RESUMEN

Wilms tumor, the most common pediatric kidney cancer, resembles embryonic renal progenitors. Currently, there are no ways to therapeutically target Wilms tumor driver mutations, such as in the microRNA processing gene DROSHA. In this study, we used a "multiomics" approach to define the effects of DROSHA mutation in Wilms tumor. We categorized Wilms tumor mutations into four mutational subclasses with unique transcriptional effects: microRNA processing, MYCN activation, chromatin remodeling, and kidney developmental factors. In particular, we find that DROSHA mutations are correlated with de-repressing microRNA target genes that regulate differentiation and proliferation and a self-renewing, mesenchymal state. We model these findings by inhibiting DROSHA expression in a Wilms tumor cell line, which led to upregulation of the cell cycle regulator cyclin D2 (CCND2). Furthermore, we observed that DROSHA mutations in Wilms tumor and DROSHA silencing in vitro were associated with a mesenchymal state with aberrations in redox metabolism. Accordingly, we demonstrate that Wilms tumor cells lacking microRNAs are sensitized to ferroptotic cell death through inhibition of glutathione peroxidase 4, the enzyme that detoxifies lipid peroxides. Implications: This study reveals genotype-transcriptome relationships in Wilms tumor and points to ferroptosis as a potentially therapeutic vulnerability in one subset of Wilms tumor.

Asunto(s)

Neoplasias Renales , Ribonucleasa III , Tumor de Wilms , Humanos , Tumor de Wilms/genética , Tumor de Wilms/patología , Tumor de Wilms/metabolismo , Ribonucleasa III/genética , Ribonucleasa III/metabolismo , Neoplasias Renales/genética , Neoplasias Renales/patología , Neoplasias Renales/metabolismo , Regulación Neoplásica de la Expresión Génica , Mutación , MicroARNs/genética , Línea Celular Tumoral

RESUMEN

Patient-derived xenografts (PDX) remain valuable models for understanding the biology and for developing novel therapeutics. To expand current PDX models of childhood leukemia, we have developed new PDX models from Hispanic patients, a subgroup with a poorer overall outcome. Of 117 primary leukemia samples obtained, successful engraftment and serial passage in mice were achieved in 82 samples (70%). Hispanic patient samples engrafted at a rate (51/73, 70%) that was similar to non-Hispanic patient samples (31/45, 70%). With a new algorithm to remove mouse contamination in multi-omics datasets including methylation data, we found PDX models faithfully reflected somatic mutations, copy-number alterations, RNA expression, gene fusions, whole-genome methylation patterns, and immunophenotypes found in primary tumor (PT) samples in the first 50 reported here. This cohort of characterized PDX childhood leukemias represents a valuable resource in that germline DNA sequencing has allowed the unambiguous determination of somatic mutations in both PT and PDX.

RESUMEN

PURPOSE: Osteosarcoma research advancement requires enhanced data integration across different modalities and sources. Current osteosarcoma research, encompassing clinical, genomic, protein, and tissue imaging data, is hindered by the siloed landscape of data generation and storage. MATERIALS AND METHODS: Clinical, molecular profiling, and tissue imaging data for 573 patients with pediatric osteosarcoma were collected from four public and institutional sources. A common data model incorporating standardized terminology was created to facilitate the transformation, integration, and load of source data into a relational database. On the basis of this database, a data commons accompanied by a user-friendly web portal was developed, enabling various data exploration and analytics functions. RESULTS: The Osteosarcoma Explorer (OSE) was released to the public in 2021. Leveraging a comprehensive and harmonized data set on the backend, the OSE offers a wide range of functions, including Cohort Discovery, Patient Dashboard, Image Visualization, and Online Analysis. Since its initial release, the OSE has experienced an increasing utilization by the osteosarcoma research community and provided solid, continuous user support. To our knowledge, the OSE is the largest (N = 573) and most comprehensive research data commons for pediatric osteosarcoma, a rare disease. This project demonstrates an effective framework for data integration and data commons development that can be readily applied to other projects sharing similar goals. CONCLUSION: The OSE offers an online exploration and analysis platform for integrated clinical, molecular profiling, and tissue imaging data of osteosarcoma. Its underlying data model, database, and web framework support continuous expansion onto new data modalities and sources.

