RESUMEN
Optic nerve injuries are severely disrupt the structural and functional integrity of the retina, often leading to visual impairment or blindness. Despite the profound impact of these injuries, the molecular mechanisms involved remain poorly understood. In this study, we performed a comprehensive whole-transcriptome analysis of mouse retina samples after optic nerve crush (ONC) to elucidate changes in gene expression and regulatory networks. Transcriptome analysis revealed a variety of molecular alterations, including 256 mRNAs, 530 lncRNAs, and 37 miRNAs, associated with metabolic, inflammatory, signaling, and biosynthetic pathways in the injured retina. The integrated analysis of co-expression and protein-protein interactions identified an active interconnected module comprising 5 co-expressed proteins (Fga, Serpina1a, Hpd, Slc38a4, and Ahsg) associated with the complement and coagulation cascades. Finally, 5 mRNAs (Fga, Serpinala, Hpd, Slc38a4, and Ahsg), 2 miRNAs (miR-671-5p and miR-3057-5p), and 6 lncRNAs (MSTRG. 1830.1, Gm10814, A530013C23Rik, Gm40634, MSTRG.9514.1, A330023F24Rik) were identified by qPCR in the injured retina, and some of them were validated as critical components of a ceRNA network active in 661W and HEK293T cells through dual-luciferase reporter assays. In conclusion, our study provides comprehensive insight into the complex and dynamic biological mechanisms involved in retinal injury responses and highlights promising potential targets to enhance neuroprotection and restore vision.
Asunto(s)
Perfilación de la Expresión Génica , Redes Reguladoras de Genes , Ratones Endogámicos C57BL , Traumatismos del Nervio Óptico , ARN Mensajero , Retina , Animales , Ratones , Traumatismos del Nervio Óptico/metabolismo , Traumatismos del Nervio Óptico/genética , Retina/metabolismo , ARN Mensajero/genética , Modelos Animales de Enfermedad , Transcriptoma , MicroARNs/genética , Regulación de la Expresión Génica/fisiología , Masculino , Humanos , ARN Largo no Codificante/genéticaRESUMEN
Uveal melanoma (UM) is the most frequent primary intraocular malignancy with high metastatic potential and poor prognosis. Macrophages represent one of the most abundant infiltrating immune cells with diverse functions in cancers. However, the cellular heterogeneity and functional diversity of macrophages in UM remain largely unexplored. In this study, we analyzed 63,264 single-cell transcriptomes from 11 UM patients and identified four transcriptionally distinct macrophage subsets (termed MΦ-C1 to MΦ-C4). Among them, we found that MΦ-C4 exhibited relatively low expression of both M1 and M2 signature genes, loss of inflammatory pathways and antigen presentation, instead demonstrating enhanced signaling for proliferation, mitochondrial functions and metabolism. We quantified the infiltration abundance of MΦ-C4 from single-cell and bulk transcriptomes across five cohorts and found that increased MΦ-C4 infiltration was relevant to aggressive behaviors and may serve as an independent prognostic indicator for poor outcomes. We propose a novel subtyping scheme based on macrophages by integrating the transcriptional signatures of MΦ-C4 and machine learning to stratify patients into MΦ-C4-enriched or MΦ-C4-depleted subtypes. These two subtypes showed significantly different clinical outcomes and were validated through bulk RNA sequencing and immunofluorescence assays in both public multicenter cohorts and our in-house cohort. Following further translational investigation, our findings highlight a potential therapeutic strategy of targeting macrophage subsets to control metastatic disease and consistently improve the outcome of patients with UM.
Asunto(s)
Melanoma , Neoplasias de la Úvea , Humanos , Macrófagos/metabolismo , Melanoma/genética , Melanoma/metabolismo , Neoplasias de la Úvea/genética , Neoplasias de la Úvea/metabolismo , Neoplasias de la Úvea/patologíaRESUMEN
Uveal melanoma (UM) is the most common primary intraocular malignancy in adults. However, challenges in early diagnosis, high risk of liver metastasis, and lack of effective targeted therapy lead to poor prognosis and high mortality of UM. Therefore, generating an effective molecular tool for UM diagnosis and targeted treatment is of great significance. In this study, a UM-specific DNA aptamer, PZ-1, was successfully developed, which could specifically distinguish molecular differences between UM cells and noncancerous cells with nanomolar-range affinity and presented excellent recognition ability for UM in vivo and clinical UM tissues. Subsequently, the binding target of PZ-1 on UM cells was identified as JUP (junction plakoglobin) protein, which held great potential as a biomarker and therapeutic target for UM. Meanwhile, the strong stability and internalization capacity of PZ-1 were also determined, and a UM-specific aptamer-guided "nanoship" was engineered to load and selectively release doxorubicin (Dox) to targeted UM cells, with lower toxicity to nontumor cells. Taken together, the UM-specific aptamer PZ-1 could serve as a molecular tool to discover the potential biomarker for UM and to achieve the targeted therapy of UM.
