The Role Of FOXO3a On The Acquired Radioresistance Of Nasopharyngeal Carcinoma Cell Lines

Part? Effect of FOXO3 a on radiosensitivity of nasopharyngeal carcinoma cell linesObjective: To explore the effect of knockdown of FOXO3 a on the radiosensitivity of nasopharyngeal carcinoma cells(NPC).Methods: To examine the expression of FOXO3 a in NPC cells(CNE1,CNE2,HNE1,and CNE1-LMP1),western blotting and q RT-PCR were performed.Western blotting and q RT-PCR were applied to detect FOXO3 a expression in irradiated NPC cells by 6Gy irradiation at different time points.We generated CNE2 and HNE1 cell lines by transfecting cells with mock sh RNA,sh RNA1,sh RNA2,and sh RNA3.Western blotting and q RT-PCR were then performed to confirm transfection efficiency.Engineered cell lines were exposed to different doses of radiation for colony formation.Western blotting and immunofluorescence was performed to detect alterations in EMT marker proteins after transfection with sh RNA3 and mock sh RNA.Scratch tests and transwell invasion experiments were also performed to verify the migration and invasion abilities of the transfected cells.Results: The expression of FOXO3 a was very similar in NPC cells.Western blottin g and q RT-PCR found that FOXO3 a expression decreased gradually after irradiation,and reach to the peak after 24 h.CNE2 and HNE1 were transfected by sh RNA,w estern blotting and q RT-PCR showed that NPC cells transfected with FOXO3 a sh RN A3 could significantly inhibit the expression of FOXO3 a protein.Therefore,cells tra nsfected with sh RNA3(CNE2-sh RNA3? HNE1-sh RNA3)and mock sh RNA(CNE2-Mock?HNE1-Mock)were chosen for further experiments.CNE2-sh RNA3 ? HNE1-sh RNA3 cells had relatively higher colony survival rates compared to mock cells af ter irradiation which suggested that silencing FOXO3 a induces radioresistance in NPC cells.To investigate the association between FOXO3 a and EMT,western blotting was performed to detect alterations in EMT marker proteins after transfection with s h RNA3 and mock sh RNA.Epithelial marker protein E-cadherin levels were reduced,whereas the levels of mesenchymal marker proteins vimentin and N-cadherin were increased in cells transfected with sh RNA3.We also used immunofluorescence to in vestigate the location of EMT marker proteins.We found that the amount of E-cadh erin located in the cell membrane was reduced,whereas that of vimentin located in the cytoplasm was increased in cells transfected with sh RNA3.These results illustr ate that silencing of FOXO3 a may induce EMT in NPC cells.Scratch tests and tra nswell invasion experiments found that silencing of FOXO3 a cound enhance the abi lity of invasion and migration in NPC cells.Conclusion: knockdown of FOXO3 a could induce radioresistance in NPC cells.Silencing of FOXO3 a could downregulate epithelial marker protein E-cadherin,upregulate mesenchymal marker proteins vimentin and N-cadherin,induce EMT and then enhance the ability of invasion and migration in NPC cells.Part ? The study of the mechanism on tumor radioresistance of nasopharyngeal carcinoma cell lines modulated by FOXO3 a silencing.Objective: To explore the underlying mechanism of tumor radioresistance of NPC modulated by FOXO3 a silencing.Methods: Western blotting and immunofluorescence were used to detect the expression of E-cadherin and ?-catenin after 6Gy irradiation.Wnt/?-catenin signaling pathway-related genes were detected by western blotting.Flow cytometry was used to detect the apoptosis in each NPC groups at 48 h or 72 h cells after 6Gy radiation.Flow cytometry was used to detect the cell cycle distribution at 24 h or 48 h after 6Gy radiation.?-Catenin sh RNA was transfected in NPC cell lines in which FOXO3 a was silenced,and colony formation assay was performed.The expression levels of Wnt/?-catenin signaling pathway-and EMT-associated proteins were determined by western blotting and immunofluorescence.Results: Western blotting found that E-cadherin levels decreased after irradiation,whereas ?-catenin levels in the nucleus increased,in sh RNA3 FOXO3 a cells compared with mock cells.After FOXO3 a knockdown,E-cadherin levels decreased more obviously,and ?-catenin levels in the nucleus increased more significantly than in mock cells.These results were confirmed by immunofluorescence.Wnt/?