Asunto(s)

Manejo de Datos , Osteosarcoma , Niño , Humanos , Bases de Datos Factuales , Genómica , Osteosarcoma/diagnóstico por imagen , Osteosarcoma/genética

RESUMEN

Wilms tumor, the most common kidney cancer in pediatrics, arises from embryonic renal progenitors. Although many patients are cured with multimodal therapy, outcomes remain poor for those with high-risk features. Recent sequencing efforts have provided few biological or clinically actionable insights. Here, we performed DNA and RNA sequencing on 94 Wilms tumors to understand how Wilms tumor mutations transform the transcriptome to arrest differentiation and drive proliferation. We show that most Wilms tumor mutations fall into four classes, each with unique transcriptional signatures: microRNA processing, MYCN activation, chromatin remodeling, and kidney development. In particular, the microRNA processing enzyme DROSHA is one of the most commonly mutated genes in Wilms tumor. We show that DROSHA mutations impair pri-microRNA cleavage, de-repress microRNA target genes, halt differentiation, and overexpress cyclin D2 (CCND2). Several mutational classes converge to drive CCND2 overexpression, which could render them susceptible to cell-cycle inhibitors.

RESUMEN

Low-grade oncocytic tumor (LOT) of the kidney is a recently described entity with poorly understood pathogenesis. Using next-generation sequencing (NGS) and complementary approaches, we provide insight into its biology. We describe 22 LOT corresponding to 7 patients presenting with a median age of 75 years (range 63-86 years) and male to female ratio 2:5. All 22 tumors demonstrated prototypical microscopic features. Tumors were well-circumscribed and solid. They were composed of sheets of tumor cells in compact nests. Tumor cells had eosinophilic cytoplasm, round to oval nuclei (without nuclear membrane irregularities), focal subtle perinuclear halos, and occasional binucleation. Sharply delineated edematous stromal islands were often observed. Tumor cells were positive for PAX8, negative for CD117, and exhibited diffuse and strong cytokeratin-7 expression. Six patients presented with pT1 tumors. At a median follow-up of 29 months, four patients were alive without recurrence (three patients had died from unrelated causes). All tumors were originally classified as chromophobe renal cell carcinoma, eosinophilic variant (chRCC-eo). While none of the patients presented with known syndromic features, one patient with multiple bilateral LOTs was subsequently found to have a likely pathogenic germline TSC1 mutation. Somatic, likely activating, mutations in MTOR and RHEB were identified in all other evaluable LOTs. As assessed by phospho-S6 and phospho-4E-BP1, mTOR complex 1 (mTORC1) was activated across all cases but to different extent. MTOR mutant LOT exhibited lower levels of mTORC1 activation, possibly related to mTORC1 dimerization and the preservation of a wild-type MTOR copy (retained chromosome 1). Supporting its distinction from related entities, gene expression analyses showed that LOT clustered separately from classic chRCC, chRCC-eo, and RO. In summary, converging mTORC1 pathway mutations, mTORC1 complex activation, and a distinctive gene expression signature along with characteristic phenotypic features support LOT designation as a distinct entity with both syndromic and non-syndromic cases associated with an indolent course.

