摘要
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 2O 2)–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 2O 2‐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 2O 2. 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 2O 2‐induced oxidative damage by active Keap‐1/HO‐1/Nrf2, Akt, and MAPK signaling.
摘要译文
本研究的目的是研究NYD-SP15在ARPE-19细胞的生长和氧化应激反应中的作用。通过慢病毒转染建立过表达针对NYD-SP15的野生型或RNA干扰的ARPE-19细胞系。使用细胞计数试剂盒-8测定法，伤口划痕测定法和流式细胞术分别监测细胞生长和增殖，迁移，细胞凋亡和细胞周期进展。使用caspase-3/8/9测定试剂盒检查Caspase-3/8/9活性。使用过氧化氢（H 2 O 2）诱导的氧化应激损伤模型通过检查活性氧（ROS）的活性来研究NYD-SP15敲低的影响。 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的表达均为通过Western印迹分析检测。 NYD-SP15过表达ARPE-19细胞的mRNA芯片以及对照通过一个阵列加工艺进行。 NYD-SP15的过表达（OE）抑制ARPE-19细胞的增殖和迁移，并导致细胞凋亡和胱天蛋白酶-3/9活化。 NYD-SP15的OE抑制MAPK和Akt信号传导。 NYD-SP15的下调对具有10％胎牛血清的正常培养的ARP19细胞的生长没有影响，但是在饥饿攻击的存在下促进了ARP19细胞的生长。基因芯片显示，NYD-SP15的OE导致254个基因的下调和57个基因的上调。 NYD-SP15的下调也对H 2 O 2诱导的细胞凋亡和ROS发挥保护作用。 NYD-SP15下调导致响应于200μMH2 O 2的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，促进细胞凋亡。 NYD-SP15的下调可通过活性Keap-1 / HO-1 / Nrf2，Akt和MAPK信号传导保护ARPE-19细胞免受H 2 O 2诱导的氧化损伤。
Yidan Xu[1];Linnong Wang[1];Liu Cao[1];Lixun Chen[1];Qinghuai Liu[2]. Involvement of NYD‐SP15 in growth and oxidative‐stress responses of ARPE‐19[J]. Journal of Cellular Biochemistry, 2019,120(2): 1362-1375