Ping Huang1, Dongdong Tong2, Jing Sun3, Qing Li2, Fenghe Zhang4. 1. Department of Gynecology, Qilu Hospital, Shandong University, Shandong Provincial Key Laboratory of Oral Tissue Regeneration, Shandong University, Jinan, Shandong 250012, PR China. 2. Department of Oral and Maxillofacial Surgery, Shandong Provincial Key Laboratory of Oral Tissue Regeneration, Shandong University, Jinan, Shandong 250012, PR China. 3. Department of Bone Metabolism, Shandong Provincial Key Laboratory of Oral Tissue Regeneration, Shandong University, Jinan, Shandong 250012, PR China. 4. Department of Oral and Maxillofacial Surgery, Shandong Provincial Key Laboratory of Oral Tissue Regeneration, Shandong University, Jinan, Shandong 250012, PR China. Electronic address: zfengh@sdu.edu.cn.
Abstract
OBJECTIVE: To 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 METHODS: The 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. RESULTS: Compared 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. CONCLUSION: These 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.
OBJECTIVE: To investigate the importance of the p75 neurotrophin receptor (p75NTR) in humantongue 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 METHODS: The 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. RESULTS: Compared 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. CONCLUSION: These 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.