Asunto(s)

Adenoma Oxifílico , Carcinoma de Células Renales , Neoplasias Renales , Adenoma Oxifílico/genética , Adenoma Oxifílico/patología , Anciano , Anciano de 80 o más Años , Biomarcadores de Tumor/análisis , Biomarcadores de Tumor/genética , Carcinoma de Células Renales/genética , Carcinoma de Células Renales/patología , Femenino , Células Germinativas/química , Células Germinativas/patología , Humanos , Riñón/patología , Neoplasias Renales/genética , Neoplasias Renales/patología , Masculino , Persona de Mediana Edad , Mutación , Serina-Treonina Quinasas TOR/genética

RESUMEN

Metastasis is the principal cause of cancer related deaths. Tumor invasion is essential for metastatic spread. However, determinants of invasion are poorly understood. We addressed this knowledge gap by leveraging a unique attribute of kidney cancer. Renal tumors invade into large vessels forming tumor thrombi (TT) that migrate extending sometimes into the heart. Over a decade, we prospectively enrolled 83 ethnically-diverse patients undergoing surgical resection for grossly invasive tumors at UT Southwestern Kidney Cancer Program. In this study, we perform comprehensive histological analyses, integrate multi-region genomic studies, generate in vivo models, and execute functional studies to define tumor invasion and metastatic competence. We find that invasion is not always associated with the most aggressive clone. Driven by immediate early genes, invasion appears to be an opportunistic trait attained by subclones with diverse oncogenomic status in geospatial proximity to vasculature. We show that not all invasive tumors metastasize and identify determinants of metastatic competency. TT associated with metastases are characterized by higher grade, mTOR activation and a particular immune contexture. Moreover, TT grade is a better predictor of metastasis than overall tumor grade, which may have implications for clinical practice.

Asunto(s)

Carcinoma de Células Renales/secundario , Regulación Neoplásica de la Expresión Génica , Neoplasias Renales/patología , Trombosis/genética , Anciano , Animales , Carcinoma de Células Renales/complicaciones , Carcinoma de Células Renales/diagnóstico , Carcinoma de Células Renales/genética , Línea Celular Tumoral , Variaciones en el Número de Copia de ADN , Femenino , Humanos , Riñón/irrigación sanguínea , Riñón/patología , Neoplasias Renales/complicaciones , Neoplasias Renales/diagnóstico , Neoplasias Renales/genética , Masculino , Ratones , Persona de Mediana Edad , Clasificación del Tumor , Invasividad Neoplásica/genética , Invasividad Neoplásica/patología , Estudios Prospectivos , RNA-Seq , Factores de Riesgo , Trombosis/patología , Secuenciación del Exoma , Ensayos Antitumor por Modelo de Xenoinjerto

RESUMEN

Germ cell tumors (GCTs) are considered a rare disease but are the most common solid tumors in adolescents and young adults, accounting for 15% of all malignancies in this age group. The rarity of GCTs in some groups, particularly children, has impeded progress in treatment and biologic understanding. The most effective GCT research will result from the interrogation of data sets from historical and prospective trials across institutions. However, inconsistent use of terminology among groups, different sample-labeling rules, and lack of data standards have hampered researchers' efforts in data sharing and across-study validation. To overcome the low interoperability of data and facilitate future clinical trials, we worked with the Malignant Germ Cell International Consortium (MaGIC) and developed a GCT clinical data model as a uniform standard to curate and harmonize GCT data sets. This data model will also be the standard for prospective data collection in future trials. Using the GCT data model, we developed a GCT data commons with data sets from both MaGIC and public domains as an integrated research platform. The commons supports functions, such as data query, management, sharing, visualization, and analysis of the harmonized data, as well as patient cohort discovery. This GCT data commons will facilitate future collaborative research to advance the biologic understanding and treatment of GCTs. Moreover, the framework of the GCT data model and data commons will provide insights for other rare disease research communities into developing similar collaborative research platforms.

