The Molecular Mechanism Of Redioresistance And Radiation-induced Epithelial-mesenchymal In Hypopharyngeal Carcinoma

Purpose:The aim of this study was to explore the role of ionizing radiation(IR)in EMT in hypopharyngeal cancer cells FaDu,the mechanisms and effects of acquired radioresistance and EMT induced by IR in the radioresistance human hypopharyngeal carcinoma cells FaDuRR.It is expected to find out a novel therapeutic target for reversing IR-induced EMT and radioresistance in hypopharyngeal carcinoma.Methods:1.the morphology of hypopharyngeal cancer FaDu cells after treatment with IR and FaDuRR(radiotherapy-resistant FaDu)cells were observed by using Microscope.Wound-healing assay and Transwell assay were used to measure the abilities of cell migration and invasiveness in FaDu cells after treatment with IR and FaDuRR cells.Western blot and Quantitative real-time PCR were used to detect the expression of EMT phenotypes and AKT/GSK-3β/Snail signaling pathway related proteins inFaDu cells after treatment with IR and FaduRR cells.Immunohistochemistry was used to assess the expression of EMT marker proteins and Snail in 80 hypopharyngeal carcinoma patient samples from radiosensitivity and radioresistance groups.Colony forming assay was used to detect radiosensitivity in FaDu cells after treatment with IR and FaduRR cells.2.RNA interference(RNAi)was used to silence the expression of Snail in FaDuRR cells.Western blot and Quantitative real-time PCR were used to detect the expression of EMT markers,Snail and apoptosis-related proteins in FaDuRR cells.Wound-healing assay and Transwell assay were used to measure the abilities of cell migration and invasiveness in FaDuRR cells treated with si-Snail.Colony forming assay was used to detect radiosensitivity in FaDuRR cells after treatment with si-Snail.Flow cytometry was used to detect the apoptosis FaDuRR cells treated with IR and si-Snail.Results:1.The morphology of FaDu after treatment with IR and FaDuRR cells turned from brick shaped and with tight cell-cell adhesion into irregular and narrow phenotype,and a loss of cell polarity and adhesion.Compared with the parental cells,the migration and invasive capacity of Fadu cells after treatment with IR and FaDuRR cells was enhanced.Compared with the parental cells,the expression of E-cadherin declined,whilst the expressionof N-cadherin,Vimentin,P-AKT,P-GSK-3β and Snail were enhanced,and no significant differences between AKT and GSK-3β were observed in Fadu cells after treatment with IR and FaDuRR cells.Compared to the radiosensitivity group,epithelial biomarker E-cadherin expression was down-regulated,however the mesenchymal biomarkers N-cadherin,Vimentin and transcription factor Snail were up-regulated in the radioresistance group.Compared with the parental cells,radioresistance was enhanced in Fadu cells after treatment with IR and FaDuRR cells.2.Compared with the FaDuRR/si-NC group,the expression of E-cadherin was enhanced,whilst the expression of N-cadherin and Vimentin were decreased in the FaDuRR/si-Snail group.Compared with the FaDuRR/si-NC group,the capacity of migration and invasive were decreased in the FaDuRR/si-Snail group.Compared with the FaDuRR/si-NC group,radiosensitivity was enhanced in the FaDuRR/si-Snail group.Compared with the FaDuRR/si-NC group,apoptosis rate of the FaDuRR/si-NC group was increased,the expression of apoptosis protein Bax and Cleaved caspase3 were increased,the expression of anti-apoptosis protein Bcl-2 was decreased in the FaDuRR/si-Snail group.Conclusion:1.IR can induce EMT and increase the ability of invasion and migration in the hypopharyngeal cancer FaDu and FaduRR cells.2.IR Promotes EMT by activating AKT/GSK-3β/Snail signalingpathway in FaduRR cells.3.Silencing Snail can reverse EMT and radioresistance ability in FaduRR cells.4.Snail silencing reverses acquired radioresistance by promoting apoptosis in FaduRR cells.