The Role Of NYD-SP15 In Age-related Macular Degeneration

Background and purpose:Age-related macular degeneration (AMD) is an irreversibly degenerative deisease that affects primarily the retinal pigment epithelial (RPE) cells. NYD-SP15 is a novel cytidine deaminase that has a potential role on cell growth inhibition. In this present study, we aim to investigate whether NYD-SP15 has the effects on function of ARPE-19 cells, and provide the basis for NYD-SP15 in pathogenesis and protection of AMD.Methods:Lentivirus stably transfected ARPE-19 cells with over-expressed NYD-SP15 and NYD-SP15 knockdown were generated and identified. Cell growth and proliferation, migration, apoptosis and cell cycle were performed by CCK-8 assay, wound scratch assay and flow cytometry. caspase-3, caspase-8 and caspase-9 activity were determined by the caspase-3/8/9 assay kit and western blot. The mRNA chip of NYD-SP15 over-expressed ARPE-19 cells as well as controls were performed by one array plus process. H2O2 induced oxidative stress damage model was used for NYD-SP15 knockdown research, and cell apoptosis and ROS activity was detected. Keap-1/Nrf2/HO-1 pathway, Akt and MAPKs pathway were determined by western blot.Results:NYD-SP15 inhibited the proliferation, migration of ARPE-19 cells, and could lead to cell apoptosis via a caspase-3 and caspase-9-mediated mitochondrial pathway. Results from gene chip showed that over-expression of NYD-SP15 might cause 257 genes down-regulation and only 57 genes up-regulation. GO assay showed that NYD-SP15 was more likely to work on the transcript regulation, and Akt as well as MAPKs pathway were significantly linked with NYD-SP15. Indeed, western blot revealed that NYD-SP15 inhibited the expression of Akt and MAPKs pathway including Erkl/2, JNK and p38, which were the key pathway on normal cell growth maintaining. Furthermore, down-regulation of NYD-SP15 had no effect on the growth of normal cultured ARPE-19 cells with 10%FBS, but could promote the ARPE-19 cells proliferation from the damage caused by starve situation. A knockdown of NYD-SP15 in AREP-19 cells could be protective against H2O2-induced oxidative stress damage by decreasing cell apoptosis and intracellular reactive oxygen species (ROS) level. Additionally, NYD-SP15 gene silence could induce an increased activation of nuclear Nrf2 and HO-1,as well as phosphorylation level of Akt, Erkl/2 and p38 pathway in response to 200μM H2O2 in ARPE-19 cells, while JNK activation were inhibited.Conclusions:High expression of NYD-SP15 might inhibit the growth, proliferation and migration of ARPE-19 cells by blocking the expression of Akt and MAPKs pathway, and might even cause mitochondria-mediated cell apoptosis. Besides, ARPE-19 cells lacking NYD-SP15 display an increased Akt-dependent activation of Nrf2/HO-1 pathway and are less susceptive to oxidative stress induced apoptosis. It is implied that NYD-SP 15 might be a potential pathogenic gene to AMD, and down-regulation of pathological NYD-SP15 expression could protect RPE cells against oxidative stress.