<p>Construction of a GLUT-1 and HIF-1&alpha; gene knockout cell model in HEp-2 cells using the CRISPR/Cas9 technique</p>

Abstract

BackgroundGlucose 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 HIF-1α and GLUT-1 are no longer expressed can provide important information about carcinogenesis in laryngeal carcinoma.PurposeIn 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.MethodsHigh-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.ResultsIn HIF-1α and GLUT-1 double gene knockout HEp-2 cells, a 171-bp deletion in the HIF-1α genomic sequence was detected, whereas multiple base insertions resulted in frameshift mutations in the GLUT-1 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 GLUT-1 double gene knockout using CRISPR/Cas9 technique lead to reduction of glucose uptake and lactic acid generation.ConclusionOur HIF-1α and GLUT-1 double gene knockout HEp-2 cell model, obtained using a CRISPR/Cas9-based system, may facilitate studies of the pathogenesis of laryngeal carcinoma.

<p>Autophagy-Related Beclin 1 and Head and Neck Cancers</p>

“…Abnormal Beclin 1 expression was associated with chemo-radiotherapy resistance in head and neck cancer. In combination with our previous studies, [125][126][127][128] we will continue to investigate the role of Beclin 1-mediated autophagy in laryngeal carcinoma as well as the role of Beclin1 in glucose transporter-1 (GLUT1)-mediated radioresistance. Furthermore, future studies should be designed to test the effects of Beclin1 activity on GLUT-1 expression as a pathway to improve the radiosensitivity of laryngeal carcinoma.…”

Effect of GLUT1 Inhibition and Autophagy Modulation on the Growth and Migration of Laryngeal Carcinoma Stem Cells Under Hypoxic and Low-Glucose Conditions

“…OncoTargets and Therapy 2021:14 overexpressed in many cancers, including laryngeal carcinoma, and is associated with their metastasis, drug resistance, and poor prognosis. 6,7,[31][32][33][34][35][36][37] In the present study, we investigated the role of GLUT-1 in CD133 + laryngeal carcinoma cells under hypoxic and low-glucose conditions.…”

Effect of Inhibition of GLUT1 Expression and Autophagy Modulation on the Growth and Migration of Laryngeal Carcinoma Stem Cells under Hypoxic and Low-Glucose Conditions

“…Glucose is the main energy source for tumor cells and GLUT-1 is a key transporter of extracellular glucose [6,7,29]. GLUT-1 is overexpressed in many cancers, including laryngeal carcinoma, and is associated with their metastasis, drug resistance, and prognosis [6,7,[30][31][32][33][34][35][36]. We investigated the role of GLUT-1 in Tu212…”