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2.
Immunity ; 42(3): 457-70, 2015 Mar 17.
Artículo en Inglés | MEDLINE | ID: mdl-25769609

RESUMEN

Little is known about the role of negative regulators in controlling natural killer (NK) cell development and effector functions. Foxo1 is a multifunctional transcription factor of the forkhead family. Using a mouse model of conditional deletion in NK cells, we found that Foxo1 negatively controlled NK cell differentiation and function. Immature NK cells expressed abundant Foxo1 and little Tbx21 relative to mature NK cells, but these two transcription factors reversed their expression as NK cells proceeded through development. Foxo1 promoted NK cell homing to lymph nodes by upregulating CD62L expression and inhibited late-stage maturation and effector functions by repressing Tbx21 expression. Loss of Foxo1 rescued the defect in late-stage NK cell maturation in heterozygous Tbx21(+/-) mice. Collectively, our data reveal a regulatory pathway by which the negative regulator Foxo1 and the positive regulator Tbx21 play opposing roles in controlling NK cell development and effector functions.


Asunto(s)
Factores de Transcripción Forkhead/inmunología , Regulación Neoplásica de la Expresión Génica , Células Asesinas Naturales/inmunología , Neoplasias Pulmonares/genética , Melanoma Experimental/genética , Neoplasias Cutáneas/genética , Proteínas de Dominio T Box/inmunología , Animales , Diferenciación Celular , Proteína Forkhead Box O1 , Factores de Transcripción Forkhead/genética , Heterocigoto , Células Asesinas Naturales/patología , Selectina L/genética , Selectina L/inmunología , Pulmón/inmunología , Pulmón/patología , Neoplasias Pulmonares/inmunología , Neoplasias Pulmonares/secundario , Ganglios Linfáticos/inmunología , Ganglios Linfáticos/patología , Depleción Linfocítica , Melanoma Experimental/inmunología , Melanoma Experimental/patología , Ratones , Ratones Noqueados , Trasplante de Neoplasias , Transducción de Señal , Neoplasias Cutáneas/inmunología , Neoplasias Cutáneas/secundario , Proteínas de Dominio T Box/genética
3.
Nature ; 542(7641): 362-366, 2017 02 16.
Artículo en Inglés | MEDLINE | ID: mdl-28178232

RESUMEN

Malignant neoplasms evolve in response to changes in oncogenic signalling. Cancer cell plasticity in response to evolutionary pressures is fundamental to tumour progression and the development of therapeutic resistance. Here we determine the molecular and cellular mechanisms of cancer cell plasticity in a conditional oncogenic Kras mouse model of pancreatic ductal adenocarcinoma (PDAC), a malignancy that displays considerable phenotypic diversity and morphological heterogeneity. In this model, stochastic extinction of oncogenic Kras signalling and emergence of Kras-independent escaper populations (cells that acquire oncogenic properties) are associated with de-differentiation and aggressive biological behaviour. Transcriptomic and functional analyses of Kras-independent escapers reveal the presence of Smarcb1-Myc-network-driven mesenchymal reprogramming and independence from MAPK signalling. A somatic mosaic model of PDAC, which allows time-restricted perturbation of cell fate, shows that depletion of Smarcb1 activates the Myc network, driving an anabolic switch that increases protein metabolism and adaptive activation of endoplasmic-reticulum-stress-induced survival pathways. Increased protein turnover renders mesenchymal sub-populations highly susceptible to pharmacological and genetic perturbation of the cellular proteostatic machinery and the IRE1-α-MKK4 arm of the endoplasmic-reticulum-stress-response pathway. Specifically, combination regimens that impair the unfolded protein responses block the emergence of aggressive mesenchymal subpopulations in mouse and patient-derived PDAC models. These molecular and biological insights inform a potential therapeutic strategy for targeting aggressive mesenchymal features of PDAC.


Asunto(s)
Mesodermo/patología , Neoplasias Pancreáticas/patología , Animales , Carcinoma Ductal Pancreático/tratamiento farmacológico , Carcinoma Ductal Pancreático/metabolismo , Carcinoma Ductal Pancreático/patología , Desoxicitidina/análogos & derivados , Desoxicitidina/farmacología , Desoxicitidina/uso terapéutico , Estrés del Retículo Endoplásmico/genética , Femenino , Genes myc , Genes ras , Humanos , MAP Quinasa Quinasa 4/metabolismo , Sistema de Señalización de MAP Quinasas , Masculino , Mesodermo/metabolismo , Ratones , Mosaicismo , Proteína Oncogénica p55(v-myc)/metabolismo , Neoplasias Pancreáticas/tratamiento farmacológico , Neoplasias Pancreáticas/metabolismo , Proteolisis , Proteínas Proto-Oncogénicas p21(ras)/genética , Proteínas Proto-Oncogénicas p21(ras)/metabolismo , Proteína SMARCB1/deficiencia , Proteína SMARCB1/metabolismo , Transcriptoma/genética , Gemcitabina
4.
Mol Cell ; 57(4): 708-720, 2015 Feb 19.
Artículo en Inglés | MEDLINE | ID: mdl-25639470