Asunto(s)

Neoplasias de Células Germinales y Embrionarias , Neoplasias , Adolescente , Estudios de Cohortes , Humanos , Difusión de la Información , Neoplasias de Células Germinales y Embrionarias/epidemiología , Neoplasias de Células Germinales y Embrionarias/terapia

RESUMEN

Lack of responsiveness to checkpoint inhibitors is a central problem in the modern era of cancer immunotherapy. Tumor neoantigens are critical targets of the host antitumor immune response, and their presence correlates with the efficacy of immunotherapy treatment. Many studies involving assessment of tumor neoantigens principally focus on total neoantigen load, which simplistically treats all neoantigens equally. Neoantigen load has been linked with treatment response and prognosis in some studies but not others. We developed a Cauchy-Schwarz index of Neoantigens (CSiN) score to better account for the degree of concentration of immunogenic neoantigens in truncal mutations. Unlike total neoantigen load determinations, CSiN incorporates the effect of both clonality and MHC binding affinity of neoantigens when characterizing tumor neoantigen profiles. By analyzing the clinical responses in 501 treated patients with cancer (with most receiving checkpoint inhibitors) and the overall survival of 1978 patients with cancer at baseline, we showed that CSiN scores predict treatment response to checkpoint inhibitors and prognosis in patients with melanoma, lung cancer, and kidney cancer. CSiN score substantially outperformed prior genetics-based prediction methods of responsiveness and fills an important gap in research involving assessment of tumor neoantigen burden.

Asunto(s)

Antígenos de Neoplasias/inmunología , Células Clonales/inmunología , Células Clonales/patología , Inmunoterapia , Neoplasias/patología , Neoplasias/terapia , Anciano , Antígenos de Neoplasias/genética , Estudios de Cohortes , Femenino , Perfilación de la Expresión Génica , Humanos , Neoplasias Renales/genética , Neoplasias Renales/inmunología , Neoplasias Renales/patología , Neoplasias Renales/terapia , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/inmunología , Neoplasias Pulmonares/patología , Neoplasias Pulmonares/terapia , Masculino , Melanoma/diagnóstico , Melanoma/genética , Melanoma/inmunología , Melanoma/patología , Mutación , Neoplasias/inmunología , Resultado del Tratamiento

RESUMEN

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

RESUMEN

We recently developed a novel computational algorithm that incorporates Bayesian methodology to identify rhabdomyosarcoma disease genes whose expression level correlates with copy-number variations, and we identified PLAG1 as a candidate oncogenic driver. Although PLAG1 has been shown to contribute to other type of cancers, its role in rhabdomyosarcoma has not been elucidated. We observed that PLAG1 mRNA is highly expressed in rhabdomyosarcoma and is associated with PLAG1 gene copy-number gain. Knockdown of PLAG1 dramatically decreased cell accumulation and induced apoptosis in rhabdomyosarcoma cells, whereas its ectopic expression increased cell accumulation in vitro and as a xenograft and promoted G1 to S-phase cell-cycle progression. We found that PLAG1 regulates IGF2 expression and influences AKT and MAPK pathways in rhabdomyosarcoma, and IGF2 partially rescues cell death triggered by PLAG1 knockdown. The expression level of PLAG1 correlated with the IC50 of rhabdomyosarcoma cells to BMS754807, an IGF receptor inhibitor. IMPLICATIONS: Our data demonstrate that PLAG1 contributes to proliferation and survival of rhabdomyosarcoma cells at least partially by inducing IGF2, and this new understanding may have the potential for clinical translation.

Asunto(s)

Proteínas de Unión al ADN/metabolismo , Rabdomiosarcoma/genética , Animales , Apoptosis/fisiología , Teorema de Bayes , Línea Celular Tumoral , Proliferación Celular/fisiología , Proteínas de Unión al ADN/biosíntesis , Proteínas de Unión al ADN/genética , Dosificación de Gen , Xenoinjertos , Humanos , Ratones , Ratones Endogámicos NOD , Ratones SCID , ARN Mensajero/genética , ARN Mensajero/metabolismo , Rabdomiosarcoma/metabolismo , Rabdomiosarcoma/patología , Análisis de Supervivencia

