Your browser doesn't support javascript.
loading
Mostrar: 20 | 50 | 100
Resultados 1 - 20 de 10.372
Filtrar
1.
Nat Commun ; 12(1): 2198, 2021 04 13.
Artículo en Inglés | MEDLINE | ID: mdl-33850160

RESUMEN

Cancer is initiated by somatic mutations in oncogenes or tumor suppressor genes. However, additional alterations provide selective advantages to the tumor cells to resist treatment and develop metastases. Their identification is of paramount importance. Reduced expression of EFA6B (Exchange Factor for ARF6, B) is associated with breast cancer of poor prognosis. Here, we report that loss of EFA6B triggers a transcriptional reprogramming of the cell-to-ECM interaction machinery and unleashes CDC42-dependent collective invasion in collagen. In xenograft experiments, MCF10 DCIS.com cells, a DCIS-to-IDC transition model, invades faster when knocked-out for EFA6B. In addition, invasive and metastatic tumors isolated from patients have lower expression of EFA6B and display gene ontology signatures identical to those of EFA6B knock-out cells. Thus, we reveal an EFA6B-regulated molecular mechanism that controls the invasive potential of mammary cells; this finding opens up avenues for the treatment of invasive breast cancer.


Asunto(s)
Neoplasias de la Mama/genética , Neoplasias de la Mama/metabolismo , Regulación Neoplásica de la Expresión Génica , Factores de Intercambio de Guanina Nucleótido/genética , Factores de Intercambio de Guanina Nucleótido/metabolismo , Animales , Carcinoma Ductal de Mama/genética , Carcinoma Ductal de Mama/metabolismo , Línea Celular Tumoral , Femenino , Perfilación de la Expresión Génica , Técnicas de Inactivación de Genes , Humanos , Ratones , Ratones Desnudos , Transcriptoma , Proteína de Unión al GTP cdc42
2.
Int J Mol Sci ; 22(6)2021 Mar 14.
Artículo en Inglés | MEDLINE | ID: mdl-33799339

RESUMEN

In the last two decades, interest has grown significantly in the investigation of the role of trace amines and their receptors in mammalian physiology and pathology. Trace amine-associated receptor 9 (TAAR9) is one of the least studied members of this receptor family with unidentified endogenous ligands and an unknown role in the central nervous system and periphery. In this study, we generated two new TAAR9 knockout (TAAR9-KO) rat strains by CRISPR-Cas9 technology as in vivo models to evaluate the role of TAAR9 in mammalian physiology. In these mutant rats, we performed a comparative analysis of a number of hematological and biochemical parameters in the blood. Particularly, we carried out a complete blood count, erythrocyte osmotic fragility test, and screening of a panel of basic biochemical parameters. No significant alterations in any of the hematological and most biochemical parameters were found between mutant and WT rats. However, biochemical studies revealed a significant decrease in total and low-density lipoprotein cholesterol levels in the blood of both strains of TAAR9-KO rats. Such role of TAAR9 in cholesterol regulation not only brings a new understanding of mechanisms and biological pathways of lipid exchange but also provides a new potential drug target for disorders involving cholesterol-related pathology, such as atherosclerosis.


Asunto(s)
Aterosclerosis/genética , Colesterol/sangre , Receptores Acoplados a Proteínas G/genética , Animales , Aterosclerosis/sangre , Aterosclerosis/patología , Sistemas CRISPR-Cas , Sistema Nervioso Central/metabolismo , Sistema Nervioso Central/patología , Colesterol/genética , Modelos Animales de Enfermedad , Eritrocitos/metabolismo , Eritrocitos/patología , Técnicas de Inactivación de Genes , Humanos , Ligandos , Fragilidad Osmótica/genética , Ratas
3.
Gene ; 786: 145621, 2021 Jun 20.
Artículo en Inglés | MEDLINE | ID: mdl-33798680

RESUMEN

KPNA4 (also called importin-α3) belongs to the importin α adaptor proteins family, which orchestrates classical nuclear transport processes, importin-α/importin-ß1 pathway, and involves in cellular homeostasis. Disruption of balanced transport pathways may result in ectopic nuclear proteins and eventually cause diseases, mainly under the situation of cellular stress, such as oxidative stress. Little evidence is available on its cellular functions for high specific expression in lens. We firstly studied the role of KPNA4 in cataract formation. Lens defects were observed at an early age in kpna4 gene knockout zebrafish, generated by the CRISPR/Cas9 system. Those phenotype, including cloudy center part of the lens, via bright field microscopy, and the thinning of the LE layer, wider space between the adjacent LE and LF cells, irregular cells morphology and the increased number of holes inside the LE cells, which were detected by transmission electron microscopy, recapitulate the clinical features of cataract patients. As the p53-specific adaptor of the nuclear import, KPNA4 upregulated with the same pattern of p53 in hydrogen peroxide-induced apoptosis in human lens epithelia cells. Furthermore, the loss of Kpna4 resulted in the accumulation of p53 in the center of lens. Taken together, we showed that KPNA4 was involved in the formation of cataract, likely by mediating p53 nuclear transport.