RESUMEN

mTOR senses nutrient and energy status to regulate cell survival and metabolism in response to environmental changes. Surprisingly, targeted mutation of Tsc1, a negative regulator of mTORC1, caused a broad reduction in miRNAs due to Drosha degradation. Conversely, targeted mutation of Raptor, an essential component of mTORC1, increased miRNA biogenesis. mTOR activation increased expression of Mdm2, which is hereby identified as the necessary and sufficient ubiquitin E3 ligase for Drosha. Drosha was induced by nutrient and energy deprivation and conferred resistance to glucose deprivation. Using a high-throughput screen of a miRNA library, we identified four miRNAs that were necessary and sufficient to protect cells against glucose-deprivation-induced apoptosis. These miRNA was regulated by glucose through the mTORC1-MDM2-DROSHA axis. Taken together, our data reveal an mTOR-Mdm2-Drosha pathway in mammalian cells that broadly regulates miRNA biogenesis as a response to alteration in cellular environment.


Asunto(s)
MicroARNs/biosíntesis , Complejos Multiproteicos/fisiología , Proteínas Proto-Oncogénicas c-mdm2/fisiología , Ribonucleasa III/fisiología , Serina-Treonina Quinasas TOR/fisiología , Aminoácidos/metabolismo , Animales , Regulación de la Expresión Génica , Glucosa/metabolismo , Células HeLa , Humanos , Diana Mecanicista del Complejo 1 de la Rapamicina , Ratones Endogámicos C57BL , Complejos Multiproteicos/genética , Complejos Multiproteicos/metabolismo , Proteolisis , Proteínas Proto-Oncogénicas c-mdm2/genética , Proteínas Proto-Oncogénicas c-mdm2/metabolismo , Ribonucleasa III/genética , Ribonucleasa III/metabolismo , Serina-Treonina Quinasas TOR/genética , Serina-Treonina Quinasas TOR/metabolismo , Proteína p53 Supresora de Tumor/fisiología , Ubiquitinación
5.
J Immunol ; 201(8): 2492-2501, 2018 10 15.
Artículo en Inglés | MEDLINE | ID: mdl-30171165

RESUMEN

Neutrophils are the first responders to sites of inflammation when the intestinal epithelial barrier is breached and the gut microbiota invade. Despite current efforts in understanding the role of neutrophils in intestinal homeostasis, the complex interactions between neutrophils and intestinal epithelial cells (IECs) is still not well characterized. In this study, we demonstrated that neutrophils enhanced production of amphiregulin (AREG), a member of the EGFR ligand family, by IECs, which promoted IEC barrier function and tissue repair. Depletion of neutrophils resulted in more severe colitis in mice because of decreased AREG production by IECs upon dextran sodium sulfate (DSS) insult. Administration of AREG restored epithelial barrier function and ameliorated colitis. Furthermore, neutrophil-derived TGF-ß promoted AREG production by IECs. Mechanistically, TGF-ß activated MEK1/2 signaling, and inhibition of MEK1/2 abrogated TGF-ß-induced AREG production by IECs. Collectively, these findings reveal that neutrophils play an important role in the maintenance of IEC barrier function and homeostasis.


Asunto(s)
Anfirregulina/metabolismo , Colitis/inmunología , Enfermedades Inflamatorias del Intestino/inmunología , Mucosa Intestinal/fisiología , Neutrófilos/fisiología , Factor de Crecimiento Transformador beta/metabolismo , Animales , Células Cultivadas , Colitis/inducido químicamente , Sulfato de Dextran , Modelos Animales de Enfermedad , Femenino , Homeostasis , Humanos , MAP Quinasa Quinasa 1/metabolismo , Masculino , Ratones , Ratones Endogámicos C57BL , Transducción de Señal
6.
Proc Natl Acad Sci U S A ; 114(30): 8071-8076, 2017 07 25.
Artículo en Inglés | MEDLINE | ID: mdl-28696308

RESUMEN

Small, noncoding RNAs are short untranslated RNA molecules, some of which have been associated with cancer development. Recently we showed that a class of small RNAs generated during the maturation process of tRNAs (tRNA-derived small RNAs, hereafter "tsRNAs") is dysregulated in cancer. Specifically, we uncovered tsRNA signatures in chronic lymphocytic leukemia and lung cancer and demonstrated that the ts-4521/3676 cluster (now called "ts-101" and "ts-53," respectively), ts-46, and ts-47 are down-regulated in these malignancies. Furthermore, we showed that tsRNAs are similar to Piwi-interacting RNAs (piRNAs) and demonstrated that ts-101 and ts-53 can associate with PiwiL2, a protein involved in the silencing of transposons. In this study, we extended our investigation on tsRNA signatures to samples collected from patients with colon, breast, or ovarian cancer and cell lines harboring specific oncogenic mutations and representing different stages of cancer progression. We detected tsRNA signatures in all patient samples and determined that tsRNA expression is altered upon oncogene activation and during cancer staging. In addition, we generated a knocked-out cell model for ts-101 and ts-46 in HEK-293 cells and found significant differences in gene-expression patterns, with activation of genes involved in cell survival and down-regulation of genes involved in apoptosis and chromatin structure. Finally, we overexpressed ts-46 and ts-47 in two lung cancer cell lines and performed a clonogenic assay to examine their role in cell proliferation. We observed a strong inhibition of colony formation in cells overexpressing these tsRNAs compared with untreated cells, confirming that tsRNAs affect cell growth and survival.


