VEGFgene polymorphisms regulate human retinal vascular endothelial cell proliferation and apoptosis through ASF/SF2-associated alternative splicing

This study investigated the effects of single nucleotide polymorphisms (SNPs) of the VEGF（vascular endothelial growth factor） gene, which are associated with susceptibility to age-related macular degeneration (AMD), on the expression of VEGF proteins (VEGF165and VEGF165b) and their role in cell proliferation and apoptosis in human retinal vascular endothelial cells (hRVECs). Cell viability and VEGF165and VEGF165bexpressions were evaluated in hRVECs transfected with VEGFgenes containing different SNPs (rs3025039, rs3025033, and rs10434). The Cell Counting Kit 8 assay, quantitative real-time PCR, western blotting, TUNEL assay, and enzyme-linked immunosorbent assay were used to examine the effects of VEGFgene SNPs on cell viability, VEGF165and VEGF165bexpressions, and cell apoptosis in hRVECs. The interaction and localization of the RNA-binding protein alternative splicing factor/splicing factor 2 (ASF/SF2) were assessed using RNA pull-down. Although VEGF165expression decreased, VEGF165blevels increased significantly in hRVECs transfected with rs3025039, which decreased cell viability and induced apoptosis. The SNPs rs3025033 and rs10434 had no significant effects on VEGF165bprotein production and apoptosis; however, they promoted cell proliferation. SNPs affected the interaction between RNA and ASF/SF2, a splicing factor for intron retention. Insulin-like growth factor-1 treatment induced the expression of VEGF165, but not VEGF165b, whereas SRPIN340 treatment, an inhibitor of ASF/SF2, increased VEGF165bprotein levels. VEGFgene sequence variations affected hRVEC proliferation and apoptosis via alternative gene splicing. Thus, the regulation of splicing via ASF/SF2 could be a potential strategy in treating pathological neovascularization in patients with AMD.