Asunto(s)
Catarata/diagnóstico por imagen , Proteína p53 Supresora de Tumor/metabolismo , alfa Carioferinas/genética , alfa Carioferinas/metabolismo , Transporte Activo de Núcleo Celular , Animales , Catarata/genética , Catarata/metabolismo , Línea Celular , Núcleo Celular/metabolismo , Supervivencia Celular/efectos de los fármacos , Modelos Animales de Enfermedad , Células Epiteliales/citología , Células Epiteliales/efectos de los fármacos , Células Epiteliales/metabolismo , Técnicas de Inactivación de Genes , Humanos , Peróxido de Hidrógeno/efectos adversos , Cristalino/citología , Cristalino/efectos de los fármacos , Cristalino/metabolismo , Microscopía Electrónica de Transmisión , Pez Cebra
4.
Science ; 372(6538)2021 04 09.
Artículo en Inglés | MEDLINE | ID: mdl-33833093

RESUMEN

DNA methylation is essential to mammalian development, and dysregulation can cause serious pathological conditions. Key enzymes responsible for deposition and removal of DNA methylation are known, but how they cooperate to regulate the methylation landscape remains a central question. Using a knockin DNA methylation reporter, we performed a genome-wide CRISPR-Cas9 screen in human embryonic stem cells to discover DNA methylation regulators. The top screen hit was an uncharacterized gene, QSER1, which proved to be a key guardian of bivalent promoters and poised enhancers of developmental genes, especially those residing in DNA methylation valleys (or canyons). We further demonstrate genetic and biochemical interactions of QSER1 and TET1, supporting their cooperation to safeguard transcriptional and developmental programs from DNMT3-mediated de novo methylation.


Asunto(s)
Metilación de ADN , ADN/metabolismo , Células Madre Embrionarias Humanas/metabolismo , Sistemas CRISPR-Cas , ADN (Citosina-5-)-Metiltransferasas/metabolismo , Elementos de Facilitación Genéticos , Regulación del Desarrollo de la Expresión Génica , Técnicas de Sustitución del Gen , Técnicas de Inactivación de Genes , Genoma Humano , Humanos , Oxigenasas de Función Mixta/genética , Oxigenasas de Función Mixta/metabolismo , Regiones Promotoras Genéticas , Unión Proteica , Proteínas Proto-Oncogénicas/genética , Proteínas Proto-Oncogénicas/metabolismo , Transcripción Genética
5.
Int J Mol Sci ; 22(5)2021 Mar 04.
Artículo en Inglés | MEDLINE | ID: mdl-33806448

RESUMEN

Lambda interferons mediate antiviral immunity by inducing interferon-stimulated genes (ISGs) in epithelial tissues. A common variant rs368234815TT/∆G creating functional gene from an IFNL4 pseudogene is associated with the expression of major ISGs in the liver but impaired clearance of hepatitis C. To explain this, we compared Halo-tagged and non-tagged IFNL3 and IFNL4 signaling in liver-derived cell lines. Transfection with non-tagged IFNL3, non-tagged IFNL4 and Halo-tagged IFNL4 led to a similar degree of JAK-STAT activation and ISG induction; however, the response to transfection with Halo-tagged IFNL3 was lower and delayed. Transfection with non-tagged IFNL3 or IFNL4 induced no transcriptome change in the cells lacking either IL10R2 or IFNLR1 receptor subunits. Cytosolic overexpression of signal peptide-lacking IFNL3 or IFNL4 in wild type cells did not interfere with JAK-STAT signaling triggered by interferons in the medium. Finally, expression profile changes induced by transfection with non-tagged IFNL3 and IFNL4 were highly similar. These data do not support the hypothesis about IFNL4-specific non-canonical signaling and point out that functional studies conducted with tagged interferons should be interpreted with caution.