Asunto(s)
Neoplasias/metabolismo , ARN Pequeño no Traducido/metabolismo , Células A549 , Estudios de Casos y Controles , Células HEK293 , Humanos , Oncogenes
7.
Nature ; 497(7449): 383-7, 2013 May 16.
Artículo en Inglés | MEDLINE | ID: mdl-23636329

RESUMEN

MicroRNAs (miRNAs) are generated by two-step processing to yield small RNAs that negatively regulate target gene expression at the post-transcriptional level. Deregulation of miRNAs has been linked to diverse pathological processes, including cancer. Recent studies have also implicated miRNAs in the regulation of cellular response to a spectrum of stresses, such as hypoxia, which is frequently encountered in the poorly angiogenic core of a solid tumour. However, the upstream regulators of miRNA biogenesis machineries remain obscure, raising the question of how tumour cells efficiently coordinate and impose specificity on miRNA expression and function in response to stresses. Here we show that epidermal growth factor receptor (EGFR), which is the product of a well-characterized oncogene in human cancers, suppresses the maturation of specific tumour-suppressor-like miRNAs in response to hypoxic stress through phosphorylation of argonaute 2 (AGO2) at Tyr 393. The association between EGFR and AGO2 is enhanced by hypoxia, leading to elevated AGO2-Y393 phosphorylation, which in turn reduces the binding of Dicer to AGO2 and inhibits miRNA processing from precursor miRNAs to mature miRNAs. We also identify a long-loop structure in precursor miRNAs as a critical regulatory element in phospho-Y393-AGO2-mediated miRNA maturation. Furthermore, AGO2-Y393 phosphorylation mediates EGFR-enhanced cell survival and invasiveness under hypoxia, and correlates with poorer overall survival in breast cancer patients. Our study reveals a previously unrecognized function of EGFR in miRNA maturation and demonstrates how EGFR is likely to function as a regulator of AGO2 through novel post-translational modification. These findings suggest that modulation of miRNA biogenesis is important for stress response in tumour cells and has potential clinical implications.


Asunto(s)
Proteínas Argonautas/química , Proteínas Argonautas/metabolismo , Hipoxia de la Célula/fisiología , Receptores ErbB/metabolismo , MicroARNs/metabolismo , Neoplasias de la Mama/genética , Neoplasias de la Mama/metabolismo , Neoplasias de la Mama/mortalidad , Neoplasias de la Mama/patología , Hipoxia de la Célula/genética , Línea Celular Tumoral , Supervivencia Celular , Femenino , Regulación Neoplásica de la Expresión Génica , Humanos , MicroARNs/biosíntesis , MicroARNs/química , MicroARNs/genética , Invasividad Neoplásica , Conformación de Ácido Nucleico , Fosforilación , Fosfotirosina/metabolismo , Pronóstico , Unión Proteica , Precursores del ARN/química , Precursores del ARN/genética , Precursores del ARN/metabolismo , Ribonucleasa III/metabolismo , Análisis de Supervivencia
8.
Mol Cell ; 44(5): 770-84, 2011 Dec 09.
Artículo en Inglés | MEDLINE | ID: mdl-22152480

RESUMEN

Both H4K16 acetylation and H3K4 trimethylation are required for gene activation. However, it is still largely unclear how these modifications are orchestrated by transcriptional factors. Here, we analyzed the mechanism of the transcriptional activation by FOXP3, an X-linked suppressor of autoimmune diseases and cancers. FOXP3 binds near transcriptional start sites of its target genes. By recruiting MOF and displacing histone H3K4 demethylase PLU-1, FOXP3 increases both H4K16 acetylation and H3K4 trimethylation at the FOXP3-associated chromatins of multiple FOXP3-activated genes. RNAi-mediated silencing of MOF reduced both gene activation and tumor suppression by FOXP3, while both somatic mutations in clinical cancer samples and targeted mutation of FOXP3 in mouse prostate epithelial cells disrupted nuclear localization of MOF. Our data demonstrate a pull-push model in which a single transcription factor orchestrates two epigenetic alterations necessary for gene activation and provide a mechanism for somatic inactivation of the FOXP3 protein function in cancer cells.


Asunto(s)
Factores de Transcripción Forkhead/metabolismo , Histona Acetiltransferasas/metabolismo , Histonas/metabolismo , Histona Demetilasas con Dominio de Jumonji/metabolismo , Proteínas Nucleares/metabolismo , Proteínas Represoras/metabolismo , Acetilación , Neoplasias de la Mama/genética , Neoplasias de la Mama/metabolismo , Neoplasias de la Mama/patología , Línea Celular Tumoral , Núcleo Celular/metabolismo , Femenino , Factores de Transcripción Forkhead/genética , Regulación de la Expresión Génica , Células HEK293 , Humanos , Metilación , Mutación
9.
Proc Natl Acad Sci U S A ; 113(18): 5071-6, 2016 May 03.
Artículo en Inglés | MEDLINE | ID: mdl-27071132

