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1.
Proc Natl Acad Sci U S A ; 118(10)2021 03 09.
Artículo en Inglés | MEDLINE | ID: mdl-33649241

RESUMEN

α1-Antitrypsin (AAT) deficiency is a common genetic disease presenting with lung and liver diseases. AAT deficiency results from pathogenic variants in the SERPINA1 gene encoding AAT and the common mutant Z allele of SERPINA1 encodes for Z α1-antitrypsin (ATZ), a protein forming hepatotoxic polymers retained in the endoplasmic reticulum of hepatocytes. PiZ mice express the human ATZ and are a valuable model to investigate the human liver disease of AAT deficiency. In this study, we investigated differential expression of microRNAs (miRNAs) between PiZ and control mice and found that miR-34b/c was up-regulated and its levels correlated with intrahepatic ATZ. Furthermore, in PiZ mouse livers, we found that Forkhead Box O3 (FOXO3) driving microRNA-34b/c (miR-34b/c) expression was activated and miR-34b/c expression was dependent upon c-Jun N-terminal kinase (JNK) phosphorylation on Ser574 Deletion of miR-34b/c in PiZ mice resulted in early development of liver fibrosis and increased signaling of platelet-derived growth factor (PDGF), a target of miR-34b/c. Activation of FOXO3 and increased miR-34c were confirmed in livers of humans with AAT deficiency. In addition, JNK-activated FOXO3 and miR-34b/c up-regulation were detected in several mouse models of liver fibrosis. This study reveals a pathway involved in liver fibrosis and potentially implicated in both genetic and acquired causes of hepatic fibrosis.


Asunto(s)
Proteína Forkhead Box O3/metabolismo , Cirrosis Hepática , MAP Quinasa Quinasa 4/metabolismo , Regulación hacia Arriba , Animales , Modelos Animales de Enfermedad , Proteína Forkhead Box O3/genética , Cirrosis Hepática/genética , Cirrosis Hepática/metabolismo , Cirrosis Hepática/prevención & control , MAP Quinasa Quinasa 4/genética , Masculino , Ratones , Ratones Noqueados , MicroARNs/biosíntesis , MicroARNs/genética , alfa 1-Antitripsina/genética , alfa 1-Antitripsina/metabolismo
2.
Genet Med ; 24(2): 439-453, 2022 02.
Artículo en Inglés | MEDLINE | ID: mdl-34906501

RESUMEN

PURPOSE: This study aimed to describe a multisystemic disorder featuring cardiovascular, facial, musculoskeletal, and cutaneous anomalies caused by heterozygous loss-of-function variants in TAB2. METHODS: Affected individuals were analyzed by next-generation technologies and genomic array. The presumed loss-of-function effect of identified variants was assessed by luciferase assay in cells transiently expressing TAB2 deleterious alleles. In available patients' fibroblasts, variant pathogenicity was further explored by immunoblot and osteoblast differentiation assays. The transcriptomic profile of fibroblasts was investigated by RNA sequencing. RESULTS: A total of 11 individuals from 8 families were heterozygotes for a novel TAB2 variant. In total, 7 variants were predicted to be null alleles and 1 was a missense change. An additional subject was heterozygous for a 52 kb microdeletion involving TAB2 exons 1 to 3. Luciferase assay indicated a decreased transcriptional activation mediated by NF-κB signaling for all point variants. Immunoblot analysis showed a reduction of TAK1 phosphorylation while osteoblast differentiation was impaired. Transcriptomic analysis identified deregulation of multiple pleiotropic pathways, such as TGFß-, Ras-MAPK-, and Wnt-signaling networks. CONCLUSION: Our data defined a novel disorder associated with loss-of-function or, more rarely, hypomorphic alleles in a restricted linker region of TAB2. The pleiotropic manifestations in this disorder partly recapitulate the 6q25.1 (TAB2) microdeletion syndrome and deserve the definition of cardio-facial-cutaneous-articular syndrome.


