Xu Yidan, Wang Linnong, Cao Liu, Chen Lixun, Liu Qinghuai
Department of Ophthalmology, Nanjing First Hospital, Nanjing Medical University, Nanjing 210000, Jiangsu, China.
Department of Ophthalmology, The first affiliated Hospital of Nanjing Medical University, Nanjing 210000, Jiangsu, China.
J Cell Biochem. 2019 Feb;120(2):1362-1375. doi: 10.1002/jcb.27104. Epub 2018 Oct 28.
The aim of this study was to investigate the role of NYD-SP15 in the growth and oxidative-stress responses of ARPE-19 cells. ARPE-19 cell lines overexpressing wild type or RNA interference against NYD-SP15 were established via lentivirus transfection. Cell growth and proliferation, migration, apoptosis, and cell cycle progression were monitored using the Cell Counting Kit-8 assay, the wound scratch assay, and flow cytometry, respectively. Caspase-3/8/9 activity was examined using the caspase-3/8/9 assay kit. An hydrogen peroxide (H O )-induced oxidative-stress damage model was used to study the effect of NYD-SP15 knockdown by examining the activity of reactive oxygen species (ROS). Expressions of Kelch-like ECH-associated protein 1 (Keap-1)/heme oxygenase-1 (HO-1)/nuclear factor erythroid 2-related factor 2 (Nrf2), mitogen-activated protein kinase (MAPK), and Akt were detected by Western blot analysis. The mRNA chip of NYD-SP15 overexpressed ARPE-19 cells as well as controls were performed by one array plus process. Overexpression (OE) of NYD-SP15 inhibited the proliferation and migration of ARPE-19 cells, and led to apoptosis and caspase-3/9 activation. OE of NYD-SP15 inhibited MAPKs and Akt signaling. Downregulation of NYD-SP15 had no effect on the growth of normally cultured ARP19 cells with 10% fetal bovine serum, but promoted the growth of ARP19 cells in the presence of starvation challenge. Gene chip showed that OE of NYD-SP15 led to downregulation of 254 genes and upregulation of 57 genes. Downregulation of NYD-SP15 also exerted a protective effect on H O -induced cell apoptosis and ROS. NYD-SP15 downregulation led to increments in the expression of Nrf2, Keap-1, and HO-1 in response to 200 μM H O . NYD-SP15 might inhibit the growth, proliferation, and migration and promote apoptosis of ARPE-19 cells via MAPK and Akt signaling. Downregulation of NYD-SP15 could protect ARPE-19 cells from H O -induced oxidative damage by active Keap-1/HO-1/Nrf2, Akt, and MAPK signaling.
本研究旨在探讨NYD-SP15在人视网膜色素上皮细胞系（ARPE-19细胞）生长及氧化应激反应中的作用。通过慢病毒转染建立过表达野生型NYD-SP15或针对NYD-SP15进行RNA干扰的ARPE-19细胞系。分别使用细胞计数试剂盒-8法、划痕实验和流式细胞术监测细胞生长与增殖、迁移、凋亡及细胞周期进程。使用caspase-3/8/9检测试剂盒检测caspase-3/8/9活性。采用过氧化氢（H₂O₂）诱导的氧化应激损伤模型，通过检测活性氧（ROS）活性来研究敲低NYD-SP15的效果。通过蛋白质免疫印迹法检测kelch样ECH相关蛋白1（Keap-1）/血红素加氧酶-1（HO-1）/核因子红细胞2相关因子2（Nrf2）、丝裂原活化蛋白激酶（MAPK）和Akt的表达。对过表达NYD-SP15的ARPE-19细胞以及对照细胞进行一次阵列加流程的mRNA芯片检测。NYD-SP15过表达（OE）抑制ARPE-19细胞的增殖与迁移，并导致细胞凋亡及caspase-3/9激活。NYD-SP15的OE抑制MAPKs和Akt信号通路。敲低NYD-SP15对含10%胎牛血清正常培养的ARP19细胞生长无影响，但在饥饿应激条件下可促进ARP19细胞生长。基因芯片显示，NYD-SP15的OE导致254个基因下调和57个基因上调。敲低NYD-SP15对H₂O₂诱导的细胞凋亡和ROS也具有保护作用。敲低NYD-SP15导致在200 μM H₂O₂作用下Nrf2、Keap-1和HO-1的表达增加。NYD-SP15可能通过MAPK和Akt信号通路抑制ARPE-19细胞的生长、增殖和迁移并促进其凋亡。敲低NYD-SP15可通过激活Keap-1/HO-1/Nrf2、Akt和MAPK信号通路保护ARPE-19细胞免受H₂O₂诱导的氧化损伤。