RESUMEN

Chronic lymphocytic leukemia (CLL) is the most common human leukemia, and transgenic mouse studies indicate that activation of the T-cell leukemia/lymphoma 1 (TCL1) oncogene is a contributing event in the pathogenesis of the aggressive form of this disease. While studying the regulation of TCL1 expression, we identified the microRNA cluster miR-4521/3676 and discovered that these two microRNAs are associated with tRNA sequences and that this region can produce two small RNAs, members of a recently identified class of small noncoding RNAs, tRNA-derived small RNAs (tsRNAs). We further proved that miR-3676 and miR-4521 are tsRNAs using Northern blot analysis. We found that, like ts-3676, ts-4521 is down-regulated and mutated in CLL. Analysis of lung cancer samples revealed that both ts-3676 and ts-4521 are down-regulated and mutated in patient tumor samples. Because tsRNAs are similar in nature to piRNAs [P-element-induced wimpy testis (Piwi)-interacting small RNAs], we investigated whether ts-3676 and ts-4521 can interact with Piwi proteins and found these two tsRNAs in complexes containing Piwi-like protein 2 (PIWIL2). To determine whether other tsRNAs are involved in cancer, we generated a custom microarray chip containing 120 tsRNAs 16 bp or more in size. Microarray hybridization experiments revealed tsRNA signatures in CLL and lung cancer, indicating that, like microRNAs, tsRNAs may have an oncogenic and/or tumor-suppressor function in hematopoietic malignancies and solid tumors. Thus, our results show that tsRNAs are dysregulated in human cancer.


Asunto(s)
Leucemia Linfocítica Crónica de Células B/genética , Neoplasias Pulmonares/genética , Familia de Multigenes/genética , ARN Neoplásico/genética , ARN Pequeño no Traducido/genética , ARN de Transferencia/genética , Regulación Neoplásica de la Expresión Génica/genética , Marcadores Genéticos/genética , Predisposición Genética a la Enfermedad/genética , Humanos
10.
Hum Mutat ; 38(11): 1491-1499, 2017 11.
Artículo en Inglés | MEDLINE | ID: mdl-28703315

RESUMEN

Lysosomes are membrane-bound, acidic eukaryotic cellular organelles that play important roles in the degradation of macromolecules. Mutations that cause the loss of lysosomal protein function can lead to a group of disorders categorized as the lysosomal storage diseases (LSDs). Suspicion of LSD is frequently based on clinical and pathologic findings, but in some cases, the underlying genetic and biochemical defects remain unknown. Here, we performed whole-exome sequencing (WES) on 14 suspected LSD cases to evaluate the feasibility of using WES for identifying causal mutations. By examining 2,157 candidate genes potentially associated with lysosomal function, we identified eight variants in five genes as candidate disease-causing variants in four individuals. These included both known and novel mutations. Variants were corroborated by targeted sequencing and, when possible, functional assays. In addition, we identified nonsense mutations in two individuals in genes that are not known to have lysosomal function. However, mutations in these genes could have resulted in phenotypes that were diagnosed as LSDs. This study demonstrates that WES can be used to identify causal mutations in suspected LSD cases. We also demonstrate cases where a confounding clinical phenotype may potentially reflect more than one lysosomal protein defect.


Asunto(s)
Exoma , Estudios de Asociación Genética , Predisposición Genética a la Enfermedad , Enfermedades por Almacenamiento Lisosomal/diagnóstico , Enfermedades por Almacenamiento Lisosomal/genética , Adolescente , Adulto , Alelos , Sustitución de Aminoácidos , Niño , Mapeo Cromosómico , Activación Enzimática , Femenino , Marcadores Genéticos , Genómica/métodos , Genotipo , Humanos , Mutación con Pérdida de Función , Masculino , Anotación de Secuencia Molecular , Mutación , Linaje , Fenotipo , Polimorfismo de Nucleótido Simple , Secuenciación del Exoma
11.
Gut ; 65(1): 19-32, 2016 Jan.
Artículo en Inglés | MEDLINE | ID: mdl-25410163

RESUMEN

BACKGROUND: Worldwide, gastric cancer (GC) is the fourth most common malignancy and the most common cancer in East Asia. Development of targeted therapies for this disease has focused on a few known oncogenes but has had limited effects. OBJECTIVE: To determine oncogenic mechanisms and novel therapeutic targets specific for GC by identifying commonly dysregulated genes from the tumours of both Asian-Pacific and Caucasian patients. METHODS: We generated transcriptomic profiles of 22 Caucasian GC tumours and their matched non-cancerous samples and performed an integrative analysis across different GC gene expression datasets. We examined the inhibition of commonly overexpressed oncogenes and their constituent signalling pathways by RNAi and/or pharmacological inhibition. RESULTS: Hepatocyte nuclear factor-4α (HNF4α) upregulation was a key signalling event in gastric tumours from both Caucasian and Asian patients, and HNF4α antagonism was antineoplastic. Perturbation experiments in GC tumour cell lines and xenograft models further demonstrated that HNF4α is downregulated by AMPKα signalling and the AMPK agonist metformin; blockade of HNF4α activity resulted in cyclin downregulation, cell cycle arrest and tumour growth inhibition. HNF4α also regulated WNT signalling through its target gene WNT5A, a potential prognostic marker of diffuse type gastric tumours. CONCLUSIONS: Our results indicate that HNF4α is a targetable oncoprotein in GC, is regulated by AMPK signalling through AMPKα and resides upstream of WNT signalling. HNF4α may regulate 'metabolic switch' characteristic of a general malignant phenotype and its target WNT5A has potential prognostic values. The AMPKα-HNF4α-WNT5A signalling cascade represents a potentially targetable pathway for drug development.


