Snail Promotes Radioresistance Of Nasopharyngeal Carcinoma Cell Via Inducing EMT

Part Ⅰ Effect of Snail on EMT-like phenotype of nasopharyngeal carcinoma cell linesObjective:Establish Snail over-expressed nasopharyngeal carcinoma cell lines. Validate its effect on the migration and invasion in nasopharyngeal carcinoma cell in vitro.Methods:Western blot assay was used to detected the protein expression of Snail and E-cadherin. HNE-1and CNE-2cells were transfected with recombinant lentiviral transduction at the appropriate MOI; The mRNA and protein expression of Snail and EMT-related gene were detected by Real-time PCR and Western blot. Immunofluorescent assay was employed to detect the position and expression of EMT marker genes. Transwell assay and wound healing repair assay were performed to investigate the invasion and migration capability.Results:The expression of Snail was decreased in human poorly-differentiated nasopharyngeal carcinoma cell lines HNE-1and CNE-2. Real-time PCR and Western blot assay showed that the expression of Snail mRNA and protein were up-regulated in HNE-1-Snail and CNE-2-Snail cell lines than empty vector group. Down-regulation of E-cadherin and up-regulation of Vimentin, N-cadherin and MMP-2were observed in HNE-1-Snail and CNE-2-Snail cells. Immunofluorescent assay indicated that lose of E-cadherin at the cell-cell contact site and increase of Vimentin in Snail up-regulated group. The invasion and migration capability of HNE-1-Snail and CNE-2-Snail cells were increased compare to empty-vector group. The difference was statistically significant (P<0.01).Conclusion:Snail promotes the invasion and migration via inducing epithelial-mesenchymal transition in nasopharyngeal carcinoma cells in vitro. Part Ⅱ Effect of Snail on radiosensitivity of nasopharyngeal carcinoma cells in vitroObjective:To investigate the effect of Snail on radiosensitivity in nasopharyngeal carcinoma cells.Methods:Cloning formation experiment assay was performed to measure the survival fraction of nasopharyngeal carcinoma cells. Radiation biology parameters were calculated to compare the radiosensitivity. Flow cytometry was used to measure the apoptosis48or72hours after radiotherapy. Residual y-H2AX foci was determined by immunofloresence assay. Western Blot was performed to evaluate the y-H2AX, p-EGFR, p-DNA-PK protein expression after radiotherapy in HNE-1-Snail and CNE-2-Snail cells.Results:The colony forming assay demonstrated that the colony formation efficiency was significant increased in HNE-1-Snail and CNE-2-Snail cells. The value of SF2, Do, Dq, and N of Snail up-regulated cell lines were significant higher than empty vector group cells. Snail induced apoptosis decrease and radioresistance in nasopharyngeal carcinoma cell lines. The amount of residual γ-H2AX foci after irradiation was decreased in HNE-1-Snail and CNE-2-Snail. Nuclear accumulation of EGFR and phosporylation of EGFR and DNA-PKcs was increased in Snail up-regulated group. The difference was statistically significant (P<0.05).Conclusion:EMT induced by Snail may play a key role in increasing IR-induced EGFR nuclear transport and as a consequence promoting DNA-PKcs phosphorylation at the Thr-2609cluster. These molecular events are associated with enhanced DNA repair activity and resulted in an increase in clonogenic survival after irradiation. Part Ⅲ Effect of Snail on radiosensitivity of nasopharyngeal carcinoma cells in vivoObjective:To identify the effects of Snail on radiosensitivity of nasopharyngeal carcinoma cells in vivo.Methods:In order to construct subcutaneous xenograft tumors modle, CNE-2cells transfected with Snail over-expression lentivirus or empty vectors was subcutaneously inject into the right hind limbs of BALB/c nude mice, respectively. The effects of Snail on tumor formation was investigated and tumor volume was measured every2days after10Gy radiotherapy. VEGF and apoptosis-related protein caspase-3was detected by immunohistochemical assay.Results:Tumor growth delay was significantly shortened for mice injected with Snail up-regulated NPC cells. Morever, treatment with10Gy IR resulted in a significant reduction of tumor volume in the empty vector group compared to Snail up-regulated tumor-bearing mice. Immunohistochemical assay indicated that up-regulation of Snail resulted in increase of caspase-3and decrease of VEGF expression in tumor tissue. Over-expression of Snail promoted caspase-3and alleviated VEGF after IR. Conclusion:Snail over-expressed NPC cells have a greater capacity for xenograft formation. Mesenchymal-like characteristics and reduction of IR-induced apoptosis was observed in Snail over-expressed xenograft tissue. Snail promoted radioresistance in NPC in vivo.