Background and purpose: Notch signaling pathway plays a critical rol

e in the development of nasopharyngeal carcinoma (NPC) and self-renewal of cancer stem cells (CSC). Complete loss of biological functions of specific gene is difficult to achieve by previous methods in Notch pathway. This study aimed to establish Notch1 knockout NPC CNE2 cell line using CRISPRCas9 gene editing technology and to explore the effects of Notch1 on the proliferation and radiosensitivity of CNE2. Methods: Western blot was applied to determine the protein level of Notch1 in NPC cell line CNE2 and normal nasopharyngeal epithelial cell line NP69. SgRNA online design tool was used to design sgRNA for Notch1. PX459 plasmid was utilized to construct knockout vector containing the sgRNA named PX459-Notch1-sgRNA. Notch1 knockout CNE2 cell line was constructed through transfection

drug screening and cloning

followed by verification via Western blot and immunofluorescence (IF). Then Cell Count Kit-8 (CCK-8)

colony formation assay and flow cytometry were used respectively to detect cell proliferation

radiation sensitivity and sidepopulation cell ratio in group with Notch1 knockout (Notch1-KO)

group without transfection (Parental) and group only transfected with PX459 (Ctrl). Results: Notch1 protein level was higher in CNE2 than NP69 (P0.001). Notch1 was not detected by Western blot and IF in Notch1-KO CNE2 cell line. Notch1 knockout inhibited cell proliferation of CNE2 cells (P0.05). The values of D

0

D

q

and SF2 in Notch1-KO group were 1.160

1.881 and 0.630 Gy

respectively

obviously lower than those in Parental group (1.176

2.533 and 0.824 Gy) (P0.001) and those in Ctrl group (1.182

2.516 and 0.819 Gy) (P0.001). There was significant difference in side population cell ratio between the Notch1-KO ［(1.13±0.01)%］ group and the Parental group［(3.81±0.03)%］ (P0.001) or the Ctrl group ［(3.70±0.03)%］(P0.001). Conclusion: CRISPR-Cas9 technology can successfully knockout Notch1 gene of CNE2 cell line. Notch1 knockout results in suppression of CNE2 cell proliferation

radiation sensitization

and down-regulation of side population cell ratio.