Asunto(s)
Proteínas Quinasas Activadas por AMP/genética , Adenocarcinoma/genética , Biomarcadores de Tumor/genética , Regulación Neoplásica de la Expresión Génica , Factor Nuclear 4 del Hepatocito/genética , Proteínas Proto-Oncogénicas/genética , Neoplasias Gástricas/genética , Proteínas Wnt/genética , Proteínas Quinasas Activadas por AMP/metabolismo , Adenocarcinoma/etnología , Adenocarcinoma/metabolismo , Adenocarcinoma/patología , Animales , Pueblo Asiatico , Biomarcadores de Tumor/metabolismo , Western Blotting , Estudios de Casos y Controles , Línea Celular Tumoral , Regulación hacia Abajo , Femenino , Factor Nuclear 4 del Hepatocito/metabolismo , Humanos , Inmunohistoquímica , Ratones , Ratones Endogámicos BALB C , Trasplante de Neoplasias , Proteínas Proto-Oncogénicas/metabolismo , Distribución Aleatoria , Estudios Retrospectivos , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Neoplasias Gástricas/etnología , Neoplasias Gástricas/metabolismo , Neoplasias Gástricas/patología , Regulación hacia Arriba , Población Blanca , Proteínas Wnt/metabolismo , Vía de Señalización Wnt , Proteína Wnt-5a
12.
Mod Pathol ; 29(4): 330-46, 2016 Apr.
Artículo en Inglés | MEDLINE | ID: mdl-26916073

RESUMEN

Inflammatory breast cancer is the most aggressive form of breast cancer. Identifying new biomarkers to be used as therapeutic targets is in urgent need. Messenger RNA expression profiling studies have indicated that inflammatory breast cancer is a transcriptionally heterogeneous disease, and specific molecular targets for inflammatory breast cancer have not been well established. We performed microRNA expression profiling in inflammatory breast cancer in comparison with locally advanced noninflammatory breast cancer in this study. Although many microRNAs were differentially expressed between normal breast tissue and tumor tissue, most of them did not show differential expression between inflammatory and noninflammatory tumor samples. However, by microarray analysis, quantitative reverse transcription PCR, and in situ hybridization, we showed that microRNA-205 expression was decreased not only in tumor compared with normal breast tissue, but also in inflammatory breast cancer compared with noninflammatory breast cancer. Lower expression of microRNA-205 correlated with worse distant metastasis-free survival and overall survival in our cohort. A small-scale immunohistochemistry analysis showed coexistence of decreased microRNA-205 expression and decreased E-cadherin expression in some ductal tumors. MicroRNA-205 may serve as a therapeutic target in advanced breast cancer including inflammatory breast cancer.


Asunto(s)
Biomarcadores de Tumor/genética , Neoplasias Inflamatorias de la Mama/genética , MicroARNs/biosíntesis , Adulto , Anciano , Biomarcadores de Tumor/análisis , Femenino , Perfilación de la Expresión Génica , Humanos , Inmunohistoquímica , Hibridación in Situ , Neoplasias Inflamatorias de la Mama/mortalidad , Estimación de Kaplan-Meier , MicroARNs/análisis , Persona de Mediana Edad , Análisis de Secuencia por Matrices de Oligonucleótidos , Pronóstico , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa
13.
J Biol Chem ; 289(40): 27526-39, 2014 Oct 03.
Artículo en Inglés | MEDLINE | ID: mdl-25118289

RESUMEN

IL-6/Stat3 is associated with the regulation of transcription of key cellular regulatory genes (microRNAs) during different types of liver injury. This study evaluated the role of IL-6/Stat3 in regulating miRNA and miR-21 in alcoholic liver disease. By microarray, we identified that ethanol feeding significantly up-regulated 0.8% of known microRNAs in mouse liver compared with controls, including miR-21. Similarly, the treatment of normal human hepatocytes (N-Heps) and hepatic stellate cells (HSCs) with ethanol and IL-6 significantly increased miR-21 expression. Overexpression of miR-21 decreased ethanol-induced apoptosis in both N-Heps and HSCs. The expression level of miR-21 was significantly increased after Stat3 activation in N-Heps and HSCs, in support of the concept that the 5'-promoter region of miR-21 is regulated by Stat3. Using real time PCR, we confirmed that miR-21 activation is associated with ethanol-linked Stat3 binding of the miR-21 promoter. A combination of bioinformatics, PCR array, dual-luciferase reporter assay, and Western blot analysis revealed that Fas ligand (TNF superfamily, member 6) (FASLG) and death receptor 5 (DR5) are the direct targets of miR-21. Furthermore, inhibition of miR-21 by specific Vivo-Morpholino and knock-out of IL-6 in ethanol-treated mice also increased the expression of DR5 and FASLG in vivo during alcoholic liver injury. The identification of miR-21 as an important regulator of hepatic cell survival, transformation, and remodeling in vitro, as well as its upstream modulators and downstream targets, will provide insight into the involvement of altered miRNA expression in contributing to alcoholic liver disease progression and testing novel therapeutic approaches for human alcoholic liver diseases.