Asunto(s)
Hepatocitos/inmunología , Hepatocitos/metabolismo , Interferones/genética , Interferones/metabolismo , Interleucinas/genética , Interleucinas/metabolismo , Línea Celular , Expresión Génica , Técnicas de Inactivación de Genes , Células Hep G2 , Humanos , Factores Reguladores del Interferón/genética , Factores Reguladores del Interferón/metabolismo , Interferones/deficiencia , Subunidad beta del Receptor de Interleucina-10/deficiencia , Subunidad beta del Receptor de Interleucina-10/genética , Subunidad beta del Receptor de Interleucina-10/metabolismo , Interleucinas/deficiencia , ARN Mensajero/genética , ARN Mensajero/metabolismo , Receptores de Interferón/deficiencia , Receptores de Interferón/genética , Receptores de Interferón/metabolismo , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Transducción de Señal , Transfección
6.
World J Surg Oncol ; 19(1): 131, 2021 Apr 21.
Artículo en Inglés | MEDLINE | ID: mdl-33882945

RESUMEN

BACKGROUND: Radiotherapy is a main therapeutic method for cancers, including colon cancer. In the current study, we aim to explore the effects of circular RNA (circRNA) circ_0055625 in the progression and radiosensitivity of colon cancer and the underlying mechanism. METHODS: The expression of circ_0055625 and musashi homolog 1 (MSI1) mRNA was detected by quantitative real-time polymerase chain reaction (qRT-PCR). MSI1 protein expression was determined by Western blot. Cell proliferation was assessed by cell counting kit-8 (CCK-8) and colony formation assays. Cell survival fraction, apoptosis, and invasion were investigated by colony formation assay, flow cytometry analysis, and transwell invasion assay, respectively. Cell migration was detected by wound-healing and transwell migration assays. The binding relationship between microRNA-338-3p (miR-338-3p) and circ_0055625 or MSI1 was predicted by online databases and identified by Dual-Luciferase Reporter Assay. The effects of circ_0055625 silencing on the tumor formation and radiosensitivity of colon cancer in vivo were explored by in vivo tumor formation assay. RESULTS: Circ_0055625 and MSI1 were upregulated in colon cancer tissues and cells relative to control groups. Radiation treatment apparently increased the expression of circ_0055625 and MSI1 in colon cancer cells. Circ_0055625 knockdown or MSI1 silencing repressed cell proliferation, migration, and invasion and promoted cell apoptosis and radiosensitivity in colon cancer. Also, circ_0055625 silencing-mediated effects were attenuated by MSI1 overexpression. Additionally, circ_0055625 silencing reduced MSI1 expression, which could be attenuated by miR-338-3p inhibitor. Mechanically, circ_0055625 acted as a sponge for miR-338-3p to regulate MSI1. Furthermore, circ_0055625 knockdown hindered tumor growth and improved radiosensitivity in vivo. CONCLUSION: Circ_0055625 repression inhibited the progression and radioresistance of colon cancer by downregulating MSI1 through sponging miR-338-3p. This result might provide a theoretical basis for improving the therapy of colon cancer with radiation.


Asunto(s)
Neoplasias del Colon , MicroARNs/genética , Proteínas del Tejido Nervioso/genética , ARN Circular/genética , Proteínas de Unión al ARN/genética , Carcinogénesis/genética , Línea Celular Tumoral , Neoplasias del Colon/genética , Neoplasias del Colon/radioterapia , Regulación Neoplásica de la Expresión Génica/genética , Técnicas de Inactivación de Genes , Silenciador del Gen , Humanos , Proteínas del Tejido Nervioso/biosíntesis , Pronóstico , Proteínas de Unión al ARN/biosíntesis , Tolerancia a Radiación/genética , Tolerancia a Radiación/efectos de la radiación , Transfección
7.
Molecules ; 26(7)2021 Mar 24.
Artículo en Inglés | MEDLINE | ID: mdl-33805024

RESUMEN

Depression and anxiety disorders are widespread diseases, and they belong to the leading causes of disability and greatest burdens on healthcare systems worldwide. It is expected that the numbers will dramatically rise during the COVID-19 pandemic. Established medications are not sufficient to adequately treat depression and are not available for everyone. Plants from traditional medicine may be promising alternatives to treat depressive symptoms. The model organism Chaenorhabditis elegans was used to assess the stress reducing effects of methanol/dichlormethane extracts from plants used in traditional medicine. After initial screening for antioxidant activity, nine extracts were selected for in vivo testing in oxidative stress, heat stress, and osmotic stress assays. Additionally, anti-aging properties were evaluated in lifespan assay. The extracts from Acanthopanax senticosus, Campsis grandiflora, Centella asiatica, Corydalis yanhusuo, Dan Zhi, Houttuynia cordata, Psoralea corylifolia, Valeriana officinalis, and Withaniasomnifera showed antioxidant activity of more than 15 Trolox equivalents per mg extract. The extracts significantly lowered ROS in mutants, increased resistance to heat stress and osmotic stress, and the extended lifespan of the nematodes. The plant extracts tested showed promising results in increasing stress resistance in the nematode model. Further analyses are needed, in order to unravel underlying mechanisms and transfer results to humans.


