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

Abstract

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 (VEGF₁₆₅ and VEGF_165b) and their role in cell proliferation and apoptosis in human retinal vascular endothelial cells (hRVECs). Cell viability and VEGF₁₆₅ and VEGF_165b expressions were evaluated in hRVECs transfected with VEGF genes 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 VEGF gene SNPs on cell viability, VEGF₁₆₅ and VEGF_165b expressions, 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 VEGF₁₆₅ expression decreased, VEGF_165b levels increased significantly in hRVECs transfected with rs3025039, which decreased cell viability and induced apoptosis. The SNPs rs3025033 and rs10434 had no significant effects on VEGF_165b protein 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 VEGF_165b, whereas SRPIN340 treatment, an inhibitor of ASF/SF2, increased VEGF_165b protein levels. VEGF gene 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.