carcinoma and the potential molecular mechanisms for improving the therapeutic effect and prognosis of patients with nasopharyngeal carcinoma.Vascular endothelial growth factor(VEGF)is an important proangiogenic factor that promotes tumor angiogenesis.Many studies have shown that VEGF contributes to the development and metastasis of cancer.In addition,VEGF can also lead to excessive DNA replication and increased synthesis of radioresistance genes in tumor cells.Studies have shown that the radioresistance of esophageal cancer is related to the abnormal secretion of VEGF induced by radiation.VEGF protects tumor blood vessels from radiation damage,thus increasing the radioresistance of tumors.In summary,we hypothesized that VEGF is also involved in radioresistance and metastasis of nasopharyngeal carcinoma cells.Therefore,it is of great significance to further explore the influence of VEGF on the radiosensitivity and metastasis mechanism of nasopharyngeal carcinoma.Objective This study first verified the high expression of VEGF in nasopharyngeal carcinoma cells through experiments,and observed the changes of radiosensitivity and metastasis of nasopharyngeal carcinoma cells after VEGF silenced through a series of experiments in vivo and in vitro,and then further studied the regulatory mechanism between VEGF and radiosensitivity and metastasis of nasopharyngeal carcinoma cells.This study will provide some references for the exploration of effective therapeutic targets for nasopharyngeal carcinoma.Methods 1.Western blot and qPCR were used to verify the difference in the expression of VEGF between NPC radioresistant cell line CNE-2R and its parent radiation sensitive cell line CNE-2,and the expression of VEGF was silenced by lentivirus transfection in NPC radioresistant cell line CNE-2R.After irradiation,the changes of radiosensitivity of CNE-2R cells after VEGF silencing were verified by clonal formation,CCK8,flow cytometry and other experiments.The changes of autophagy-related proteins in CNE-2R cells after VEGF silences were verified by Western Blot.After irradiation,the changes of radiosensitivity of CNE-2R cells were verified by clone formation and CCK8 assay after the administration of autophagy activator of VEGF silencing.Western Blot and Coimmunoprecipitation experiments were performed to verify the changes of m TOR pathway protein in CNE-2R cells after VEGF silencing.2.Western Blot and qPCR were used to verify the difference in the expression of VEGF between NPC cell lines CNE-2 and 5-8F and immortified nasopharyngeal epithelial cells NP-69,and the expression of VEGF was silenced by lentivirus transfection in NPC cell lines CNE-2 and 5-8F.The changes of invasion and migration ability of nasopharyngeal carcinoma cells after VEGF silencing were verified by scratch and Transwell experiments.The changes of EMT and MMPs in nasopharyngeal carcinoma cells after VEGF silenced were verified by Western Blot.The effect of VEGF secreted by nasopharyngeal carcinoma cells on angiogenesis was verified by ELISA and tubular experiments.The regulation of VEGF-VEGFR2 autocrine pathway on EMT and MMPs in nasopharyngeal carcinoma cells was verified by Western Blot.3.The effects of silencing VEGF on the radiosensitivity and lung metastasis of transplanted tumor were observed by constructing the xenograft model in nude mice.The number of apoptotic cells in the transplanted tumor after VEGF silencing was detected by TUNEL assay.The changes of autophagy-related proteins,EMT,MMPs and other related proteins in vivo were detected by immunohistochemistry after VEGF silence.Results: 1.Western Blot and qPCR confirmed that the expression of VEGF in CNE-2R cells was higher than that in CNE-2 cells.Cloning,CCK8 and flow cytometry experiments confirmed that downregulation of VEGF increased the radiosensitivity of CNE-2R cells.Western Blot assay confirmed that VEGF silencing could inhibit the expression of autophagy in CNE-2R cells,as well as autophagy induced by irradiation.We used clone formation and CCK8 assays confirmed that the activation of autophagy can reverse the radiosensitivity induced by silencing VEGF.Western Blot and Co-immunoprecipitation experiments confirmed that VEGF regulates autophagy related proteins through m TOR pathway.2.Western Blot and qPCR confirmed that the expression of VEGF in NPC cells CNE-2 and 5-8F was higher than that in NP-69 cells.The scratch and Transwell experiments confirmed that the invasion and migration ability of nasopharyngeal carcinoma cells decreased after VEGF silencing.Western Blot confirmed that VEGF silenced the expression of N-cadherin,Vimentin,MMP2,MMP9 and other proteins in NPC cells,and increased the expression of E-cadherin protein.ELISA assay showed that the silencing of VEGF could inhibit the secretion of VEGF in nasopharyngeal carcinoma cells,and tubular assay showed that the silencing of VEGF could reduce the formation of blood vessels.Western Blot confirmed the existence of VEGF-VEGFR2 autocrine pathway in nasopharyngeal carcinoma cells to regulate EMT,MMPs and other related proteins.3.The tumor transplantation experiment in nude mice showed that the silencing of VEGF inhibited tumor growth,and the inhibition effect was more obvious after 8Gy irradiation.In lung metastatic models,silencing VEGF reduces the number of pulmonary nodules.The TUNEL assay also detected the number of tumor apoptosis in vivo,which also indicated that the silencing of VEGF would increase the rate of apoptosis.The results of immunohistochemistry were consistent with those of in vitro Western Blot.Conclusion This study demonstrated the internal mechanism between VEGF and radiosensitivity and metastasis of nasopharyngeal carcinoma from two aspects.First,we found that VEGF was highly expressed in radioresistant nasopharyngeal carcinoma cells(CNE-2R).Through lentiviral mediation and functional experiments,we found that VEGF silencing can increase radiosensitivity of CNE-2R cells.Through further exploration,we found that silencing VEGF can activate m TOR pathway to inhibit autophagy and enhance radiosensitivity of CNE-2R cells.Second,we also found that VEGF in nasopharyngeal carcinoma cells CNE-2 and 5-8F showed higher expression than that in NP69,through a series of experiments,we found that the silencing VEGF can restrain the invasion of nasopharyngeal carcinoma cell migration ability and EMT and the expression of MMPs related proteins,finally we found in nasopharyngeal carcinoma cells exist in the VEGF-VEGFR2 autocrine loop to regulate the transfer occurred.Therefore,VEGF plays an important role in the radioresistance and metastasis of nasopharyngeal carcinoma cells.Explore the mechanism of action of VEGF will provide new ideas for the treatment of nasopharyngeal carcinoma.