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bioRxiv · 10.1101/2020.02.24.962449

shRNA Glut-1 inhibits cell viability, apoptosis and migration of laryngeal carcinoma HEp-2 cells through regulating Beclin-1-mediated autophagy

Wang, W.-D.·Zhu, J.-L.·Zhou, S.-H.·Fan, J.·Bao, Y.-Y.
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Abstract

ObjectiveGlut-1 is a key regulator in the process of glucose uptake. Previous studies have shown that Glut-1 affects autophagy. However, it is unclear whether there is a correlation between Glut-1 and autophagy in the progression of laryngeal carcinoma. This study was performed to investigate the role of Glut-1 in the development of laryngeal carcinoma. MethodsA stable HEp-2 cell model was constructed by Glut-1 and Beclin-1 shRNA lentiviral infection. The autophagosome was measured by transmission electron microscopy. Protein levels of LC3, ATG5, CyclinD1, Bcl-2, Caspase-3, and c-Myc were determined by Western blotting. CCK8 assay and Transwell assays were used to determine cell viability and migration rate of HEp-2 cells, respectively. Flow cytometry was performed to analyze the rate of apoptosis. Immunofluorescence was performed to determine the expression distribution of LC3. ResultsGlut-1 knockdown significantly promoted autophagosome formation by upregulating the ratio of LC3-II/LC3-I as well as the role of rapamycin (RAP) and Beclin-1 overexpression on autophagy flux in HEp-2 cells. Glut-1 inhibition also reduced the viability of HEp-2 cells followed by the decreases in expression of cyclinD1 and c-Myc. In addition, Glut-1 depletion increased the number of apoptotic HEp-2 cells accompanied by activation of caspase-3 and downregulation of Bcl-2. Glut-1 knockdown also reduced the migration rate of HEp-2 cells by promoting the expression of N-cadherin and inhibiting the expression of E-cadherin. Beclin-1 consumption significantly reversed Gult-1 knockdown-mediated autophagy activation, resulting in promotion of both proliferation and migration and inhibition of apoptosis. ConclusionsGlut-1 knockdown-induced autophagy inhibits the proliferation and migration of HEp-2 cells, and promotes apoptosis of HEp-2 cells partly by regulating autophagy.

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Wang, W.-D., Zhu, J.-L., Zhou, S.-H., Fan, J., Bao, Y.-Y.. 2020-02-24. shRNA Glut-1 inhibits cell viability, apoptosis and migration of laryngeal carcinoma HEp-2 cells through regulating Beclin-1-mediated autophagy. https://doi.org/10.1101/2020.02.24.962449

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