Asunto(s)
Apoptosis , Hepatopatías Alcohólicas/metabolismo , Hepatopatías Alcohólicas/fisiopatología , MicroARNs/metabolismo , Animales , Células Estrelladas Hepáticas/citología , Células Estrelladas Hepáticas/metabolismo , Humanos , Interleucina-6/genética , Interleucina-6/metabolismo , Hígado/citología , Hígado/metabolismo , Hepatopatías Alcohólicas/genética , Ratones , Ratones Endogámicos C57BL , MicroARNs/genética , Factor de Transcripción STAT3/genética , Factor de Transcripción STAT3/metabolismo , Transducción de Señal , Regulación hacia Arriba
14.
Infect Immun ; 83(3): 1068-77, 2015 Mar.
Artículo en Inglés | MEDLINE | ID: mdl-25561708

RESUMEN

Two-component gene regulatory systems (TCSs) are a major mechanism by which bacteria respond to environmental stimuli and thus are critical to infectivity. For example, the control of virulence regulator/sensor kinase (CovRS) TCS is central to the virulence of the major human pathogen group A Streptococcus (GAS). Here, we used a combination of quantitative in vivo phosphorylation assays, isoallelic strains that varied by only a single amino acid in CovS, and transcriptome analyses to characterize the impact of CovS on CovR phosphorylation and GAS global gene expression. We discovered that CovS primarily serves to phosphorylate CovR, thereby resulting in the repression of virulence factor-encoding genes. However, a GAS strain selectively deficient in CovS phosphatase activity had a distinct transcriptome relative to that of its parental strain, indicating that both CovS kinase and phosphatase activities influence the CovR phosphorylation status. Surprisingly, compared to a serotype M3 strain, serotype M1 GAS strains had high levels of phosphorylated CovR, low transcript levels of CovR-repressed genes, and strikingly different responses to environmental cues. Moreover, the inactivation of CovS in the serotype M1 background resulted in a greater decrease in phosphorylated CovR levels and a greater increase in the transcript levels of CovR-repressed genes than did CovS inactivation in a serotype M3 strain. These data clarify the influence of CovS on the CovR phosphorylation status and provide insight into why serotype M1 GAS strains have high rates of spontaneous mutations in covS during invasive GAS infection, thus providing a link between TCS molecular function and the epidemiology of deadly bacterial infections.


Asunto(s)
Proteínas Bacterianas/genética , Regulación Bacteriana de la Expresión Génica , Péptidos y Proteínas de Señalización Intracelular/genética , ARN Mensajero/genética , Proteínas Represoras/genética , Streptococcus pyogenes/genética , Transcriptoma , Sustitución de Aminoácidos , Proteínas Bacterianas/metabolismo , Perfilación de la Expresión Génica , Histidina Quinasa , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Mutación , Fosforilación , ARN Mensajero/metabolismo , Proteínas Represoras/metabolismo , Serogrupo , Streptococcus pyogenes/metabolismo
15.
Eur J Immunol ; 44(3): 673-82, 2014 Mar.
Artículo en Inglés | MEDLINE | ID: mdl-24293139

RESUMEN

Commensal flora plays an important role in the development of the mucosal immune system and in maintaining intestinal homeostasis. However, the mechanisms involved in regulation of host-microbiota interaction are still not completely understood. In this study, we examined how microbiota and intestinal inflammatory conditions regulate host microRNA expression and observed lower microRNA-107 (miR-107) expression in the inflamed intestines of colitic mice, compared with that in normal control mice. miR-107 was predominantly reduced in epithelial cells and CD11c(+) myeloid cells including dendritic cells and macrophages in the inflamed intestines. We demonstrate that IL-6, IFN-γ, and TNF-α downregulated, whereas TGF-ß promoted, miR-107 expression. In addition, miR-107 expression was higher in the intestines of germ-free mice than in mice housed under specific pathogen-free conditions, and the presence of microbiota downregulated miR-107 expression in DCs and macrophages in a MyD88- and NF-κB-dependent manner. We determined that the ectopic expression of miR-107 specifically repressed the expression of IL-23p19, a key molecule in innate immune responses to commensal bacteria. We concluded that regulation of miR-107 by intestinal microbiota and proinflammatory cytokine serve as an important pathway for maintaining intestinal homeostasis.