Asunto(s)
Antidepresivos/farmacología , Caenorhabditis elegans/efectos de los fármacos , Caenorhabditis elegans/fisiología , Extractos Vegetales/farmacología , Plantas Medicinales/química , Envejecimiento/efectos de los fármacos , Envejecimiento/fisiología , Animales , Antioxidantes/farmacología , Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/genética , Técnicas de Inactivación de Genes , Respuesta al Choque Térmico/efectos de los fármacos , Longevidad/efectos de los fármacos , Longevidad/genética , Longevidad/fisiología , Mutación , Presión Osmótica/efectos de los fármacos , Estrés Oxidativo/efectos de los fármacos , Extractos Vegetales/química , Especies Reactivas de Oxígeno/metabolismo
8.
Nat Commun ; 12(1): 1464, 2021 03 05.
Artículo en Inglés | MEDLINE | ID: mdl-33674610

RESUMEN

The interpretation of high throughput sequencing data is limited by our incomplete functional understanding of coding and non-coding transcripts. Reliably predicting the function of such transcripts can overcome this limitation. Here we report the use of a consensus independent component analysis and guilt-by-association approach to predict over 23,000 functional groups comprised of over 55,000 coding and non-coding transcripts using publicly available transcriptomic profiles. We show that, compared to using Principal Component Analysis, Independent Component Analysis-derived transcriptional components enable more confident functionality predictions, improve predictions when new members are added to the gene sets, and are less affected by gene multi-functionality. Predictions generated using human or mouse transcriptomic data are made available for exploration in a publicly available web portal.


Asunto(s)
Perfilación de la Expresión Génica/métodos , Transcriptoma , Animales , Biología Computacional , Técnicas de Inactivación de Genes , Secuenciación de Nucleótidos de Alto Rendimiento , Humanos , Ratones , ARN Mensajero/metabolismo
9.
Nat Plants ; 7(3): 353-364, 2021 03.
Artículo en Inglés | MEDLINE | ID: mdl-33686223

RESUMEN

Plant roots acquire nutrients and water while managing interactions with the soil microbiota. The root endodermis provides an extracellular diffusion barrier through a network of lignified cell walls called Casparian strips, supported by subsequent formation of suberin lamellae. Whereas lignification is thought to be irreversible, suberin lamellae display plasticity, which is crucial for root adaptative responses. Although suberin is a major plant polymer, fundamental aspects of its biosynthesis and turnover have remained obscure. Plants shape their root system via lateral root formation, an auxin-induced process requiring local breaking and re-sealing of endodermal lignin and suberin barriers. Here, we show that differentiated endodermal cells have a specific, auxin-mediated transcriptional response dominated by cell wall remodelling genes. We identified two sets of auxin-regulated GDSL lipases. One is required for suberin synthesis, while the other can drive suberin degradation. These enzymes have key roles in suberization, driving root suberin plasticity.


Asunto(s)
Proteínas de Arabidopsis/metabolismo , Arabidopsis/metabolismo , Hidrolasas de Éster Carboxílico/metabolismo , Lípidos , Dominios Proteicos , Arabidopsis/enzimología , Proteínas de Arabidopsis/genética , Hidrolasas de Éster Carboxílico/genética , Conjuntos de Datos como Asunto , Endodermo/metabolismo , Técnicas de Inactivación de Genes , Ácidos Indolacéticos/metabolismo , Lípidos/genética , Células Vegetales/metabolismo , Raíces de Plantas/metabolismo , Polimerizacion , Proteolisis
10.
Nat Commun ; 12(1): 1918, 2021 03 26.
Artículo en Inglés | MEDLINE | ID: mdl-33771997

RESUMEN

The RNA-binding protein SFPQ plays an important role in neuronal development and has been associated with several neurodegenerative disorders, including amyotrophic lateral sclerosis (ALS), frontotemporal dementia (FTD), and Alzheimer's disease. Here, we report that loss of sfpq leads to premature termination of multiple transcripts due to widespread activation of previously unannotated cryptic last exons (CLEs). These SFPQ-inhibited CLEs appear preferentially in long introns of genes with neuronal functions and can dampen gene expression outputs and/or give rise to short peptides interfering with the normal gene functions. We show that one such peptide encoded by the CLE-containing epha4b mRNA isoform is responsible for neurodevelopmental defects in the sfpq mutant. The uncovered CLE-repressive activity of SFPQ is conserved in mouse and human, and SFPQ-inhibited CLEs are found expressed across ALS iPSC-derived neurons. These results greatly expand our understanding of SFPQ function and uncover a gene regulation mechanism with wide relevance to human neuropathologies.


