Genome-Scale CRISPR-Cas9 Knockout Screening in Nasopharyngeal Carcinoma for Radiosensitive and Radioresistant Genes.

Abstract

PURPOSE/OBJECTIVE(S)\nRadiotherapy is the primary treatment for nasopharyngeal carcinoma (NPC), and resistance of cancer cells to radiation is one of the major causes of treatment failure. In this study, the genome-wide CRISPR/Cas9-sgRNA library virus was used for the first time to screen for the functional genes associated with radiosensitivity and radioresistance in nasopharyngeal carcinoma, which lays the foundation for new research aiming to explain the mechanisms behind the radiosensitivity of nasopharyngeal carcinoma and makes some contribution to the discovery of new therapeutic targets and the development of new drugs to increase the sensitivity of NPC cells to radiotherapy.\n\n\nMATERIALS/METHODS\nCRISPR/Cas9 library lentivirus screening in radiation-treated nasopharyngeal carcinoma cells was combined with second-generation sequencing technology to identify specific sgRNAs. A set of samples as contrast, and early library infection was the starting control for genetic change trend analysis. Two groups were exposed to doses of 0 and 2 Gy, and cell samples were collected 7 and 14 days after irradiation. The genomic DNA of living cells was used for PCR amplification in the coding region of sgRNA, and then high-throughput sequencing analysis was performed. The radiosensitivity or radioresistance of the screened functional genes in nasopharyngeal carcinoma were further verified by colony formation and cell proliferation experiments using a cell counting kit in a stable cell line with single gene knockout and qPCR analysis in radioresistant nasopharyngeal carcinoma cells.\n\n\nRESULTS\nEleven radiosensitive and radioresistant function genes were selected based on high-throughput sequencing and bioinformatics analysis. Compared with the control group, after the single gene FBLN5, FAM3C, MUS81, or DNAJC17 knockout, the colony formation and proliferation ability of nasopharyngeal carcinoma cells C666-1 and CNE1 were significantly promoted, while after the single gene CDKN2AIP and SP1 knockout, the colony formation and proliferation ability were significantly lower (P < 0.05). Compared to the negative control cells, the expression levels of FBLN5, FAM3C, MUS81, and DNAJC17 were significantly lower in the C666-1-Rs and 5-8F-Rs cell lines (P < 0.05), while the expression levels of TOMM20, CDKN2AIP, SNX22, and SP1 were significantly higher (P < 0.05). KEGG enrichment analysis showed that the potential signaling pathway contributed to radiosensitivity or radioresistance in NPC, including the Fanconi anemia pathway and the TGF-beta signaling pathway.\n\n\nCONCLUSION\nWe found nine genes through CRISPR/Cas9-mediated genome-wide knockout involved in the radiosensitivity or radioresistance of NPC cells: four genes for radiosensitivity, FBLN5, FAM3C, MUS81, and DNAJC17; two genes for radioresistance: CDKN2AIP, SP1, while TOMM20 and SNX22 are potential radioresistant genes, and CALD1 is a potential radiosensitive gene.