Asunto(s)
Subunidad p19 de la Interleucina-23/genética , Mucosa Intestinal/metabolismo , Intestinos/microbiología , MicroARNs/genética , Microbiota , Células Mieloides/metabolismo , Animales , Bacterias/inmunología , Bacterias/metabolismo , Emparejamiento Base , Secuencia de Bases , Colitis/genética , Colitis/inmunología , Citocinas/metabolismo , Citocinas/farmacología , Células Dendríticas/efectos de los fármacos , Células Dendríticas/inmunología , Células Dendríticas/metabolismo , Regulación de la Expresión Génica , Mediadores de Inflamación/metabolismo , Mediadores de Inflamación/farmacología , Subunidad p19 de la Interleucina-23/química , Subunidad p19 de la Interleucina-23/metabolismo , Intestinos/inmunología , Ligandos , Macrófagos/efectos de los fármacos , Macrófagos/inmunología , Macrófagos/metabolismo , Ratones , Ratones Transgénicos , MicroARNs/química , Células Mieloides/efectos de los fármacos , Células Mieloides/inmunología , Receptores Toll-Like/metabolismo
16.
Genome Res ; 22(11): 2120-9, 2012 Nov.
Artículo en Inglés | MEDLINE | ID: mdl-23028188

RESUMEN

Endometrial cancer is the most common gynecological malignancy, with more than 280,000 cases occurring annually worldwide. Although previous studies have identified important common somatic mutations in endometrial cancer, they have primarily focused on a small set of known cancer genes and have thus provided a limited view of the molecular basis underlying this disease. Here we have developed an integrated systems-biology approach to identifying novel cancer genes contributing to endometrial tumorigenesis. We first performed whole-exome sequencing on 13 endometrial cancers and matched normal samples, systematically identifying somatic alterations with high precision and sensitivity. We then combined bioinformatics prioritization with high-throughput screening (including both shRNA-mediated knockdown and expression of wild-type and mutant constructs) in a highly sensitive cell viability assay. Our results revealed 12 potential driver cancer genes including 10 tumor-suppressor candidates (ARID1A, INHBA, KMO, TTLL5, GRM8, IGFBP3, AKTIP, PHKA2, TRPS1, and WNT11) and two oncogene candidates (ERBB3 and RPS6KC1). The results in the "sensor" cell line were recapitulated by siRNA-mediated knockdown in endometrial cancer cell lines. Focusing on ARID1A, we integrated mutation profiles with functional proteomics in 222 endometrial cancer samples, demonstrating that ARID1A mutations frequently co-occur with mutations in the phosphatidylinositol 3-kinase (PI3K) pathway and are associated with PI3K pathway activation. siRNA knockdown in endometrial cancer cell lines increased AKT phosphorylation supporting ARID1A as a novel regulator of PI3K pathway activity. Our study presents the first unbiased view of somatic coding mutations in endometrial cancer and provides functional evidence for diverse driver genes and mutations in this disease.


Asunto(s)
Neoplasias Endometriales/genética , Exoma , Genes Supresores de Tumor , Genómica , Secuenciación de Nucleótidos de Alto Rendimiento , Oncogenes , Estudios de Casos y Controles , Transformación Celular Neoplásica/genética , Femenino , Humanos , Mutación , Fosfatidilinositol 3-Quinasa/metabolismo , Fosforilación , ARN Interferente Pequeño , Análisis de Secuencia de ADN , Biología de Sistemas
17.
BMC Med Genet ; 16: 46, 2015 Jul 03.
Artículo en Inglés | MEDLINE | ID: mdl-26138095

RESUMEN

BACKGROUND: P19 H-Ras, a second product derived from the H-Ras gene by alternative splicing, induces a G1/S phase delay, thereby maintaining cells in a reversible quiescence state. When P21 H-Ras is mutated in tumour cells, the alternative protein P19 H-Ras is also mutated. The H-Ras mutation Q61L is frequently detected in different tumours, which acts as constitutive activator of Ras functions and is considered to be a strong activating mutant. Additionally, a rare congenital disorder named Costello Syndrome, is described as a H-Ras disorder in children, mainly due to mutation G12S in p19 and p21 H-Ras proteins, which is present in 90 % of the Costello Syndrome patients. Our aim is to better understand the role of p19 and p21 H-Ras proteins in the cancer and Costello Syndrome development, concerning the miRNAs expression. METHODS: Total miRNAs expression regulated by H-Ras proteins were first analyzed in human miRNA microarrays assays. Previously selected miRNAs, were further analyzed in developed cell lines containing H-Ras protein mutants, that included the G12S Costello Syndrome mutant, with PCR Real-Time Taq Man miRNA Assays primers. RESULTS: This study describes how p19 affects the RNA world and shows that: i) miR-342, miR-206, miR-330, miR-138 and miR-99b are upregulated by p19 but not by p19W164A mutant; ii) anti-miR-206 can restore the G2 phase in the presence of p19; iii) p19 and p21Q61L regulate their own alternative splicing; iv) miR-206 and miR-138 are differentially regulated by p19 and p21 H-Ras and v) P19G12S Costello mutants show a clear upregulation of miR-374, miR-126, miR-342, miR-330, miR-335 and let-7. CONCLUSIONS: These results allow us to conclude that the H-Ras G12S mutation plays an important role in miRNA expression and open up a new line of study to understand the consequences of this mutation on Costello syndrome. Furthermore, they suggest that oncogenes may have a sufficiently important impact on miRNA expression to promote the development of numerous cancers.


