ABSTRACT
Retinoblastoma generally affects children and causes permanent vision failure or even death. MicroRNAs (miRs) have recently gained much attention during recent years. The miR-708 acts as a tumor suppressor in several human cancers, but the former has not been functionally characterized in human retinoblastoma. The present study was designed to investigate the role of miR-708 in human retinoblastoma. The results showed that miR-708 is significantly (P<0.05) downregulated in retinoblastoma cell lines. MiR-708 overexpression significantly (P<0.05) inhibited retinoblastoma cell growth and proliferation by inducing apoptosis. Furthermore, retinoblastoma cells overexpressing miR-708 exhibited a markedly lower migratory rate and invasiveness compared to negative control cells. The bioinformatics and dual luciferase assay revealed a RAS oncogene family protein, RAP2B, which acts as the regulatory target and functional mediator of the molecular role of miR-708 in retinoblastoma. Together, the present study revealed the tumor suppressor role of miR-708 and pointed to the therapeutic implications of miR-708/RAP2B in the treatment of retinoblastoma.
Subject(s)
MicroRNAs , Retinal Neoplasms , Retinoblastoma , rap GTP-Binding Proteins , Child , Humans , Apoptosis/genetics , Cell Line, Tumor , Cell Movement/genetics , Cell Proliferation/genetics , Gene Expression Regulation, Neoplastic , Genes, ras , MicroRNAs/metabolism , Neoplasm Invasiveness/genetics , rap GTP-Binding Proteins/genetics , rap GTP-Binding Proteins/metabolism , Retinal Neoplasms/genetics , Retinal Neoplasms/pathology , Retinoblastoma/genetics , Retinoblastoma/pathologyABSTRACT
Ultra-violet (UV) radiation (UVR) causes significant oxidative injury to retinal pigment epithelium (RPE) cells. Obacunone is a highly oxygenated triterpenoid limonoid compound with various pharmacological properties. Its potential effect in RPE cells has not been studied thus far. Here in ARPE-19 cells and primary murine RPE cells, obacunone potently inhibited UVR-induced reactive oxygen species accumulation, mitochondrial depolarization, lipid peroxidation and single strand DNA accumulation. UVR-induced RPE cell death and apoptosis were largely alleviated by obacunone. Obacunone activated Nrf2 signaling cascade in RPE cells, causing Keap1-Nrf2 disassociation, Nrf2 protein stabilization and nuclear translocation. It promoted transcription and expression of antioxidant responsive element-dependent genes. Nrf2 silencing or CRISPR/Cas9-induced Nrf2 knockout almost reversed obacunone-induced RPE cytoprotection against UVR. Forced activation of Nrf2 cascade, by Keap1 knockout, similarly protected RPE cells from UVR. Importantly, obacunone failed to offer further RPE cytoprotection against UVR in Keap1-knockout cells. In vivo, intravitreal injection of obacunone largely inhibited light-induced retinal damage. Collectively, obacunone protects RPE cells from UVR-induced oxidative injury through activation of Nrf2 signaling cascade.
Subject(s)
Benzoxepins/pharmacology , Limonins/pharmacology , Macular Degeneration/drug therapy , Oxidative Stress/drug effects , Retinal Pigment Epithelium/drug effects , Ultraviolet Rays/adverse effects , Animals , Apoptosis/drug effects , Apoptosis/radiation effects , Benzoxepins/therapeutic use , Cell Line , Cell Survival/drug effects , Cell Survival/radiation effects , DNA, Single-Stranded/drug effects , DNA, Single-Stranded/radiation effects , Disease Models, Animal , Drug Evaluation, Preclinical , Gene Expression Regulation/drug effects , Gene Knockout Techniques , Humans , Intravitreal Injections , Kelch-Like ECH-Associated Protein 1/metabolism , Limonins/therapeutic use , Lipid Peroxidation/drug effects , Lipid Peroxidation/radiation effects , Macular Degeneration/etiology , Macular Degeneration/pathology , Mice , Mitochondrial Membranes/drug effects , NF-E2-Related Factor 2/genetics , NF-E2-Related Factor 2/metabolism , Oxidative Stress/genetics , Oxidative Stress/radiation effects , Primary Cell Culture , Reactive Oxygen Species/metabolism , Retinal Pigment Epithelium/cytology , Retinal Pigment Epithelium/pathology , Retinal Pigment Epithelium/radiation effects , Signal Transduction/drug effects , Signal Transduction/genetics , Signal Transduction/radiation effectsABSTRACT
AMP-activated protein kinase (AMPK) signaling activation can inhibit Ultra-violet (UV) radiation (UVR)-induced retinal pigment epithelium (RPE) cell injuries. LB-100 is a novel inhibitor of protein phosphatase 2A (PP2A), the AMPKα1 phosphatase. Here, our results demonstrated that LB-100 significantly inhibited UVR-induced viability reduction, cell death and apoptosis in established ARPE-19â¯cells and primary murine RPE cells. LB-100 activated AMPK, nicotinamide adenine dinucleotide phosphate (NADPH) and Nrf2 (NF-E2-related factor 2) signalings, inhibiting UVR-induced oxidative injuries and DNA damage in RPE cells. Conversely, AMPK inhibition, by AMPKα1-shRNA, -CRISPR/Cas9 knockout or -T172A mutation, almost blocked LB-100-induced RPE cytoprotection against UVR. Importantly, CRISPR/Cas9-mediated PP2A knockout mimicked and nullified LB-100-induced anti-UVR activity in RPE cells. Collectively, these results show that PP2A inhibition by LB-100 protects RPE cells from UVR via activation of AMPK signaling.
