Construction of a GLUT-1 and HIF-1α gene knockout cell model in HEp-2 cells using the CRISPR/Cas9 technique.
AKT
CRISPR
Cas9 system
HEp-2 cells
PI3K
glucose transporter-1
hypoxia-inducible factor-1α
laryngeal carcinoma
mTOR pathway
Journal
Cancer management and research
ISSN: 1179-1322
Titre abrégé: Cancer Manag Res
Pays: New Zealand
ID NLM: 101512700
Informations de publication
Date de publication:
2019
2019
Historique:
entrez:
19
3
2019
pubmed:
19
3
2019
medline:
19
3
2019
Statut:
epublish
Résumé
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 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 Our
Sections du résumé
BACKGROUND
BACKGROUND
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
PURPOSE
OBJECTIVE
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.
METHODS
METHODS
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.
RESULTS
RESULTS
In HIF-1α and GLUT-1 double gene knockout HEp-2 cells, a 171-bp deletion in the
CONCLUSION
CONCLUSIONS
Our
Identifiants
pubmed: 30881132
doi: 10.2147/CMAR.S183859
pii: cmar-11-2087
pmc: PMC6413817
doi:
Types de publication
Journal Article
Langues
eng
Pagination
2087-2096Déclaration de conflit d'intérêts
Disclosure The authors report no conflicts of interest in this work.
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