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Open AccessArticle

DRAM1 Protects Neuroblastoma Cells from Oxygen-Glucose Deprivation/Reperfusion-Induced Injury via Autophagy

Department of Neurosurgery, Second Xiangya Hospital of Central South University, Changsha 410011, China
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Int. J. Mol. Sci. 2014, 15(10), 19253-19264; https://doi.org/10.3390/ijms151019253
Received: 15 July 2014 / Revised: 18 September 2014 / Accepted: 9 October 2014 / Published: 23 October 2014
(This article belongs to the Section Biochemistry)
DNA damage-regulated autophagy modulator protein 1 (DRAM1), a multi-pass membrane lysosomal protein, is reportedly a tumor protein p53 (TP53) target gene involved in autophagy. During cerebral ischemia/reperfusion (I/R) injury, DRAM1 protein expression is increased, and autophagy is activated. However, the functional significance of DRAM1 and the relationship between DRAM1 and autophagy in brain I/R remains uncertain. The aim of this study is to investigate whether DRAM1 mediates autophagy activation in cerebral I/R injury and to explore its possible effects and mechanisms. We adopt the oxygen-glucose deprivation and reperfusion (OGD/R) Neuro-2a cell model to mimic cerebral I/R conditions in vitro, and RNA interference is used to knock down DRAM1 expression in this model. Cell viability assay is performed using the LIVE/DEAD viability/cytotoxicity kit. Cell phenotypic changes are analyzed through Western blot assays. Autophagy flux is monitored through the tandem red fluorescent protein–Green fluorescent protein–microtubule associated protein 1 light chain 3 (RFP–GFP–LC3) construct. The expression levels of DRAM1 and microtubule associated protein 1 light chain 3II/I (LC3II/I) are strongly up-regulated in Neuro-2a cells after OGD/R treatment and peaked at the 12 h reperfusion time point. The autophagy-specific inhibitor 3-Methyladenine (3-MA) inhibits the expression of DRAM1 and LC3II/I and exacerbates OGD/R-induced cell injury. Furthermore, DRAM1 knockdown aggravates OGD/R-induced cell injury and significantly blocks autophagy through decreasing autophagosome-lysosome fusion. In conclusion, our data demonstrate that DRAM1 knockdown in Neuro-2a cells inhibits autophagy by blocking autophagosome-lysosome fusion and exacerbated OGD/R-induced cell injury. Thus, DRAM1 might constitute a new therapeutic target for I/R diseases. View Full-Text
Keywords: DNA damage-regulated autophagy modulator protein 1 (DRAM1); oxygen-glucose deprivation and reperfusion (OGD/R); cerebral ischemia and reperfusion (I/R) injury; autophagy DNA damage-regulated autophagy modulator protein 1 (DRAM1); oxygen-glucose deprivation and reperfusion (OGD/R); cerebral ischemia and reperfusion (I/R) injury; autophagy
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Yu, M.; Jiang, Y.; Feng, Q.; Ouyang, Y.; Gan, J. DRAM1 Protects Neuroblastoma Cells from Oxygen-Glucose Deprivation/Reperfusion-Induced Injury via Autophagy. Int. J. Mol. Sci. 2014, 15, 19253-19264.

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