In vitro protective effect of recombinant prominin-1 combined with microRNA-29b on N-methyl-D-aspartate-induced excitotoxicity in retinal ganglion cells.

Abstract

To determine the in vitro protective effect of recombinant prominin-1 (Prominin-1)+microRNA-29b (P1M29) on N-methyl-D-aspartate (NMDA)-induced excitotoxicity in retinal ganglion cells (RGCs). RGC-5 cells were cultured, and NMDA-induced excitotoxicity at the range of 100-800 µmol/L was assessed using the MTT assay. NMDA (800 µmol/L) was selected as the appropriate concentration for preparing the cell model. To evaluate the protective effect of P1M29 on the cell model, Prominin-1 was added at the concentration of 1-6 ng/mL for 48h, and the cell survival was investigated with/without microRNA-29b. After obtaining the appropriate concentration and time of P1M29 at 48h, real-time polymerase chain reaction (PCR) was utilized to detect the relative mRNA expression of vascular endothelial growth factor (VEGF) and transforming growth factor (TGF)-β2. Western blot detection was applied to measure the phosphorylation levels of protein kinase B (AKT) and extracellular regulated protein kinases (ERK) in RGC-5 cells after treatment with Prominin-1. Apoptosis study of the cell model was conducted by flow cytometry for estimating the anti-apoptotic effect of P1M29. Immunofluorescence analysis was used to analyze the expression levels of VEGF and TGF-β2. MTT cytotoxicity assays demonstrated that P1M29 group had significantly higher cell survival rate than Prominin-1 group (P<0.05). Real-time PCR data indicated that the expression levels of VEGF were significantly increased in both Prominin-1 and P1M29 groups compared NMDA and microRNA-29b group (P<0.05), while TGF-β2 were significantly decreased in both microRNA-29b and P1M29 groups compared NMDA and Prominin-1 group (P<0.05). Western blot results showed that both Prominin-1 and P1M29 groups significantly increased the phosphorylation levels of AKT and ERK compared to NMDA and microRNA-29b groups (P<0.05). Flow cytometry analysis revealed that P1M29 could prevent RGC-5 cell apoptosis in the early stage of apoptosis, while immunofluorescence results showed that P1M29 group had higher expression of VEGF and lower expression of TGF-β2 with a stronger green fluorescence than NMDA group. Prominin-1 combined with microRNA-29b can provide a suitable therapeutic option for ameliorating NMDA-induced excitotoxicity in RGC-5 cells.