RESUMEN

BACKGROUND: Clear cell renal cell carcinoma (ccRCC) is a particularly challenging tumor type because of its extensive phenotypic variability as well as intra-tumoral heterogeneity (ITH). Clinically, this complexity has been reduced to a handful of pathological variables such as stage, grade and necrosis, but these variables fail to capture the breadth of the disease. How different phenotypes affect patient prognosis and influence therapeutic response is poorly understood. Extensive ITH illustrates remarkable plasticity, providing a framework to study tumor evolution. While multiregional genomic analyses have shown evolution from an ancient clone that acquires metastatic competency over time, these studies have been conducted agnostic to morphological cues and phenotypic plasticity. METHODS: We established a systematic ontology of ccRCC phenotypic variability by developing a multi-scale framework along three fundamental axes: tumor architecture, cytology and the microenvironment. We defined 33 parameters, which we comprehensively evaluated in 549 consecutive ccRCCs retrospectively. We systematically evaluated the impact of each parameter on patient outcomes, and assessed their contribution through multivariate analyses. We measured therapeutic impact in the context of anti-angiogenic therapies. We applied dimensionality reduction by t-distributed stochastic neighbor embedding (t-SNE) algorithms to tumor architectures for the study of tumor evolution superimposing tumor size and grade vectors. Evolutionary models were refined through empirical analyses of directed evolution of tumor intravascular extensions, and metastatic competency (as determined by tumor reconstitution in a heterologous host). FINDINGS: We discovered several novel ccRCC phenotypes, developed an integrated taxonomy, and identified features that improve current prognostic models. We identified a subset of ccRCCs refractory to anti-angiogenic therapies. We developed a model of tumor evolution, which revealed converging evolutionary trajectories into an aggressive type. INTERPRETATION: This work serves as a paradigm for deconvoluting tumor complexity and illustrates how morphological analyses can improve our understanding of ccRCC pleiotropy. We identified several subtypes associated with aggressive biology, and differential response to targeted therapies. By analyzing patterns of spatial and temporal co-occurrence, intravascular tumor extensions and metastatic competency, we were able to identify distinct trajectories of convergent phenotypic evolution.

Asunto(s)

Carcinoma de Células Renales/clasificación , Carcinoma de Células Renales/patología , Neoplasias Renales/clasificación , Neoplasias Renales/patología , Adulto , Anciano , Anciano de 80 o más Años , Inhibidores de la Angiogénesis/farmacología , Inhibidores de la Angiogénesis/uso terapéutico , Animales , Carcinoma de Células Renales/irrigación sanguínea , Carcinoma de Células Renales/tratamiento farmacológico , Supervivencia sin Enfermedad , Femenino , Heterogeneidad Genética , Humanos , Neoplasias Renales/irrigación sanguínea , Neoplasias Renales/tratamiento farmacológico , Masculino , Ratones Endogámicos NOD , Ratones SCID , Análisis Multivariante , Invasividad Neoplásica , Estadificación de Neoplasias , Neovascularización Patológica/patología , Fenotipo , Pronóstico , Factores de Riesgo , Procesos Estocásticos , Microambiente Tumoral/efectos de los fármacos , Ensayos Antitumor por Modelo de Xenoinjerto , Adulto Joven

