RESUMEN
BACKGROUND: 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 αvß6 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. METHODS: 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. RESULTS: 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. CONCLUSIONS: 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.
Asunto(s)
Sistemas CRISPR-Cas , Carcinoma de Células Escamosas , Movimiento Celular , Proliferación Celular , Cadenas beta de Integrinas , Neoplasias de la Boca , Humanos , Movimiento Celular/genética , Proliferación Celular/genética , Neoplasias de la Boca/genética , Neoplasias de la Boca/patología , Línea Celular Tumoral , Carcinoma de Células Escamosas/genética , Carcinoma de Células Escamosas/patología , Cadenas beta de Integrinas/genética , Técnicas de Inactivación de Genes , Carcinoma de Células Escamosas de Cabeza y Cuello/genética , Carcinoma de Células Escamosas de Cabeza y Cuello/patología , Invasividad Neoplásica/genética , Regulación Neoplásica de la Expresión GénicaRESUMEN
Despite great efforts in improving existing therapies, the outcome of patients with advanced radioresistant HPV-negative head and neck squamous cell carcinoma (HNSCC) remains poor. The chromatin remodeler Chromodomain helicase DNA binding protein 4 (CHD4) is involved in different DNA-repair mechanisms, but the role and potential in HNSCC has not been explored yet. In the present study, we evaluated the prognostic significance of CHD4 expression using in silico analysis of the pan-cancer dataset. Furthermore, we established a monoclonal HNSCC CHD4 knockdown cell clone utilizing the CRISPR/Cas9 system. Effects of lower CHD4 expression on radiosensitivity after increasing doses of ionizing radiation were characterized using clonogenic assays and cell numbers. The in silico analysis revealed that high CHD4 expression is associated with significant poorer overall survival of HPV-negative HNSCC patients. Additionally, the knockdown of CHD4 significantly increased the radiosensitivity of HNSCC cells. Therefore, CHD4 might be involved in promoting radioresistance in hard-to-treat HPV-negative HNSCC entities. We conclude that CHD4 could serve as a prognostic factor in HPV-negative HNSCC tumors and is a potential target protein overcoming radioresistance in HNSCC. Our results and the newly established cell clone laid the foundation to further characterize the underlying mechanisms and ultimately use CHD4 in HNSCC therapies.
Asunto(s)
Neoplasias de Cabeza y Cuello , Infecciones por Papillomavirus , Humanos , Carcinoma de Células Escamosas de Cabeza y Cuello/genética , Carcinoma de Células Escamosas de Cabeza y Cuello/radioterapia , Carcinoma de Células Escamosas de Cabeza y Cuello/complicaciones , Neoplasias de Cabeza y Cuello/genética , Neoplasias de Cabeza y Cuello/radioterapia , Neoplasias de Cabeza y Cuello/complicaciones , Pronóstico , Línea Celular Tumoral , Complejo Desacetilasa y Remodelación del Nucleosoma Mi-2RESUMEN
CRISPR-Cas9 is a powerful technology for accurate and optimizable genome editing. Here, we present a protocol for generating monoclonal knockout (KO) cell lines using CRISPR-Cas9, ribonucleoprotein complexes (RNPs), and lipofection in adherent HNSCC cells from start to finish. We describe steps for choosing the suitable guide and primer design, preparation of guide-RNA (gRNA), lipofection of RNP complexes in HN cells, and single-cell cloning with limiting dilution. We then detail PCR and DNA purification and the selection and verification of monoclonal KO cell lines.