Asunto(s)
Esclerosis Amiotrófica Lateral/genética , Codón sin Sentido , Exones/genética , Factor de Empalme Asociado a PTB/genética , Animales , Secuencia de Bases , Embrión no Mamífero/embriología , Embrión no Mamífero/metabolismo , Regulación del Desarrollo de la Expresión Génica , Técnicas de Inactivación de Genes , Humanos , Hibridación in Situ/métodos , Intrones/genética , Ratones , Neuronas/metabolismo , Pez Cebra/embriología , Pez Cebra/genética
11.
Nat Genet ; 53(3): 332-341, 2021 03.
Artículo en Inglés | MEDLINE | ID: mdl-33649592

RESUMEN

Resistance to immune checkpoint inhibitors (ICIs) is a key challenge in cancer therapy. To elucidate underlying mechanisms, we developed Perturb-CITE-sequencing (Perturb-CITE-seq), enabling pooled clustered regularly interspaced short palindromic repeat (CRISPR)-Cas9 perturbations with single-cell transcriptome and protein readouts. In patient-derived melanoma cells and autologous tumor-infiltrating lymphocyte (TIL) co-cultures, we profiled transcriptomes and 20 proteins in ~218,000 cells under ~750 perturbations associated with cancer cell-intrinsic ICI resistance (ICR). We recover known mechanisms of resistance, including defects in the interferon-γ (IFN-γ)-JAK/STAT and antigen-presentation pathways in RNA, protein and perturbation space, and new ones, including loss/downregulation of CD58. Loss of CD58 conferred immune evasion in multiple co-culture models and was downregulated in tumors of melanoma patients with ICR. CD58 protein expression was not induced by IFN-γ signaling, and CD58 loss conferred immune evasion without compromising major histocompatibility complex (MHC) expression, suggesting that it acts orthogonally to known mechanisms of ICR. This work provides a framework for the deciphering of complex mechanisms by large-scale perturbation screens with multimodal, single-cell readouts, and discovers potentially clinically relevant mechanisms of immune evasion.


Asunto(s)
Antígenos CD58/inmunología , Resistencia a Antineoplásicos/inmunología , Melanoma/patología , Análisis de la Célula Individual/métodos , Escape del Tumor , Antígenos CD58/genética , Antígenos CD58/metabolismo , Sistemas CRISPR-Cas , Técnicas de Cocultivo , Biología Computacional/métodos , Resistencia a Antineoplásicos/efectos de los fármacos , Resistencia a Antineoplásicos/genética , Epítopos/genética , Técnicas de Inactivación de Genes , Humanos , Interferón gamma/inmunología , Interferón gamma/metabolismo , Linfocitos Infiltrantes de Tumor/patología , Melanoma/tratamiento farmacológico , Melanoma/inmunología , Análisis de Secuencia de ARN , Escape del Tumor/genética
12.
Methods Mol Biol ; 2269: 255-268, 2021.
Artículo en Inglés | MEDLINE | ID: mdl-33687685

RESUMEN

The modulation of gene expression is essential for the investigation of function or involved pathway of a single gene of interest, in particular in the developmental/stem cell biology. The temporary knock down of gene expression via siRNA is a well-established but with a residual expression connected modulation method. The chapter describes the complete knockout of a defined target and allows a comprehensive study of different gene like the stem cell gene LGR4 (Leucine-rich repeat-containing G-protein-coupled receptor 4) using the new developed CRISPR/Cas method (clustered regularly interspaced short palindromic repeats).


Asunto(s)
Sistemas CRISPR-Cas , Edición Génica , Receptores Acoplados a Proteínas G/genética , Células Madre/metabolismo , Técnicas de Inactivación de Genes , Células HEK293 , Humanos , Receptores Acoplados a Proteínas G/metabolismo
13.
Methods Mol Biol ; 2265: 25-46, 2021.
Artículo en Inglés | MEDLINE | ID: mdl-33704703

RESUMEN

Recent advances in the treatment of metastatic melanoma have emerged only from advances in our understanding of melanoma development and progression at the cellular and molecular levels. Despite the impact that such advances have made on the clinical management of this cancer over the last decade, additional insights into factors that promote melanoma progression and therapeutic resistance are needed to combat this disease. CRISPR-Cas9 gene editing technology is a powerful tool for studying gene function in a timely and cost-effective manner, enabling the manipulation of specific DNA sequences via a targeted approach. Herein, we describe a protocol for generating functional gene knockouts in melanoma cell lines by CRISPR-Cas9 gene editing, and we present an example application of this protocol for the successful knockout of the Foxc2 transcription factor-encoding gene in the B16-F1 murine melanoma cell line.