Subject(s)
AMP-Activated Protein Kinases/genetics , Bridged Bicyclo Compounds, Heterocyclic/pharmacology , Enzyme Inhibitors/pharmacology , Epithelial Cells/drug effects , Piperazines/pharmacology , Protein Phosphatase 2/genetics , Sunscreening Agents/pharmacology , AMP-Activated Protein Kinases/metabolism , Animals , CRISPR-Cas Systems , Cell Line , Cell Survival/drug effects , Cell Survival/radiation effects , Enzyme Activation , Epithelial Cells/cytology , Epithelial Cells/metabolism , Epithelial Cells/radiation effects , Gene Editing , Gene Expression Regulation , Humans , Mice , NADP/metabolism , Primary Cell Culture , Protein Phosphatase 2/antagonists & inhibitors , Protein Phosphatase 2/metabolism , RNA, Small Interfering/genetics , RNA, Small Interfering/metabolism , Reactive Oxygen Species/metabolism , Retinal Pigment Epithelium/cytology , Retinal Pigment Epithelium/drug effects , Retinal Pigment Epithelium/metabolism , Retinal Pigment Epithelium/radiation effects , Signal Transduction , Ultraviolet Rays/adverse effectsABSTRACT
Nrf2 activation would efficiently protect retinal cells from UV radiation (UVR). Recent studies have developed a Nrf2-targeting thiazole-containing compound MIND4-17, which activates Nrf2 through blocking its association with Keap1. In the current study, we demonstrated that pretreatment with MIND4-17 efficiently protected retinal pigment epithelium (RPE) cells (RPEs) and retinal ganglion cells (RGCs) from UVR. UVR-induced apoptosis in the retinal cells was also largely attenuated by MIND4-17 pretreatment. MIND4-17 presumably separated Nrf2 from Keap1, allowing its stabilization and accumulation in retinal cells, which then translocated to cell nuclei and promoted transcription of ARE-dependent anti-oxidant genes, including HO1, NQO1 and GCLM. Significantly, shRNA-mediated knockdown of Nrf2 almost completely abolished MIND4-17-induced cytoprotection against UVR. Further studies showed that MIND4-17 largely ameliorated UVR-induced ROS production, lipid peroxidation and DNA damages in RPEs and RGCs. Together, MIND4-17 protects retinal cells from UVR by activating Nrf2 signaling.
ABSTRACT
Sorghum is a food and feed cereal crop adapted to heat and drought and a staple for 500 million of the world's poorest people. Its small diploid genome and phenotypic diversity make it an ideal C4 grass model as a complement to C3 rice. Here we present high coverage (16-45 × ) resequenced genomes of 44 sorghum lines representing the primary gene pool and spanning dimensions of geographic origin, end-use and taxonomic group. We also report the first resequenced genome of S. propinquum, identifying 8 M high-quality SNPs, 1.9 M indels and specific gene loss and gain events in S. bicolor. We observe strong racial structure and a complex domestication history involving at least two distinct domestication events. These assembled genomes enable the leveraging of existing cereal functional genomics data against the novel diversity available in sorghum, providing an unmatched resource for the genetic improvement of sorghum and other grass species.