Asunto(s)
Síndrome de Costello/genética , Síndrome de Costello/patología , MicroARNs/genética , Neoplasias/genética , Proteínas Proto-Oncogénicas p21(ras)/fisiología , Proteínas ras/fisiología , Animales , Transformación Celular Neoplásica/genética , Células Cultivadas , Embrión de Mamíferos , Regulación Neoplásica de la Expresión Génica , Células HeLa , Humanos , Ratones , Modelos Biológicos , Proteínas Mutantes/genética , Proteínas Mutantes/metabolismo , Neoplasias/metabolismo , Isoformas de Proteínas/genética , Isoformas de Proteínas/fisiología , Proteínas Proto-Oncogénicas p21(ras)/genética , Proteínas ras/genética
18.
Cancer Cell ; 12(3): 215-29, 2007 Sep.
Artículo en Inglés | MEDLINE | ID: mdl-17785203

RESUMEN

Noncoding RNA (ncRNA) transcripts are thought to be involved in human tumorigenesis. We report that a large fraction of genomic ultraconserved regions (UCRs) encode a particular set of ncRNAs whose expression is altered in human cancers. Genome-wide profiling revealed that UCRs have distinct signatures in human leukemias and carcinomas. UCRs are frequently located at fragile sites and genomic regions involved in cancers. We identified certain UCRs whose expression may be regulated by microRNAs abnormally expressed in human chronic lymphocytic leukemia, and we proved that the inhibition of an overexpressed UCR induces apoptosis in colon cancer cells. Our findings argue that ncRNAs and interaction between noncoding genes are involved in tumorigenesis to a greater extent than previously thought.


Asunto(s)
Carcinoma/genética , Leucemia/genética , ARN no Traducido/química , Secuencia de Bases , Análisis por Conglomerados , Secuencia Conservada , Perfilación de la Expresión Génica , Regulación Neoplásica de la Expresión Génica , Humanos , MicroARNs/fisiología , Datos de Secuencia Molecular , Oncogenes/fisiología , Análisis de Secuencia de ARN
19.
J Pathol ; 229(3): 449-59, 2013 Feb.
Artículo en Inglés | MEDLINE | ID: mdl-23007860

RESUMEN

Altered expression of oncogenic and tumour-suppressing microRNAs (miRNAs) is widely associated with tumourigenesis. However, the regulatory mechanisms underlying these alterations are poorly understood. We sought to shed light on the deregulation of miRNA biogenesis promoting the aberrant miRNA expression profiles identified in these tumours. Using sequencing technology to perform both whole-transcriptome and small RNA sequencing of glioma patient samples, we examined precursor and mature miRNAs to directly evaluate the miRNA maturation process, and examined expression profiles for genes involved in the major steps of miRNA biogenesis. We found that ratios of mature to precursor forms of a large number of miRNAs increased with the progression from normal brain to low-grade and then to high-grade gliomas. The expression levels of genes involved in each of the three major steps of miRNA biogenesis (nuclear processing, nucleo-cytoplasmic transport, and cytoplasmic processing) were systematically altered in glioma tissues. Survival analysis of an independent data set demonstrated that the alteration of genes involved in miRNA maturation correlates with survival in glioma patients. Direct quantification of miRNA maturation with deep sequencing demonstrated that deregulation of the miRNA biogenesis pathway is a hallmark for glioma genesis and progression.


Asunto(s)
Neoplasias Encefálicas/genética , Glioma/genética , MicroARNs/biosíntesis , Análisis de Secuencia de ARN/métodos , Transcriptoma , Neoplasias Encefálicas/mortalidad , Neoplasias Encefálicas/patología , Línea Celular Tumoral , Progresión de la Enfermedad , Perfilación de la Expresión Génica , Técnicas de Silenciamiento del Gen , Silenciador del Gen , Glioblastoma/genética , Glioblastoma/patología , Glioma/mortalidad , Glioma/patología , Humanos , Tasa de Supervivencia , Texas/epidemiología
20.
Cancer Cell ; 9(3): 189-98, 2006 Mar.
Artículo en Inglés | MEDLINE | ID: mdl-16530703

RESUMEN

MicroRNA (miRNA) expression profiles for lung cancers were examined to investigate miRNA's involvement in lung carcinogenesis. miRNA microarray analysis identified statistical unique profiles, which could discriminate lung cancers from noncancerous lung tissues as well as molecular signatures that differ in tumor histology. miRNA expression profiles correlated with survival of lung adenocarcinomas, including those classified as disease stage I. High hsa-mir-155 and low hsa-let-7a-2 expression correlated with poor survival by univariate analysis as well as multivariate analysis for hsa-mir-155. The miRNA expression signature on outcome was confirmed by real-time RT-PCR analysis of precursor miRNAs and cross-validated with an independent set of adenocarcinomas. These results indicate that miRNA expression profiles are diagnostic and prognostic markers of lung cancer.


Asunto(s)
Adenocarcinoma/genética , Biomarcadores de Tumor/genética , Neoplasias Pulmonares/genética , MicroARNs/análisis , Adenocarcinoma/mortalidad , Adenocarcinoma/patología , Anciano , Femenino , Perfilación de la Expresión Génica , Humanos , Neoplasias Pulmonares/mortalidad , Neoplasias Pulmonares/patología , Masculino , Persona de Mediana Edad , Análisis de Secuencia por Matrices de Oligonucleótidos , Pronóstico , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Análisis de Supervivencia
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