Silencing neuroglobin enhances neuronal vulnerability to oxidative injury by down-regulating 14-3-3γ
Abstract
Aim: To explore the protective role and mechanism of endogenous neuroglobin (Ngb) in neuronal cells under oxidative stress.
Methods: A stable N2a neuroblastoma cell line expressing the Ngb-siRNA plasmid (N2a/Ngb-siRNA) was established by neomycin screening. Reverse transcription (RT)-PCR and Western blot analysis were used to detect Ngb gene and protein levels. Hydrogen peroxide was used to induce oxidative stress in N2a cells. Cytotoxicity and cell viability were measured by lactate dehydrogenase (LDH) and WST-8 assays. Apoptotic cells were detected by Hoechst staining.
Results: Cotransfection of Ngb-siRNA with Ngb-GFP plasmids suppressed the expression of Ngb-GFP in N2a cells. RT-PCR and Western blot analysis showed that the expression of endogenous Ngb was successfully knocked down to about 20% in N2a/Ngb-siRNA cells compared with control cells. A WST-8 assay demonstrated that viability was significantly decreased in N2a/Ngb-siRNA cells and N2a cells transiently transfected with Ngb-siRNA plasmids compared with controls following hydrogen peroxide treatment. An LDH assay demonstrated a time-dependent increase in the death of Ngb-siRNA-transfected N2a cells following hydrogen peroxide treatment. Hoechst staining demonstrated that the quantity of apoptotic cells among N2a/Ngb-siRNA cells following hydrogen peroxide treatment significantly increased compared with controls. In N2a/Ngb-siRNA cells, the expression level of activated caspase-3 significantly increased, whereas the expression of 14-3-3γ decreased compared with that of N2a/vec cells. Transfection of 14-3-3γ plasmids significantly enhanced the viability of N2a/Ngb-siRNA cells following hydrogen peroxide treatment compared with vector controls.
Conclusion: Ngb contributes to neuronal defensive machinery against oxidative injuries by regulating 14-3-3γ expression.
Keywords:
Methods: A stable N2a neuroblastoma cell line expressing the Ngb-siRNA plasmid (N2a/Ngb-siRNA) was established by neomycin screening. Reverse transcription (RT)-PCR and Western blot analysis were used to detect Ngb gene and protein levels. Hydrogen peroxide was used to induce oxidative stress in N2a cells. Cytotoxicity and cell viability were measured by lactate dehydrogenase (LDH) and WST-8 assays. Apoptotic cells were detected by Hoechst staining.
Results: Cotransfection of Ngb-siRNA with Ngb-GFP plasmids suppressed the expression of Ngb-GFP in N2a cells. RT-PCR and Western blot analysis showed that the expression of endogenous Ngb was successfully knocked down to about 20% in N2a/Ngb-siRNA cells compared with control cells. A WST-8 assay demonstrated that viability was significantly decreased in N2a/Ngb-siRNA cells and N2a cells transiently transfected with Ngb-siRNA plasmids compared with controls following hydrogen peroxide treatment. An LDH assay demonstrated a time-dependent increase in the death of Ngb-siRNA-transfected N2a cells following hydrogen peroxide treatment. Hoechst staining demonstrated that the quantity of apoptotic cells among N2a/Ngb-siRNA cells following hydrogen peroxide treatment significantly increased compared with controls. In N2a/Ngb-siRNA cells, the expression level of activated caspase-3 significantly increased, whereas the expression of 14-3-3γ decreased compared with that of N2a/vec cells. Transfection of 14-3-3γ plasmids significantly enhanced the viability of N2a/Ngb-siRNA cells following hydrogen peroxide treatment compared with vector controls.
Conclusion: Ngb contributes to neuronal defensive machinery against oxidative injuries by regulating 14-3-3γ expression.