| Purpose : Radiation therapy plays an important role in the treatment of patients with non-small cell lung cancer(NSCLC).However,the radiocurability is greatly limited by reason of radioresistance which leads to treatment failure,tumor recurrence and metastasis.Cancer stem cell(CSC)has been identified as the main factor that contributes to radiation resistance.SOX2,one of the transcription factors specifically expressed in CSC,is involved in tumorigenesis,progression and maintenance of cell stemness.But the association between SOX2 and NSCLC radioresistance is not clear now.Here we mainly verified SOX2 expression level in NSCLC radioresistant cells,and explored the effect of SOX2 on radiosensitivity and cell stemness in NSCLC and discussed the possible mechanism between them.Methods:1.H460 cell line was selected for our study.H460 parent cells(H460-PT)were treated with single dose of 6Gy X-ray once a week,and the acquired radioresistant cells were induced by a total of 72 Gy irradiation(H460-RR).Colony formation assay was performed to detect the radiosensitivity of cells,and western blot and immunofluorescence were used to identify γH2AX expression level to verify the DNA repair ability of radioresistant cells.2.The protein and m RNA expression level of CD133,ALDH and SOX2 were respectively measured by western blot and q RT-PCR.And sphere formation assay was utilized to analyze CSC-like characteristics.3.Migration property of H460-PT and H460-RR was determined by wound healing assay and Transwell assay.4.The SOX2-upregulated model was established by lentivirus transfection in H460-PT,and the change of radiosensitivity after transfection was detected by clonogenic survival assay.5.The SOX2-downregulated model was constructed by lentivirus transfection in H460-RR,and colony formation assay was used to analyze radiosensitivity after SOX2 silence.6.After up and down-regulation of SOX2,western blot and q RT-PCR were taken to measure CSC biomarker expression level.Sphere formation assay was used to detect cell stemness,wound healing assay and Transwell were used to analyze migration capacity.Results:1.Radioresistant cell line construction: H460 cells exposed to a total of 72 Gy irradiation showed higher survival fraction and DNA repair capacity,which were our expected radioresistant cells H460-RR(p<0.05).2.Radioresistant cell line showed dedifferentiation : the protein and m RNA expression level of CD133,ALDH and SOX2 in H460-RR was significantly higher than that of H460-PT,and H460-RR had increased sphere formation ability.3.Higher migration capability was observed in H460-RR compared with H460-PT.4.SOX2 was involved in the regulation of radioresistance:overexpression of SOX2 enhanced radioresistance and DNA damage repair capability of H460-PT,while down-regulation of SOX2 led to increased radiosensitivity and decreased DNA repair ability in H460-RR.5.SOX2 was involved in dedifferentiation phenotype regulation of radioresistant cells: overexpression of SOX2 in H460-PT promoted CSC biomarker(CD133,ALDH)expression and sphere formation,while down regulation of SOX2 in H460-RR decreased CD133 expression level and sphere formation ability.6.SOX2 was involved in the regulation of cell migration: up-regulation of SOX2 promoted migration ability of H40-PT,and down-regulation of SOX2 decreased migration capacity of H460-RR.Conclusion : 1.NSCLC radioresistance and increased SOX2 expression level can be induced by fraction radiation.2.SOX2 regulates radioresistance in NSCLC via promoting cell dedifferentiation.Taken together,SOX2 plays an important role in the regulation of NSCLC radiosensitivity,which may provide a new perspective to improve curative effect. |