Asunto(s)
Edición Génica/métodos , Técnicas de Inactivación de Genes/métodos , Melanoma/genética , Animales , Sistemas CRISPR-Cas , Línea Celular , Factores de Transcripción Forkhead/genética , Vectores Genéticos , Ratones , Transfección
14.
Int J Mol Sci ; 22(4)2021 Feb 19.
Artículo en Inglés | MEDLINE | ID: mdl-33669601

RESUMEN

Lead (Pb) is one of the major heavy metals that are toxic to vertebrates and usually considered as environmental pollutants. ABCC4/MRP4 is an organic anion transporter that mediates cellular efflux of a wide range of exogenous and endogenous compounds such as cyclic nucleotides and anti-cancer drugs; however, it remains unclear whether ABCC4 and its orthologs function in the detoxification and excretion of toxic lead. In this study, we found that the transcriptional and translational expression of zebrafish abcc4 was significantly induced under lead exposure in developing zebrafish embryos and adult tissues. Overexpression of zebrafish Abcc4 markedly decreased the cytotoxicity and accumulation of lead in pig renal proximal tubule cell line (LLC-PK1 cells). To further understand the functions of zebrafish Abcc4 in lead detoxification, the clustered regularly interspaced palindromic repeats (CRISPR)/Cas9 system was used to create an abcc4-/- mutant zebrafish line. In comparison with the wild-type (WT) zebrafish, the abcc4-/- mutants showed a higher death rate and lead accumulation upon exposure to lead. Furthermore, a stable abcc4-transgenic zebrafish line was successfully generated, which exerted stronger ability to detoxify and excrete lead than WT zebrafish. These findings indicate that zebrafish Abcc4 plays a crucial role in lead detoxification and cellular efflux and could be used as a potential biomarker to monitor lead contamination in a water environment.


Asunto(s)
Técnicas de Inactivación de Genes , Plomo/metabolismo , Proteínas Asociadas a Resistencia a Múltiples Medicamentos/metabolismo , Proteínas de Pez Cebra/metabolismo , Pez Cebra/genética , Animales , Animales Modificados Genéticamente , Secuencia de Bases , Transporte Biológico , Regulación del Desarrollo de la Expresión Génica , Inactivación Metabólica , Células LLC-PK1 , Proteínas Asociadas a Resistencia a Múltiples Medicamentos/genética , Mutación/genética , Porcinos , Pez Cebra/embriología , Proteínas de Pez Cebra/genética
15.
Nat Commun ; 12(1): 1720, 2021 03 19.
Artículo en Inglés | MEDLINE | ID: mdl-33741934

RESUMEN

Pulmonary arterial hypertension is a progressive fatal disease that is characterized by pathological pulmonary artery remodeling, in which endothelial cell dysfunction is critically involved. We herein describe a previously unknown role of endothelial angiocrine in pulmonary hypertension. By searching for genes highly expressed in lung microvascular endothelial cells, we identify inhibin-ß-A as an angiocrine factor produced by pulmonary capillaries. We find that excess production of inhibin-ß-A by endothelial cells impairs the endothelial function in an autocrine manner by functioning as activin-A. Mechanistically, activin-A induces bone morphogenetic protein receptor type 2 internalization and targeting to lysosomes for degradation, resulting in the signal deficiency in endothelial cells. Of note, endothelial cells isolated from the lung of patients with idiopathic pulmonary arterial hypertension show higher inhibin-ß-A expression and produce more activin-A compared to endothelial cells isolated from the lung of normal control subjects. When endothelial activin-A-bone morphogenetic protein receptor type 2 link is overdriven in mice, hypoxia-induced pulmonary hypertension was exacerbated, whereas conditional knockout of inhibin-ß-A in endothelial cells prevents the progression of pulmonary hypertension. These data collectively indicate a critical role for the dysregulated endothelial activin-A-bone morphogenetic protein receptor type 2 link in the progression of pulmonary hypertension, and thus endothelial inhibin-ß-A/activin-A might be a potential pharmacotherapeutic target for the treatment of pulmonary arterial hypertension.


Asunto(s)
Activinas/metabolismo , Receptores de Proteínas Morfogenéticas Óseas de Tipo II/genética , Receptores de Proteínas Morfogenéticas Óseas de Tipo II/metabolismo , Hipertensión Pulmonar/metabolismo , Animales , Apoptosis , Modelos Animales de Enfermedad , Endocitosis , Células Endoteliales/metabolismo , Técnicas de Inactivación de Genes , Humanos , Hipertensión Pulmonar/patología , Hipoxia , Subunidades beta de Inhibinas , Inhibinas , Pulmón/patología , Ratones , Ratones Endogámicos C57BL , Hipertensión Arterial Pulmonar , Remodelación Vascular
16.
Nat Commun ; 12(1): 1589, 2021 03 11.
Artículo en Inglés | MEDLINE | ID: mdl-33707434

