Construction of a GLUT-1 and HIF-1α gene knockout cell model in HEp-2 cells using the CRISPR/Cas9 technique

• Published in: Cancer management and research Vol. 11; pp. 2087 - 2096
• Main Authors: Lu, Zhong-Jie, Yu, Qi, Zhou, Shui-Hong, Fan, Jun, Shen, Li-Fang, Bao, Yang-Yang, Wu, Ting-Ting, Zhou, Min-Li, Huang, Ya-Ping
• Format: Journal Article
• Language: English
• Published: New Zealand Dove Medical Press 01.01.2019
• Subjects: CRISPR/Cas9 system; glucose transporter-1; HEp-2 cells; hypoxia inducible factor-1α; laryngeal carcinoma; Original Research; PI3K/AKT/mTOR pathway; CRISPR; HEp-2 cells; hypoxia-inducible factor-1α; PI3K; Cas9 system; mTOR pathway; AKT; glucose transporter-1; laryngeal carcinoma
• ISSN: 1179-1322; 1179-1322
• DOI: 10.2147/CMAR.S183859

Glucose transporter (GLUT)-mediated glucose uptake is an important process in the development of laryngeal carcinoma, one of the most common malignancies of the head and neck. GLUT-1, together with HIF-1α, is also an indicator of hypoxia. Both proteins play a critical role in glucose uptake and glycolysis in laryngeal carcinoma cells under hypoxic stress. A double gene knockout model in which and are no longer expressed can provide important information about carcinogenesis in laryngeal carcinoma. In this study we used the CRISPR/Cas 9 system to induce HIF-1α and GLUT-1 double gene knockout in HEp-2 cells and then used the knocked-out cells to study the role of these markers in laryngeal carcinoma, including in chemoradioresistance. High-grade small-guide RNAs (sgRNAs) of HIF-1α and GLUT-1 were designed using an online tool and inserted into the pUC57-T7-gRNA vector. The recombinant plasmids were transfected into HEp-2 cells and positive cells were screened using the dilution method. Gene mutation and expression were determined by sequence analysis and immunoblotting. In HIF-1α and GLUT-1 double gene knockout HEp-2 cells, a 171-bp deletion in the genomic sequence was detected, whereas multiple base insertions resulted in frameshift mutations in the gene. Neither HIF-1α nor GLUT-1 protein was expressed in positive cells. The proliferation, migration, and invasion of HEp-2 cells were significantly decreased afterward. The possible mechanism may be that the inhibition PI3K/AKT/mTOR pathway by HIF-1α and double gene knockout using CRISPR/Cas9 technique lead to reduction of glucose uptake and lactic acid generation. Our and double gene knockout HEp-2 cell model, obtained using a CRISPR/Cas9-based system, may facilitate studies of the pathogenesis of laryngeal carcinoma.