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

Abstract

ObjectiveTo investigate the importance of the p75 neurotrophin receptor (p75NTR) in human tongue squamous carcinoma cells, we exploited the CRISPR/Cas9 technology to establish a p75NTR-knockout SCC-9 cell line and to explore the effect on biological functions. Materials and methodsThe clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated endonuclease (Cas9) system was used to generate genomic deletion mutants of p75NTR in the tongue squamous carcinoma cell lines SCC-9. Single-guide RNA (sgRNA) sequences were designed to target the p75NTR genomic sequence and were cloned into plasmid pGK1.1. The linearized vector was electroporated into SCC-9 cells and p75NTR deletion was confirmed using Cruiser™ enzyme digestion and PCR amplification. SCC-9 clones with successful deletion of p75NTR were identified and verified by sequencing and selected for functional testing in cell proliferation, invasion, migration, and colony-forming assays. ResultsCompared with control cells, p75NTR-knockout SCC-9 cells showed significantly diminished abilities to proliferate, invade, migrate, and form colonies, indicating a reduction in pro-tumorigenic behavior. ConclusionThese data demonstrate, first, that the CRISPR/Cas9 system is a simplified method for generating p75NTR knockouts with relatively high efficiency, and second, that deletion of p75NTR suppresses several tumor-promoting properties of SCC-9 cells, suggesting that p75NTR is a potential target for the development of novel therapies for tongue cancer.