-catenin signaling pathway-related genes were detected by western blotting,and found to be activated after silencing of FOXO3 a.Flow cytometry showed that silencing FOXO3 a could suppress G2/ M arrest and apoptosis in NPC cells.?-Catenin sh RNA was transfected in NPC cell lines in which FOXO3 a was silenced,and colony formation assay was performed.Colony formation assay results showed that co-transfecting ?-catenin sh RNA could reverse the radioresistance induced by FOXO3 a sh RNA.Moreover,silencing of FOXO3 a increased the expression of ?-catenin,whereas silencing of ?-catenin had no effect on FOXO3 a expression levels.silencing ?-catenin reverses the EMT induced by FOXO3 a sh RNA in NPC cells.Conclusion: Silencing of FOXO3 a could promote ?-catenin into the nucleus,upregulate ?-catenin expression,upregulate its downstream target genes c-Myc and cyclin D1,activate Wnt/?-catenin pathway and then suppress G2/ M arrest and apoptosis which lead to acquired radioresisitance in NPC cells.Silencing of ?-catenin can reverse the activation of downstream target genes induced by silencing of FOXO3 a,but it has no effect on the expression of FOXO3 a,indicating that silencing FOXO3 a could modulate the Wnt/?-catenin pathway and then induce acquired radioresisitance of NPC cells in a unidirectional regulation.Silencing of ?-catenin could also reverse the EMT caused by silencing of FOXO3 a,but the specific regulatory mechanism between Wnt/?-catenin and EMT still needs further study.Part ? The effect of FOXO3 a sh RNA on radioresistance of nasopharyngeal carcinoma cell lines in vivoObjective: To study the effect of silencing of FOXO3 a on radioresistance of NPC cells in vivoMethods: A xenograft mouse model was established.The animals were randomly divided into 4 groups with 3 animals each: untreated CNE2-Mock,untreated CNE2-sh RNA3,CNE2-Mock irradiation(CNE2-Mock IR),and CNE2-sh RNA3 irradiation(CNE2-sh RNA3-IR)groups.The irradiated groups were irradiated with 8Gy of irradiation,then formation time and tumor volume were recorded.The nude mice were killed 28 days later,and transplanted tumors were taken off and weighed.Immunohistochemistry and western blotting were used to detect the expression of related proteins.Results: All of nude mice could form tumors.The average time to form tumor for CNE2-sh RNA3 group and CNE2-sh RNA3 IR group was 4 days,and the average time to form tumor for CNE2-Mock group and CNE2-Mock IR group was 6 days.When the tumor reached to 50mm3,irradiated groups were irradiated with 8 Gy of X-rays.The volume of transplanted tumor was significantly reduced and the growth rate was slower in the irradiated group than in the non irradiated group.The growth of the transplanted tumor was more rapid and the volume of the transplanted tumor was significantly larger in silencing of FOXO3 a group compared with control group.The nude mice were killed 28 days later,and transplanted tumors were taken off and weighed.The weight of the transplanted tumor in the irradiated group was significantly lighter than the non irradiated group,and the weight of the transplanted tumor was significantly larger in silencing of FOXO3 a group compared with control group.Immunohistochemistry showed that the expression of FOXO3 a was significantly reduced,the expression of E-cadherin was decreased,and the expression of N-cadherin and vimentin increased in CNE2-sh RNA3 group compared with CNE2-Mock group,indicating that silencing of FOXO3 a could induce EMT in vivo.Meanwhile,CNE2-sh RNA3 groupcompared with CNE2-Mock group,the expression of ?-catenin and its downstream c-Myc and cyclin D1 increased,the expression of GSk-3? decreased,indicating that silencing of FOXO3 a could activate the Wnt/?-catenin pathway in vivo.The results of western blotting experiment were consistent with the results of immunohistochemistry.The expression of Ki-67 was significantly higher and the apoptosis was inhibited in silencing of FOXO3 a group than in the control group.After FOXO3 a was silenced,the growth rate of xenografts increased rapidly,EMT was induced and Wnt/?-catenin pathway was activated which could promote cell proliferation and inhibit cell apoptosis,and then lead to radioresistance in vivo.Conclusion: Silencing of FOXO3 a could induce radioresistance of NPC cells in vivo.