Knockout notch1 gene can enhanced radiosensitivity of nasopharyngeal carcinoma cells

Abstract

Objective:To investigate the effect of Notch1 gene on radiosensitivity of nasopharyngeal carcinoma cells and its molecular mechanism. Method:A Notch1-knockout CNE-2 cell line was constructed using CRISPR/Cas9 system, and the expression of Notch1 gene was detected by RT-PCR and Western blot. After treatment with different doses of radiation, the survival fraction （SF） of each group was calculated, and used the GraphPad Prism 6.0 software and the Linear quadratic model were used to calculate the fitted dose survival curve and the sensitivity enhancement ratio（SER）. Taking 6 Gy as radiation dose, the experiment was divided into four groups: Notch1（+） group, Notch1（-） group, IR+Notch1（+） group and IR+Notch1（-） group. CCK-8 assay was used to detect cell proliferation in each group. Annexin V-FITC/PI double staining assay was used to detect the changes of apoptosis in each group. The expression of H2AX, CyclinD1, Bax, Bcl-2 and GAPDH proteins were detected by Western blot. Result:The CNE-2 cell line with Notch1 gene knockout was successfully constructed. The clonogenic assay showed knockout of Notch1 enhanced the radiosensitivity of NPC cells. The CCK-8 assay showed that cell proliferation and cell viability were significantly reduced in the IR+Notch1（-） group compared with the IR+Notch1（+） group（P<0.05）. Annexin V-FITC/PI double staining assay showed that the IR+Notch1（-） group had the highest apoptosis rate compared with the other groups （P<0.05）. Western blotting demonstrated that the expression of γH2AX was significantly increased after irradiation of Notch1 nasopharyngeal carcinoma cells, the expression of Cyclin-D1 was increased, and the ratio of Bax:Bcl-2 was higher. Conclusion:Knockout of Notch1 signaling molecule can effectively improve the radiosensitivity of NPC cells cultured in vitro, which may be a potential target for radiosensitization of NPC.