Inhibition of PTB-associated splicing factor on IGF-1/VEGF signaling pathway in retinal vascular endothelial cells

Background

Retinal neovascularization (RNV) is common findings of many fundus diseases.Research showed that insulin-like growth factor-1 (IGF-1) induces the proliferation of vascular endothelial cells and therefore promotes the formation of new blood vessel.Polypyrimidine tract-binding protein-associated splicing factor (PSF) is determined to play the inhibitory effect on the gene trancription in IGF-1 signal pathway, but its effect on RNV is still unelucidated.

Objective

This study was to observe the regulation of PSF on IGF-1/vascular endothelial growth factor(VEGF) signaling pathway in vitro.

Methods

RF/6A cells were cultured and divided into four groups.Pegfp-C2-PSF plasmid, Pegfp-C2 plasmid, pGenesil-PSF-RNAi plasmid and pGenesil-RNAi plasmid were transfected into the cells with the PSF dose of 0.00, 0.25, 0.50 and 1.00 μg respectively.The cells were cultured with or without IGF-1.The proliferation rates of the cells were detected by using MTT assay to determine the optimal PSF dose.Reverse-transceiption PCR (RT-PCR) was employed to assay and compare the relative expression levels of VEGF mRNA in the cells among different PSF plasmid tranfected groups or between the treated groups with and without U0126, and the levels of phosphorylized extracellular signal-regulation kinase (pERK) in the cells were detected by Western blot assay.

Results

After IGF-1 stimulation, 0.50 μg and 1.00 μg PSF appeared to be the optimal dose in the Pegfp-C2-PSF plasmid group and the pGenesil-PSF-RNAi plasmid group, respectively.The cell proliferation rates and VEGF mRNA levels in the cells were (0.22±0.02) and (0.79±0.07), respectively, in the Pegfp-C2-PSF plasmid group, which were significantly lower than 0.260±0.006 in the Pegfp-C2 plasmid group and 1.26±0.19 in the untransfected group (P=0.040, 0.016). The mean cell proliferative rate was 0.32±0.03, and the mean expression level of VEGF mRNA was 2.29±0.43 in the pGenesil-PSF-RNAi plasmid group, showing significant increases in comparison with 0.210±0.019 of the pGenesil-RNAi plasmid group and 1.26±0.19 of the untransfected group (P=0.003, 0.019). The expression levels of pERK protein in the cells were evidently reduced in the Pegfp-C2-PSF plasmid group compared with the untransfected group 1 hour, 3, 6 hours after addition of IGF-1 (P=0.017, 0.000, 0.000). After treated by U0126, the relative expression level of VEGF mRNA in the Pegfp-C2-PSF plasmid group was 0.93±0.21, which was significantly lower than 1.32±0.08 in the untransfected group (P=0.037) and 1.23±0.09 in the non-U0126 treated cells of the Pegfp-C2-PSF plasmid group (P=0.002).

Conclusions

PSF inhibits the activation of ERK signal pathway in the IGF-1-stimulated RF/6A cells and down-regulates the expression level of VEGF in RF/6A cells and thus suppresses the growth and proliferation of RF/6A cells.

Polypyrimidine tract-binding protein; RNA splicing; Insulin-like growth factor; Cell line; Endothelial cells/drug effect; Retinal vessels/cytology; Vascular endothelial growth factor; Extracellular signal-regulation kinase