Asunto(s)
Doxorrubicina , Melanoma , Humanos , Línea Celular Tumoral , Biomarcadores , Doxorrubicina/farmacología , Doxorrubicina/uso terapéutico , Melanoma/diagnóstico , Melanoma/tratamiento farmacológico , Melanoma/genéticaRESUMEN
Uveal melanoma (UM) is an aggressive intraocular malignant tumour that is closely related to autophagic dysfunction. We aimed to identify autophagy-related long noncoding RNAs (lncRNAs) to elucidate the molecular mechanism of UM. Here, we show that LINC01278 is a new potential biomarker with clinical prognostic value in UM through bioinformatics analysis. Application of an autophagy inhibitor (3-MA) and an autophagy agonist (MG-132) indicated that LINC01278 can inhibit UM cell proliferation, migration, and invasion by inducing autophagy. A xenograft nude mouse model was used to examine the tumorigenesis of UM cells in vivo. Mechanistically, LINC01278 can inhibit the mTOR signalling pathway to activate autophagy, as shown by experiments with an mTOR agonist (MHY1485) and mTOR inhibitor (rapamycin) treatment. Our findings indicate that LINC01278 functions as a tumour suppressor by inhibiting the mTOR signalling pathway to induce autophagy. Targeting the LINC01278-mTOR axis might be a novel and promising therapeutic approach for UM.
Asunto(s)
Melanoma , ARN Largo no Codificante , Transducción de Señal , Serina-Treonina Quinasas TOR , Neoplasias de la Úvea , Animales , Humanos , Ratones , Autofagia , Línea Celular Tumoral , Proliferación Celular , Transformación Celular Neoplásica , Serina-Treonina Quinasas TOR/metabolismo , ARN Largo no Codificante/genética , Neoplasias de la Úvea/genética , Neoplasias de la Úvea/patología , Melanoma/genética , Melanoma/patologíaRESUMEN
Microglial dysfunction has been identified as a key factor in the pathology of several traumatic and neurodegenerative diseases in the central nervous system. Due to the importance of microglia in various pathological processes, the development of molecular tools to target microglia may be of significance for the clinical diagnosis and treatment of these disorders. In this study, a DNA aptamer, ZH-1c, that binds microglia with high affinity was developed by cell-SELEX and truncated strategies. ZH-1c exhibits promising binding ability under physiological temperatures, high serum stability after being modified, and can be internalized by microglia. Also, the binding target of ZH-1c on microglia was identified as the transmembrane protein CD64, which increased in response to inflammatory stimulation via lipopolysaccharide and interferon-gamma, thus enhancing the affinity of ZH-1c for activated microglia. Based on the above experiments, the DNA aptamer ZH-1c exhibits great potential for the targeting of activated inflammatory microglia and may be suitable as a novel and effective molecular tool for diagnosis and microglia-targeted therapies.
Asunto(s)
Aptámeros de Nucleótidos , Microglía , Aptámeros de Nucleótidos/metabolismo , Sistema Nervioso Central , Interferón gamma , Lipopolisacáridos/farmacología , Lipopolisacáridos/metabolismo , Macrófagos , Microglía/metabolismo , Receptores de IgG/metabolismoRESUMEN
The optic nerve (ON) can get compressed in different diseases. However, the pathological and functional changes occurring in the compressed ON over time under constant compression are still unclear. In the present study, we implanted an artificial tube around the optic nerve of a rabbit to primarily create a clinically relevant persistent compressive optic nerve axonopathy (PCOA). Due to the protuberance on the inner ring of the tube, steady and persistent compressions were maintained. In this model, we investigated the thickness of ganglion cell complex (GCC), retinal ganglion cell (RGC) density, axon density of optic nerve, flash visual evoked potential (FVEP), and anterograde axonal transport at various times in four different groups viz. the no comp, 1/2 comp, 3/4 comp, and crush groups. The GCC thickness, RGC density, and axon density of ON were hierarchically and significantly decreased in 1/2 comp, 3/4 comp, and crush groups. Compared to no comp eyes, the P2 amplitude ratio of FVEP was significantly decreased in 3/4 comp but not in 1/2 comp eyes. Only a portion of the optic nerve lost the ability of anterograde axonal transport in the 1/2 comp group. However, it was evident at 2-wpo and more prominent at 4-wpo in 3/4 comp eyes. This study reveals that the compression only induces the homolateral ON axons impairment and the proportion of the affected axons maintains the same for mild compression for at least three months. Furthermore, an underlying threshold effect highlights that mild compression does not require urgent surgery, while the severe compression warrants immediate surgical intervention.