MicroRNA-708 suppresses the proliferation, migration, and invasion of human retinoblastoma cells by targeting RAP2B, a member of the RAS oncogene family.

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.

Somatostatin signalling promotes the differentiation of rod photoreceptors in human pluripotent stem cell-derived retinal organoid.

OBJECTIVES: Stem cell-derived photoreceptor replacement therapy is a promising strategy for the treatment of retinal degenerative disease. The development of 3D retinal organoids has permitted the production of photoreceptors. However, there is no strategy to enrich a specific photoreceptor subtype due to inadequate knowledge of the molecular mechanism underlying the photoreceptor fate determination. Hence, our aim is to explore the uncharacterized function of somatostatin signalling in human pluripotent stem cell-derived photoreceptor differentiation. MATERIALS AND METHODS: 3D retinal organoids were achieved from human embryonic stem cell. The published single-cell RNA-sequencing datasets of human retinal development were utilized to further investigate the transcriptional regulation of photoreceptor differentiation. The assays of immunofluorescence staining, lentivirus transfection, real-time quantitative polymerase chain reaction and western blotting were performed. RESULTS: We identified that the somatostatin receptor 2 (SSTR2)-mediated signalling was essential for rod photoreceptor differentiation at the precursor stage. The addition of the cognate ligand somatostatin in human 3D retinal organoids promoted rod photoreceptor differentiation and inhibited cone photoreceptor production. Furthermore, we found that the genesis of rod photoreceptors was modulated by endogenous somatostatin specifically secreted by developing retinal ganglion cells. CONCLUSIONS: Our study identified SSTR2 signalling as a novel extrinsic regulator for rod photoreceptor fate determination in photoreceptor precursors, which expands the repertoire of functional signalling pathways in photoreceptor development and sheds light on the optimization of the photoreceptor enrichment strategy.

Vitreous Inflammatory Cytokines and Chemokines, Not Altered After Preoperative Adjunctive Conbercept Injection, but Associated With Early Postoperative Macular Edema in Patients With Proliferative Diabetic Retinopathy.

Purpose: To investigate the influence of preoperative adjunctive anti-VEGF drug (Conbercept) on vitreous inflammatory cytokines and chemokines profiles and whether those cytokines were associated with early macular edema (ME) after surgery for patients with proliferative diabetic retinopathy (PDR). Methods: In this post hoc analysis of the CONCEPT clinical trial, subjects with PDR underwent vitrectomy were included and vitreous samples were collected at the start of vitrectomy. Levels of vitreous VEGF, 17 inflammatory cytokines, and 11 chemokines were measured using Luminex multiplex technology. Subjects were then divided into groups based on with (Pre-IV) or without (No-Pre-IV) preoperative intravitreous injection of Conbercept; with or without early ME after surgery. Results: There was no difference between Pre-IV (13/30) and No-Pre-IV (7/29) concerning the ratio of patients with early ME (p = 0.17). After preoperative intravitreous injection of Conbercept, VEGF level dramatically decreased (p = 0.001), TNF-α (p = 0.002), and IP-10 (p = 0.018) increased in Pre-IV group. In patients with early ME after surgery, however, a number of cytokines increased, including IL-1ß (p = 0.008), IL-2 (p = 0.023), IL-4 (p = 0.030), IL-9 (p = 0.02), IL-10 (p = 0.002), IL-12 (p = 0.001), IL-13 (p = 0.031), IL-17A (p = 0.008), TNF-α (p = 0.012), CXCL9 (p = 0.023), G-CSF (p = 0.019), MCP-1 (p = 0.048), and RANTES (p = 0.016). Conclusion: We found the preoperative adjunctive Conbercept injection has limited influence on the levels of vitreous inflammatory cytokines and chemokines in PDR. The elevated levels of a series of cytokines might be associated with early inflammation after vitrectomy, which may lead to postoperative ME.

IL-17A injury to retinal ganglion cells is mediated by retinal Müller cells in diabetic retinopathy.

Diabetic retinopathy (DR), the most common and serious ocular complication, recently has been perceived as a neurovascular inflammatory disease. However, role of adaptive immune inflammation driven by T lymphocytes in DR is not yet well elucidated. Therefore, this study aimed to clarify the role of interleukin (IL)-17A, a proinflammatory cytokine mainly produced by T lymphocytes, in retinal pathophysiology particularly in retinal neuronal death during DR process. Ins2Akita (Akita) diabetic mice 12 weeks after the onset of diabetes were used as a DR model. IL-17A-deficient diabetic mice were obtained by hybridization of IL-17A-knockout (IL-17A-KO) mouse with Akita mouse. Primarily cultured retinal Müller cells (RMCs) and retinal ganglion cells (RGCs) were treated with IL-17A in high-glucose (HG) condition. A transwell coculture of RGCs and RMCs whose IL-17 receptor A (IL-17RA) gene had been silenced with IL-17RA-shRNA was exposed to IL-17A in HG condition and the cocultured RGCs were assessed on their survival. Diabetic mice manifested increased retinal microvascular lesions, RMC activation and dysfunction, as well as RGC apoptosis. IL-17A-KO diabetic mice showed reduced retinal microvascular impairments, RMC abnormalities, and RGC apoptosis compared with diabetic mice. RMCs expressed IL-17RA. IL-17A exacerbated HG-induced RMC activation and dysfunction in vitro and silencing IL-17RA gene in RMCs abolished the IL-17A deleterious effects. In contrast, RGCs did not express IL-17RA and IL-17A did not further alter HG-induced RGC death. Notably, IL-17A aggravated HG-induced RGC death in the presence of intact RMCs but not in the presence of RMCs in which IL-17RA gene had been knocked down. These findings establish that IL-17A is actively involved in DR pathophysiology and particularly by RMC mediation it promotes RGC death. Collectively, we propose that antagonizing IL-17RA on RMCs may prevent retinal neuronal death and thereby slow down DR progression.

Obacunone protects retinal pigment epithelium cells from ultra-violet radiation-induced oxidative injury.

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.

PP2A inhibition by LB-100 protects retinal pigment epithelium cells from UV radiation via activation of AMPK signaling.

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-19 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.

PF-06409577 activates AMPK signaling to protect retinal pigment epithelium cells from UV radiation.

Ultra-violet (UV) radiation (UVR) to human retinas induces oxidative injury to the resident retinal pigment epithelium (RPE) cells. PF-06409577 a novel, potent and direct AMP-activated protein kinase (AMPK) activator. In ARPE-19 cells and primary murine RPE cells, PF-06409577 significantly inhibited UVR-induced viability reduction, cell death and apoptosis. PF-06409577 activated AMPK signaling in RPE cells by increasing AMPKα1-acetyl-CoA carboxylase phosphorylation and AMPK activity. AMPK inhibition, by AMPKα1-shRNA, -CRISPR/Cas9 knockout or -T172A dominant negative mutation, almost abolished PF-06409577-induced RPE cytoprotection against UVR. PF-06409577 enhanced nicotinamide adenine dinucleotide phosphate (NADPH) activity and expression levels of Nrf2-dependent genes in RPE cells. Furthermore, UVR-induced reactive oxygen species (ROS) production, lipid peroxidation and DNA damage were largely inhibited by the AMPK activator. In summary, PF-06409577 inhibits UVR-induced oxidative stress and RPE cell death by activating AMPK signaling.

MIND4-17 protects retinal pigment epithelium cells and retinal ganglion cells from UV.

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.