RESUMEN

Glutathione peroxidase 4 (GPX4) utilizes glutathione (GSH) to detoxify lipid peroxidation and plays an essential role in inhibiting ferroptosis. As a selenoprotein, GPX4 protein synthesis is highly inefficient and energetically costly. How cells coordinate GPX4 synthesis with nutrient availability remains unclear. In this study, we perform integrated proteomic and functional analyses to reveal that SLC7A11-mediated cystine uptake promotes not only GSH synthesis, but also GPX4 protein synthesis. Mechanistically, we find that cyst(e)ine activates mechanistic/mammalian target of rapamycin complex 1 (mTORC1) and promotes GPX4 protein synthesis at least partly through the Rag-mTORC1-4EBP signaling axis. We show that pharmacologic inhibition of mTORC1 decreases GPX4 protein levels, sensitizes cancer cells to ferroptosis, and synergizes with ferroptosis inducers to suppress patient-derived xenograft tumor growth in vivo. Together, our results reveal a regulatory mechanism to coordinate GPX4 protein synthesis with cyst(e)ine availability and suggest using combinatorial therapy of mTORC1 inhibitors and ferroptosis inducers in cancer treatment.


Asunto(s)
Cisteína/metabolismo , Cistina/metabolismo , Ferroptosis/fisiología , Diana Mecanicista del Complejo 1 de la Rapamicina/metabolismo , Fosfolípido Hidroperóxido Glutatión Peroxidasa/metabolismo , Sistema de Transporte de Aminoácidos y+/metabolismo , Línea Celular Tumoral , Técnicas de Inactivación de Genes , Glutatión/metabolismo , Células HEK293 , Humanos , Diana Mecanicista del Complejo 1 de la Rapamicina/antagonistas & inhibidores , Neoplasias/patología
17.
Nat Commun ; 12(1): 1455, 2021 03 05.
Artículo en Inglés | MEDLINE | ID: mdl-33674593

RESUMEN

T-cell exhaustion denotes a hypofunctional state of T lymphocytes commonly found in cancer, but how tumor cells drive T-cell exhaustion remains elusive. Here, we find T-cell exhaustion linked to overall survival in 675 hepatocellular carcinoma (HCC) patients with diverse ethnicities and etiologies. Integrative omics analyses uncover oncogenic reprograming of HCC methionine recycling with elevated 5-methylthioadenosine (MTA) and S-adenosylmethionine (SAM) to be tightly linked to T-cell exhaustion. SAM and MTA induce T-cell dysfunction in vitro. Moreover, CRISPR-Cas9-mediated deletion of MAT2A, a key SAM producing enzyme, results in an inhibition of T-cell dysfunction and HCC growth in mice. Thus, reprogramming of tumor methionine metabolism may be a viable therapeutic strategy to improve HCC immunity.


Asunto(s)
Carcinoma Hepatocelular/metabolismo , Neoplasias Hepáticas/metabolismo , Metionina/metabolismo , Linfocitos T/metabolismo , Animales , Biomarcadores de Tumor , Linfocitos T CD8-positivos , Sistemas CRISPR-Cas , Carcinogénesis/genética , Carcinogénesis/metabolismo , Carcinoma Hepatocelular/genética , Carcinoma Hepatocelular/inmunología , Carcinoma Hepatocelular/patología , Línea Celular Tumoral , Femenino , Regulación Neoplásica de la Expresión Génica , Técnicas de Inactivación de Genes , Humanos , Hígado/metabolismo , Hígado/patología , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/inmunología , Metionina Adenosiltransferasa/sangre , Metionina Adenosiltransferasa/genética , Metionina Adenosiltransferasa/metabolismo , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , S-Adenosilmetionina/metabolismo , Transcriptoma
18.
Int J Mol Sci ; 22(4)2021 Feb 04.
Artículo en Inglés | MEDLINE | ID: mdl-33557112

RESUMEN

Breast cancer (BCa) is one of the leading health problems among women. Although significant achievements have led to advanced therapeutic success with targeted therapy options, more efforts are required for different subtypes of tumors and according to genomic, transcriptomic, and proteomic alterations. This study underlines the role of microRNA-21 (miR-21) in metastatic MDA-MB-231 breast cancer cells. Following the knockout of miR-21 from MDA-MB-231 cells, which have the highest miR-21 expression levels compared to MCF-7 and SK-BR-3 BCa cells, a decrease in epithelial-mesenchymal transition (EMT) via downregulation of mesenchymal markers was observed. Wnt-11 was a critical target for miR-21, and the Wnt-11 related signaling axis was altered in the stable miR-21 knockout cells. miR-21 expression was associated with a significant increase in mesenchymal markers in MDA-MB-231 BCa cells. Furthermore, the release of extracellular vesicles (EVs) was significantly reduced in the miR-21 KO cells, alongside a significant reduction in relative miR-21 export in EV cargo, compared with control cells. We conclude that miR-21 is a leading factor involved in mesenchymal transition in MDA-MB-231 BCa. Future therapeutic strategies could focus on its role in the treatment of metastatic breast cancer.