RESUMEN

Disruption of the CDKN2A (INK4A/ARF) and B (INK4B) genes, which encode three function-independent tumor suppressors, is one of the most common events in human cancer. Because their relative importance in tumor prevention appears to be species- and context-specific, studying their regulation can shed light on mechanisms by which they are bypassed in malignant transformation. We previously unveiled a new pathway in which TGFß selectively induces Arf at mouse Cdkn2a in eye development and cultured fibroblasts. As TGFß signaling is often derailed in cancer development or progression, we investigated its control of CDKN2A/B in human cancer. Computational analyses of sequencing and array data from nearly 11,000 patients with cancer in TCGA showed discordant expression of ARF and INK4A in most cancer subtypes, with gene copy-number loss and promoter methylation involved in only a subset. Using HeLa cells as a model, we found that exogenous TGFß induced ARF mRNA and protein, and ARF knockdown limited TGFß-mediated growth suppression. TGFß-mediated ARF mRNA induction required SMAD2/3, p38MAPK, and SP1, and ARF mRNA was induced without added RNAPII recruitment. Chromatin immunoprecipitation unveiled a remote enhancer element engaged by TGFß by a mechanism that partially depended on p38MAPK. CRISPR-based editing of this enhancer limited induction of ARF and INK4B by TGFß, but not by oncogenic RAS. IMPLICATIONS: Our findings reveal new molecular mechanisms by which CDKN2A/B regulation is coupled to external cues, and those findings represent entry points to further explore pharmacologic strategies to restore their expression in cancer.

Asunto(s)

Inhibidor p15 de las Quinasas Dependientes de la Ciclina/genética , Inhibidor p16 de la Quinasa Dependiente de Ciclina/genética , Inhibidor p16 de la Quinasa Dependiente de Ciclina/metabolismo , Elementos de Facilitación Genéticos , Neoplasias/genética , Factor de Crecimiento Transformador beta/farmacología , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Inhibidor p15 de las Quinasas Dependientes de la Ciclina/metabolismo , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Técnicas de Silenciamiento del Gen , Células HeLa , Humanos , Modelos Biológicos , Neoplasias/metabolismo , Transducción de Señal/efectos de los fármacos , Regulación hacia Arriba

RESUMEN

In an effort to identify human endothelial cell (EC)-enriched lncRNAs,~500 lncRNAs were shown to be highly restricted in primary human ECs. Among them, lncEGFL7OS, located in the opposite strand of the EGFL7/miR-126 gene, is regulated by ETS factors through a bidirectional promoter in ECs. It is enriched in highly vascularized human tissues, and upregulated in the hearts of dilated cardiomyopathy patients. LncEGFL7OS silencing impairs angiogenesis as shown by EC/fibroblast co-culture, in vitro/in vivo and ex vivo human choroid sprouting angiogenesis assays, while lncEGFL7OS overexpression has the opposite function. Mechanistically, lncEGFL7OS is required for MAPK and AKT pathway activation by regulating EGFL7/miR-126 expression. MAX protein was identified as a lncEGFL7OS-interacting protein that functions to regulate histone acetylation in the EGFL7/miR-126 promoter/enhancer. CRISPR-mediated targeting of EGLF7/miR-126/lncEGFL7OS locus inhibits angiogenesis, inciting therapeutic potential of targeting this locus. Our study establishes lncEGFL7OS as a human/primate-specific EC-restricted lncRNA critical for human angiogenesis.

Asunto(s)

Factores de Transcripción Básicos con Cremalleras de Leucinas y Motivos Hélice-Asa-Hélice/metabolismo , Proteínas de Unión al Calcio/genética , Familia de Proteínas EGF/genética , Sitios Genéticos , MicroARNs/genética , ARN Largo no Codificante/metabolismo , Animales , Proteínas de Unión al Calcio/metabolismo , Cardiomiopatía Dilatada/genética , Movimiento Celular/genética , Proliferación Celular/genética , Familia de Proteínas EGF/metabolismo , Femenino , Regulación de la Expresión Génica , Células Endoteliales de la Vena Umbilical Humana/citología , Células Endoteliales de la Vena Umbilical Humana/metabolismo , Humanos , Ratones Endogámicos BALB C , MicroARNs/metabolismo , Modelos Biológicos , Neovascularización Fisiológica/genética , Regiones Promotoras Genéticas , ARN Largo no Codificante/genética

RESUMEN

We constructed a lung cancer-specific database housing expression data and clinical data from over 6700 patients in 56 studies. Expression data from 23 genome-wide platforms were carefully processed and quality controlled, whereas clinical data were standardized and rigorously curated. Empowered by this lung cancer database, we created an open access web resource-the Lung Cancer Explorer (LCE), which enables researchers and clinicians to explore these data and perform analyses. Users can perform meta-analyses on LCE to gain a quick overview of the results on tumor vs non-malignant tissue (normal) differential gene expression and expression-survival association. Individual dataset-based survival analysis, comparative analysis, and correlation analysis are also provided with flexible options to allow for customized analyses from the user.

