Generation and characterization of a human oral squamous carcinoma cell line SCC-9 with CRISPR/Cas9-mediated deletion of the p75 neurotrophin receptor

Objective: To investigate the importance of the p75 neurotrophin receptor (p75(NTR)) in human tongue squamous carcinoma cells, we exploited the CRISPR/Cas9 technology to establish a p75(NTR)-knockout SCC-9 cell line and to explore the effect on biological functions. Materials and methods: The clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated endonuclease (Cas9) system was used to generate genomic deletion mutants of p75(NTR) in the tongue squamous carcinoma cell lines SCC-9. Single-guide RNA (sgRNA) sequences were designed to target the p75(NTR) genomic sequence and were cloned into plasmid pGK1.1. The linearized vector was electroporated into SCC-9 cells and p75(NTR) deletion was confirmed using Cruiser (TM) enzyme digestion and PCR amplification. SCC-9 clones with successful deletion of p75(NTR) were identified and verified by sequencing and selected for functional testing in cell proliferation, invasion, migration, and colony-forming assays. Results: Compared with control cells, p75(NTR)-knockout SCC-9 cells showed significantly diminished abilities to proliferate, invade, migrate, and form colonies, indicating a reduction in pro-tumorigenic behavior. Conclusion: These data demonstrate, first, that the CRISPR/Cas9 system is a simplified method for generating p75(NTR) knockouts with relatively high efficiency, and second, that deletion of p75(NTR) suppresses several tumor promoting properties of SCC-9 cells, suggesting that p75(NTR) is a potential target for the development of novel therapies for tongue cancer.