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

Abstract

AIM: 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). METHODS: 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. RESULTS: 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. CONCLUSION: Prominin-1 combined with microRNA-29b can provide a suitable therapeutic option for ameliorating NMDA-induced excitotoxicity in RGC-5 cells.

The RNA m5C methylase NSUN4 protects retinal ganglion cells by regulating m5C methylation in the Hedgehog signaling pathway

“…Our previous study showed that the number of RGCs and the electrophysiological functions of the retina after NMDA treatment were adversely in uenced in vivo 30 . Furthermore, we determined that the expression of the lncRNA Tsix, which is mainly concentrated in the cytoplasm of the retinal GCL, was signi cantly decreased in the NMDA-induced retinal excitotoxity model 42 . These changes are consistent with previous studies 5,28 .…”