Asunto(s)

Expresión Génica/genética , Neoplasias Pulmonares/genética , Perfilación de la Expresión Génica/métodos , Estudio de Asociación del Genoma Completo/métodos , Genómica/métodos , Humanos , Análisis de Supervivencia

RESUMEN

MicroRNA miR-126 has been shown to be required for proper angiogenesis in several models. However, its expression, regulation and function in the mouse choroid remain unclear. Our previous data has shown that miR-126 expression is enriched in the endothelial cells (ECs) of the mouse choroid. Here we report that a 5.5 kb Egfl7/miR-126 promoter drives the expression of miR-126 in the choroid ECs during choroidal vascular development. The expression of miR-126 in the ECs is regulated by flow stress likely through Krüppel-like transcriptional factors. miR-126-/- mice show mildly delayed choroidal vascular development, but adult knockout mice develop periphery choroidal vascular lesions. This study suggests that miR-126 is largely dispensable for mouse choroidal development but required for maintaining choroidal vasculature integrity.

Asunto(s)

Coroides/irrigación sanguínea , Coroides/embriología , Células Endoteliales/metabolismo , Regulación del Desarrollo de la Expresión Génica/fisiología , MicroARNs/genética , Neovascularización Fisiológica/genética , Animales , Proteínas de Unión al Calcio , Células Cultivadas , Familia de Proteínas EGF , Angiografía con Fluoresceína , Células Endoteliales de la Vena Umbilical Humana , Humanos , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Plásmidos , Proteínas/genética , Reacción en Cadena en Tiempo Real de la Polimerasa

RESUMEN

The in vivo function of microRNAs (miRs) in diabetic retinopathy (DR) and age-related macular degeneration (AMD) remains unclear. We report here that let-7 family members are expressed in retinal and choroidal endothelial cells (ECs). In ECs, overexpression of let-7 by adenovirus represses EC proliferation, migration, and networking in vitro, whereas inhibition of the let-7 family with a locked nucleic acid (LNA)-anti-miR has the opposite effect. Mechanistically, silencing of the let-7 target HMGA2 gene mimics the phenotype of let-7 overexpression in ECs. let-7 transgenic (let-7-Tg) mice show features of nonproliferative DR, including tortuous retinal vessels and defective pericyte coverage. However, these mice develop significantly less choroidal neovascularization (CNV) compared to wild-type controls after laser injury. Consistently, silencing of let-7 in the eye increased laser-induced CNV in wild-type mice. Together, our data establish a causative role of let-7 in nonproliferative diabetic retinopathy and a repressive function of let-7 in pathological angiogenesis, suggesting distinct implications of let-7 in the pathogenesis of DR and AMD.

RESUMEN

Glaucoma is a major cause of irreversible blindness worldwide. Elevated intraocular pressure (IOP), which causes optic nerve damage and retinal ganglion cell death, is the primary risk factor for blindness in glaucoma patients. IOP is controlled by the balance between aqueous humor secretion from the ciliary body (CB) and its drainage through the trabecular meshwork (TM). How microRNAs (miRs) regulate IOP and glaucoma in vivo is largely unknown. Here we show that miR-143 and miR-145 expression is enriched in the smooth muscle and trabecular meshwork in the eye. Targeted deletion of miR-143/145 in mice results in significantly reduced IOP, consistent with an ~2-fold increase in outflow facilities. However, aqueous humor production in the same mice appears to be normal based on a microbeads-induced glaucoma model. Mechanistically, we found that miR-143/145 regulates actin dynamics and the contractility of TM cells, consistent with its regulation of actin-related protein complex (ARPC) subunit 2, 3, and 5, as well as myosin light chain kinase (MLCK) in these cells. Our data establish miR-143/145 as important regulators of IOP, which may have important therapeutic implications in glaucoma.

