Department of Gynecology, Qilu Hospital, Shandong University, Shandong Provincial Key Laboratory of Oral Tissue Regeneration, Shandong University, Jinan, Shandong 250012, PR China.
Department of Oral and Maxillofacial Surgery, Shandong Provincial Key Laboratory of Oral Tissue Regeneration, Shandong University, Jinan, Shandong 250012, PR China.
Arch Oral Biol. 2017 Oct;82:223-232. doi: 10.1016/j.archoralbio.2017.06.004. Epub 2017 Jun 24.
To investigate the importance of the p75 neurotrophin receptor (p75) in human tongue squamous carcinoma cells, we exploited the CRISPR/Cas9 technology to establish a p75-knockout SCC-9 cell line and to explore the effect on biological functions.
The clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated endonuclease (Cas9) system was used to generate genomic deletion mutants of p75 in the tongue squamous carcinoma cell lines SCC-9. Single-guide RNA (sgRNA) sequences were designed to target the p75 genomic sequence and were cloned into plasmid pGK1.1. The linearized vector was electroporated into SCC-9 cells and p75 deletion was confirmed using Cruiser™ enzyme digestion and PCR amplification. SCC-9 clones with successful deletion of p75 were identified and verified by sequencing and selected for functional testing in cell proliferation, invasion, migration, and colony-forming assays.
Compared with control cells, p75-knockout SCC-9 cells showed significantly diminished abilities to proliferate, invade, migrate, and form colonies, indicating a reduction in pro-tumorigenic behavior.
These data demonstrate, first, that the CRISPR/Cas9 system is a simplified method for generating p75 knockouts with relatively high efficiency, and second, that deletion of p75 suppresses several tumor-promoting properties of SCC-9 cells, suggesting that p75 is a potential target for the development of novel therapies for tongue cancer.
为了研究 p75 神经营养因子受体（p75）在人舌鳞癌细胞中的重要性，我们利用 CRISPR/Cas9 技术建立了 p75 敲除 SCC-9 细胞系，并探讨其对生物学功能的影响。
利用成簇规律间隔短回文重复序列（CRISPR）/CRISPR 相关内切酶（Cas9）系统在舌鳞癌细胞系 SCC-9 中产生 p75 的基因组缺失突变。设计靶向 p75 基因组序列的单引导 RNA（sgRNA）序列，并克隆到质粒 pGK1.1 中。将线性化载体电穿孔到 SCC-9 细胞中，并使用 Cruiser™酶消化和 PCR 扩增确认 p75 缺失。通过测序和功能测试（包括细胞增殖、侵袭、迁移和集落形成测定）鉴定和验证成功删除 p75 的 SCC-9 克隆。
与对照细胞相比，p75 敲除 SCC-9 细胞的增殖、侵袭、迁移和集落形成能力明显减弱，表明其促肿瘤行为减少。
这些数据首次表明，CRISPR/Cas9 系统是一种高效产生 p75 敲除的简化方法，其次，p75 的缺失抑制了 SCC-9 细胞的几种促肿瘤特性，表明 p75 是开发舌癌新疗法的潜在靶点。