Asunto(s)
Proteínas Adaptadoras Transductoras de Señales , FN-kappa B , Proteínas Adaptadoras Transductoras de Señales/genética , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Exones/genética , Humanos , FN-kappa B/genética , FN-kappa B/metabolismo , Fosforilación , Transducción de Señal
3.
FASEB J ; 35(4): e21357, 2021 04.
Artículo en Inglés | MEDLINE | ID: mdl-33710685

RESUMEN

First-degree relatives (FDRs) of type 2 diabetics (T2D) feature dysfunction of subcutaneous adipose tissue (SAT) long before T2D onset. miRNAs have a role in adipocyte precursor cells (APC) differentiation and in adipocyte identity. Thus, impaired miRNA expression may contribute to SAT dysfunction in FDRs. In the present work, we have explored changes in miRNA expression associated with T2D family history which may affect gene expression in SAT APCs from FDRs. Small RNA-seq was performed in APCs from healthy FDRs and matched controls and omics data were validated by qPCR. Integrative analyses of APC miRNome and transcriptome from FDRs revealed down-regulated hsa-miR-23a-5p, -193a-5p and -193b-5p accompanied by up-regulated Insulin-like Growth Factor 2 (IGF2) gene which proved to be their direct target. The expression changes in these marks were associated with SAT adipocyte hypertrophy in FDRs. APCs from FDRs further demonstrated reduced capability to differentiate into adipocytes. Treatment with IGF2 protein decreased APC adipogenesis, while over-expression of hsa-miR-23a-5p, -193a-5p and -193b-5p enhanced adipogenesis by IGF2 targeting. Indeed, IGF2 increased the Wnt Family Member 10B gene expression in APCs. Down-regulation of the three miRNAs and IGF2 up-regulation was also observed in Peripheral Blood Leukocytes (PBLs) from FDRs. In conclusion, APCs from FDRs feature a specific miRNA/gene profile, which associates with SAT adipocyte hypertrophy and appears to contribute to impaired adipogenesis. PBL detection of this profile may help in identifying adipocyte hypertrophy in individuals at high risk of T2D.


Asunto(s)
Diabetes Mellitus Tipo 2/metabolismo , Predisposición Genética a la Enfermedad , Factor II del Crecimiento Similar a la Insulina/metabolismo , MicroARNs/metabolismo , Adipogénesis , Clonación Molecular , Diabetes Mellitus Tipo 2/genética , Familia , Regulación de la Expresión Génica , Humanos , Factor II del Crecimiento Similar a la Insulina/genética , MicroARNs/genética
4.
J Biol Chem ; 295(38): 13213-13223, 2020 09 18.
Artículo en Inglés | MEDLINE | ID: mdl-32723872

RESUMEN

α1-Antitrypsin (AAT) encoded by the SERPINA1 gene is an acute-phase protein synthesized in the liver and secreted into the circulation. Its primary role is to protect lung tissue by inhibiting neutrophil elastase. The Z allele of SERPINA1 encodes a mutant AAT, named ATZ, that changes the protein structure and leads to its misfolding and polymerization, which cause endoplasmic reticulum (ER) stress and liver disease through a gain-of-function toxic mechanism. Hepatic retention of ATZ results in deficiency of one of the most important circulating proteinase inhibitors and predisposes to early-onset emphysema through a loss-of-function mechanism. The pathogenetic mechanisms underlying the liver disease are not completely understood. C/EBP-homologous protein (CHOP), a transcription factor induced by ER stress, was found among the most up-regulated genes in livers of PiZ mice that express ATZ and in human livers of patients homozygous for the Z allele. Compared with controls, juvenile PiZ/Chop-/- mice showed reduced hepatic ATZ and a transcriptional response indicative of decreased ER stress by RNA-Seq analysis. Livers of PiZ/Chop-/- mice also showed reduced SERPINA1 mRNA levels. By chromatin immunoprecipitations and luciferase reporter-based transfection assays, CHOP was found to up-regulate SERPINA1 cooperating with c-JUN, which was previously shown to up-regulate SERPINA1, thus aggravating hepatic accumulation of ATZ. Increased CHOP levels were detected in diseased livers of children homozygous for the Z allele. In summary, CHOP and c-JUN up-regulate SERPINA1 transcription and play an important role in hepatic disease by increasing the burden of proteotoxic ATZ, particularly in the pediatric population.