Asunto(s)

Glaucoma/genética , Glaucoma/patología , MicroARNs/genética , Animales , Humor Acuoso , Línea Celular , Modelos Animales de Enfermedad , Eliminación de Gen , Humanos , Presión Intraocular , Ratones , Músculo Liso/química , Malla Trabecular/química

RESUMEN

PURPOSE: MicroRNA-146a (miR-146a) has been proposed as a marker for age-associated inflammation, or "inflammaging", acting as a negative regulator of cellular senescence and pro-inflammatory signaling pathways. However, the regulation and function of miR-146 during ocular aging remains unclear. Here we propose that miR-146 is regulated during aging of the retina and choroid, and functions in retinal pigment epithelial (RPE) cells to regulate key genes involved in inflammation and angiogenesis. METHODS: The expression of miR-146a and miR-146b was examined in the neuroretina and RPE/choroid in mice aged from 2 months to 24 months. Then, the effect of synthetic miR-146a mimetic on IL-6 and VEGF-A expression was analyzed in RPE cells treated with and without TNF-α. RESULTS: miR-146a and miR-146b was upregulated during aging of RPE/choroid but not neuroretina, supporting tissue-specific regulation of aging-related miRNAs in retinal tissues. Overexpression of miR-146a by miRNA mimics inhibited VEGF-A and TNF-α-induced IL-6 expression. CONCLUSIONS: Elevation of miR-146a and miR-146b in the aging RPE/choroid but not neuroretina suggests a role for miRNAs in inflammaging in the RPE/choroid. miR-146a overexpression inhibits the expression IL-6 and VEGF-A in the RPE cells, supporting a negative feedback regulation mechanism by which inflammatory pathways may be dysregulated in RPE during aging.

RESUMEN

Parkinson's disease (PD), the second most common age-associated progressive neurodegenerative disorder, is characterized by the loss of dopaminergic (DA) neurons in the substantia nigra pars compacta (SN). The pathogenesis of PD and the mechanisms underlying the degeneration of DA neurons are still not fully understood. Our previous quantitative proteomics study revealed that hyaluronan and proteoglycan binding link protein 2 (Hapln2) is one of differentially expressed proteins in the substantia nigra tissues from PD patients and healthy control subjects. However, the potential role of Hapln2 in PD pathogenesis remains elusive. In the present study, we characterized the expression pattern of Hapln2. In situ hybridization revealed that Hapln2 mRNA was widely expressed in adult rat brain with high abundance in the substantia nigra. Immunoblotting showed that expression levels of Hapln2 were markedly upregulated in the substantia nigra of either human subjects with Parkinson's disease compared with healthy control. Likewise, there were profound increases in Hapln2 expression in neurotoxin 6-hydroxydopamine-treated rat. Overexpression of Hapln2 in vitro increased vulnerability of MES23.5 cells, a dopaminergic cell line, to 6-hydroxydopamine. Moreover, Hapln2 overexpression led to the formation of cytoplasmic aggregates which were co-localized with ubiquitin and E3 ligases including Parkin, Gp78, and Hrd1 in vitro. Endogenous α-synuclein was also localized in Hapln2-containing aggregates and ablation of Hapln2 led to a marked decrease of α-synuclein in insoluble fraction compared with control. Thus, Hapln2 is identified as a novel factor contributing to neurodegeneration in PD. Our data provides new insights into the cellular mechanism underlying the pathogenesis in PD.