Asunto(s)
Neoplasias de la Mama/genética , Neoplasias de la Mama/patología , Transición Epitelial-Mesenquimal/genética , Regulación Neoplásica de la Expresión Génica , MicroARNs/genética , Biomarcadores de Tumor , Neoplasias de la Mama/mortalidad , Línea Celular Tumoral , Femenino , Edición Génica , Técnicas de Inactivación de Genes , Humanos , Pronóstico , Interferencia de ARN , Proteínas Wnt/metabolismo
19.
Plant Mol Biol ; 105(6): 637-654, 2021 Apr.
Artículo en Inglés | MEDLINE | ID: mdl-33543390

RESUMEN

KEY MESSAGE: We demonstrate that OsNAC109 regulates senescence, growth and development via binding to the cis-element CNTCSSNNSCAVG and altering the expression of multiple senescence- and hormone-associated genes in rice. The NAC family is one of the largest transcripton factor families in plants and plays an essential role in plant development, leaf senescence and responses to biotic/abiotic stresses through modulating the expression of numerous genes. Here, we isolated and characterized a novel yellow leaf 3 (yl3) mutant exhibiting arrested-growth, increased accumulation of reactive oxygen species (ROS), decreased level of soluble proteins, increased level of malondialdehyde (MDA), reduced activities of ROS scavenging enzymes, altered expression of photosynthesis and senescence/hormone-associated genes. The yellow leaf and arrested-growth trait was controlled by a single recessive gene located to chromosome 9. A single nucleotide substitution was detected in the mutant allele leading to premature termination of its coding protein. Genetic complementation could rescue the mutant phenotype while the YL3 knockout lines displayed similar phenotype to WT. YL3 was expressed in all tissues tested and predicted to encode a transcriptional factor OsNAC109 which localizes to the nucleus. It was confirmed that OsNAC109 could directly regulate the expression of OsNAP, OsNYC3, OsEATB, OsAMTR1, OsZFP185, OsMPS and OsGA2ox3 by targeting to the highly conserved cis-element CNTCSSNNSCAVG except OsSAMS1. Our results demonstrated that OsNAC109 is essential to rice leaf senescence, growth and development through regulating the expression of senescence- and phytohormone-associated genes in rice.


Asunto(s)
Oryza/crecimiento & desarrollo , Oryza/genética , Oryza/metabolismo , Reguladores del Crecimiento de las Plantas/metabolismo , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Alelos , Cloroplastos/ultraestructura , Regulación de la Expresión Génica de las Plantas , Técnicas de Inactivación de Genes , Genes de Plantas/genética , Mutación , Fenotipo , Fotosíntesis , Hojas de la Planta/genética , Hojas de la Planta/metabolismo , Especies Reactivas de Oxígeno/metabolismo , Estrés Fisiológico , Factores de Transcripción , Transcriptoma
20.
Nat Genet ; 53(3): 269-278, 2021 03.
Artículo en Inglés | MEDLINE | ID: mdl-33558760

RESUMEN

Cancer-associated, loss-of-function mutations in genes encoding subunits of the BRG1/BRM-associated factor (BAF) chromatin-remodeling complexes1-8 often cause drastic chromatin accessibility changes, especially in important regulatory regions9-19. However, it remains unknown how these changes are established over time (for example, immediate consequences or long-term adaptations), and whether they are causative for intracomplex synthetic lethalities, abrogating the formation or activity of BAF complexes9,20-24. In the present study, we use the dTAG system to induce acute degradation of BAF subunits and show that chromatin alterations are established faster than the duration of one cell cycle. Using a pharmacological inhibitor and a chemical degrader of the BAF complex ATPase subunits25,26, we show that maintaining genome accessibility requires constant ATP-dependent remodeling. Completely abolishing BAF complex function by acute degradation of a synthetic lethal subunit in a paralog-deficient background results in an almost complete loss of chromatin accessibility at BAF-controlled sites, especially also at superenhancers, providing a mechanism for intracomplex synthetic lethalities.


Asunto(s)
Cromatina/genética , ADN Helicasas/metabolismo , Complejos Multiproteicos/genética , Proteínas Nucleares/metabolismo , Factores de Transcripción/metabolismo , Acetilación , Animales , Línea Celular , Cromatina/metabolismo , ADN Helicasas/genética , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Drosophila/citología , Elementos de Facilitación Genéticos , Técnicas de Inactivación de Genes , Histonas/genética , Histonas/metabolismo , Humanos , Complejos Multiproteicos/metabolismo , Proteínas Nucleares/genética , Factores de Transcripción/genética
SELECCIÓN DE REFERENCIAS
DETALLE DE LA BÚSQUEDA
...