Asunto(s)
Hepatopatías/metabolismo , Hígado/metabolismo , Mutación , Agregación Patológica de Proteínas/metabolismo , Proteínas Proto-Oncogénicas c-jun/metabolismo , Factor de Transcripción CHOP/metabolismo , alfa 1-Antitripsina/biosíntesis , Alelos , Animales , Estrés del Retículo Endoplásmico/genética , Humanos , Hígado/patología , Hepatopatías/genética , Hepatopatías/patología , Ratones , Ratones Noqueados , Agregación Patológica de Proteínas/genética , Agregación Patológica de Proteínas/patología , Pliegue de Proteína , Proteínas Proto-Oncogénicas c-jun/genética , Factor de Transcripción CHOP/genética , Transcripción Genética , Regulación hacia Arriba , alfa 1-Antitripsina/genética
5.
Genet Med ; 21(3): 591-600, 2019 03.
Artículo en Inglés | MEDLINE | ID: mdl-29997386

RESUMEN

PURPOSE: We studied microRNAs as potential biomarkers for Pompe disease. METHODS: We analyzed microRNA expression by small RNA-seq in tissues from the disease murine model at two different ages (3 and 9 months), and in plasma from Pompe patients. RESULTS: In the mouse model we found 211 microRNAs that were differentially expressed in gastrocnemii and 66 in heart, with a different pattern of expression at different ages. In a preliminary analysis in plasma from six patients 55 microRNAs were differentially expressed. Sixteen of these microRNAs were common to those dysregulated in mouse tissues. These microRNAs are known to modulate the expression of genes involved in relevant pathways for Pompe disease pathophysiology (autophagy, muscle regeneration, muscle atrophy). One of these microRNAs, miR-133a, was selected for further quantitative real-time polymerase chain reaction analysis in plasma samples from 52 patients, obtained from seven Italian and Dutch biobanks. miR-133a levels were significantly higher in Pompe disease patients than in controls and correlated with phenotype severity, with higher levels in infantile compared with late-onset patients. In three infantile patients miR-133a decreased after start of enzyme replacement therapy and evidence of clinical improvement. CONCLUSION: Circulating microRNAs may represent additional biomarkers of Pompe disease severity and of response to therapy.


Asunto(s)
Enfermedad del Almacenamiento de Glucógeno Tipo II/diagnóstico , Enfermedad del Almacenamiento de Glucógeno Tipo II/genética , MicroARNs/genética , Adulto , Animales , Biomarcadores/sangre , Modelos Animales de Enfermedad , Femenino , Humanos , Masculino , Ratones , Ratones Noqueados , MicroARNs/fisiología , Persona de Mediana Edad
6.
Int J Mol Sci ; 20(6)2019 Mar 16.
Artículo en Inglés | MEDLINE | ID: mdl-30884856

RESUMEN

Human mesenchymal/stromal stem cells (hMSC) are the most promising cell source for adult cell therapies in regenerative medicine. Many clinical trials have reported the use of autologous transplantation of hMSCs in several disorders, but with limited results. To exert their potential, hMSCs could exhibit efficient homing and migration toward lesion sites among other effects, but the underlying process is not clear enough. To further increase the knowledge, we studied the co-regulation between hypoxia-regulated genes and miRNAs. To this end, we investigated the miRNA expression profile of healthy hMSCs in low oxygen/nutrient conditions to mimic ischemia and compared with cells of patients suffering from critical limb ischemia (CLI). miRNAs are small, highly conserved, non-coding RNAs, skilled in the control of the target's expression level in a fine-tuned way. After analyzing the miRNOme in CLI-derived hMSC cells and healthy controls, and intersecting the results with the mRNA expression dataset under hypoxic conditions, we identified two miRNAs potentially relevant to the disease: miR-29b as a pathological marker of the disease and miR-638 as a therapeutic target. This study yielded a deeper understanding of stem cell biology and ischemic disorders, opening new potential treatments in the future.


Asunto(s)
Isquemia/genética , Células Madre Mesenquimatosas/metabolismo , MicroARNs/genética , Adulto , Diferenciación Celular/genética , Hipoxia de la Célula/genética , Movimiento Celular/genética , Femenino , Regulación de la Expresión Génica/genética , Humanos , Inflamación/genética , Inflamación/patología , Isquemia/fisiopatología , Masculino , Células Madre Mesenquimatosas/patología , Persona de Mediana Edad
7.
J Hepatol ; 69(2): 325-335, 2018 08.
Artículo en Inglés | MEDLINE | ID: mdl-29580866

