Department of Oral and Maxillofacial Surgery, Klinikum rechts der Isar der Technischen Universität München, D-81675, Munich, Germany.
Clinical Research Unit, Department of Obstetrics and Gynecology, Technische Universität München, D-81675, Munich, Germany.
Head Face Med. 2024 Jun 18;20(1):37. doi: 10.1186/s13005-024-00437-x.
The treatment of oral squamous cell carcinoma (OSCC) remains challenging and survival rates have not been improved significantly over the past decades. Integrins have been recognized driving the cancer progression and high expression levels cause poor outcomes in patients afflicted with OSCC. Integrin αvβ6 and its subunit integrin beta 6 (ITGB6) were discovered to enhance the invasiveness by providing beneficial effects on downstream pathways promoting the cancer progression. The objective of this study was to establish a CRISPR/Cas9-mediated knock out of ITGB6 in the human OSCC cell line HN and investigate the effects on the migration and proliferation ability.
ITGB6 knock out was performed using the CRISPR/Cas9-system, RNPs, and lipofection. Monoclonal cell clones were achieved by limiting dilution and knock out verification was carried out by sanger sequencing and FACS on protein level. The effects of the knock out on the proliferation and migration ability were evaluated by using MTT and scratch assays. In addition, in silico TCGA analysis was utilized regarding the effects of ITGB6 on overall survival and perineural invasion.
In silico analysis revealed a significant impact of ITGB6 mRNA expression levels on the overall survival of patients afflicted with OSCC. Additionally, a significantly higher rate of perineural invasion was discovered. CRISPR/Cas9-mediated knock out of ITGB6 was performed in the OSCC cell line HN, resulting in the generation of a monoclonal knock out clone. The knock out clone exhibited a significantly reduced migration and proliferation ability when compared to the wildtype.
ITGB6 is a relevant factor in the progression of OSCC and can be used for the development of novel treatment strategies. The present study is the first to establish a monoclonal CRISPR/Cas9-mediated ITGB6 knockout cell clone derived from an OSCC cell line. It suggests that ITGB6 has a significant impact on the proliferative and migratory capacity in vitro.
口腔鳞状细胞癌（OSCC）的治疗仍然具有挑战性，在过去几十年中，生存率并没有显著提高。整合素已被认为是推动癌症进展的因素，其高表达水平导致 OSCC 患者预后不良。整合素 αvβ6 及其亚基整合素β 6（ITGB6）被发现通过提供有利于下游通路的有益作用来增强侵袭性，从而促进癌症进展。本研究的目的是建立 CRISPR/Cas9 介导的人 OSCC 细胞系 HN 中 ITGB6 的敲除，并研究其对迁移和增殖能力的影响。
使用 CRISPR/Cas9 系统、RNP 和脂质转染进行 ITGB6 敲除。通过有限稀释获得单克隆细胞克隆，并通过桑格测序和蛋白质水平的 FACS 进行敲除验证。通过 MTT 和划痕实验评估敲除对增殖和迁移能力的影响。此外，还利用 TCGA 分析了 ITGB6 对 OSCC 患者总生存和神经周围侵犯的影响。
计算机分析显示 ITGB6 mRNA 表达水平对 OSCC 患者的总生存有显著影响。此外，还发现神经周围侵犯的发生率显著升高。在 OSCC 细胞系 HN 中进行了 CRISPR/Cas9 介导的 ITGB6 敲除，产生了一个单克隆敲除克隆。与野生型相比，敲除克隆的迁移和增殖能力显著降低。
ITGB6 是 OSCC 进展的一个相关因素，可以用于开发新的治疗策略。本研究首次建立了源自 OSCC 细胞系的单克隆 CRISPR/Cas9 介导的 ITGB6 敲除细胞克隆。这表明 ITGB6 对体外增殖和迁移能力有显著影响。