RESUMEN

BACKGROUND & AIMS: Acute liver failure is a rapidly progressive deterioration of hepatic function resulting in high mortality and morbidity. Metabolic enzymes can translocate to the nucleus to regulate histone acetylation and gene expression. METHODS: Levels and activities of pyruvate dehydrogenase complex (PDHC) and lactate dehydrogenase (LDH) were evaluated in nuclear fractions of livers of mice exposed to various hepatotoxins including CD95-antibody, α-amanitin, and acetaminophen. Whole-genome gene expression profiling by RNA-seq was performed in livers of mice with acute liver failure and analyzed by gene ontology enrichment analysis. Cell viability was evaluated in cell lines knocked-down for PDHA1 or LDH-A and in cells incubated with the LDH inhibitor galloflavin after treatment with CD95-antibody. We evaluated whether the histone acetyltransferase inhibitor garcinol or galloflavin could reduce liver damage in mice with acute liver failure. RESULTS: Levels and activities of PDHC and LDH were increased in nuclear fractions of livers of mice with acute liver failure. The increase of nuclear PDHC and LDH was associated with increased concentrations of acetyl-CoA and lactate in nuclear fractions, and histone H3 hyper-acetylation. Gene expression in livers of mice with acute liver failure suggested that increased histone H3 acetylation induces the expression of genes related to damage response. Reduced histone acetylation by the histone acetyltransferase inhibitor garcinol decreased liver damage and improved survival in mice with acute liver failure. Knock-down of PDHC or LDH improved viability in cells exposed to a pro-apoptotic stimulus. Treatment with the LDH inhibitor galloflavin that was also found to inhibit PDHC, reduced hepatic necrosis, apoptosis, and expression of pro-inflammatory cytokines in mice with acute liver failure. Mice treated with galloflavin also showed a dose-response increase in survival. CONCLUSION: PDHC and LDH translocate to the nucleus, leading to increased nuclear concentrations of acetyl-CoA and lactate. This results in histone H3 hyper-acetylation and expression of damage response genes. Inhibition of PDHC and LDH reduces liver damage and improves survival in mice with acute liver failure. Thus, PDHC and LDH are targets for therapy of acute liver failure. LAY SUMMARY: Acute liver failure is a rapidly progressive deterioration of liver function resulting in high mortality. In experimental mouse models of acute liver failure, we found that two metabolic enzymes, namely pyruvate dehydrogenase complex and lactic dehydrogenase, translocate to the nucleus resulting in detrimental gene expression. Treatment with an inhibitor of these two enzymes was found to reduce liver damage and to improve survival.


Asunto(s)
Isocumarinas/farmacología , L-Lactato Deshidrogenasa/metabolismo , Fallo Hepático Agudo , Hígado , Complejo Piruvato Deshidrogenasa/metabolismo , Animales , Línea Celular , Supervivencia Celular/efectos de los fármacos , Modelos Animales de Enfermedad , Inhibidores Enzimáticos/farmacología , Perfilación de la Expresión Génica , Hígado/efectos de los fármacos , Hígado/metabolismo , Fallo Hepático Agudo/tratamiento farmacológico , Fallo Hepático Agudo/metabolismo , Ratones , Ratones Endogámicos C57BL
8.
Hepatology ; 66(1): 124-135, 2017 07.
Artículo en Inglés | MEDLINE | ID: mdl-28295475

RESUMEN

α1 -Antitrypsin (AAT) deficiency is one of the most common genetic disorders and the liver disease due to the Z mutant of AAT (ATZ) is a prototype of conformational disorder due to protein misfolding with consequent aberrant intermolecular protein aggregation. In the present study, we found that livers of PiZ transgenic mice expressing human ATZ have altered expression of a network of hepatocyte transcriptional factors, including hepatocyte nuclear factor-4α, that is early down-regulated and induces a transcriptional repression of ATZ expression. Reduced hepatocyte nuclear factor-4α was associated with activation of ß-catenin, which regulates liver zonation. Livers of PiZ mice and human patients with AAT deficiency were both found to have a severe perturbation of liver zonation. Functionally, PiZ mice showed a severe defect of ureagenesis, as shown by increased baseline ammonia, and reduced urea production and survival after an ammonia challenge. Down-regulation of hepatocyte nuclear factor-4α expression and defective zonation in livers have not been recognized so far as features of the liver disease caused by ATZ and are likely involved in metabolic disturbances and in the increased risk of hepatocellular carcinoma in patients with AAT deficiency. CONCLUSION: The findings of this study are consistent with the concept that abnormal AAT protein conformation and intrahepatic accumulation have broad effects on metabolic liver functions. (Hepatology 2017;66:124-135).


Asunto(s)
Carcinoma Hepatocelular/genética , Regulación Neoplásica de la Expresión Génica , Factor Nuclear 4 del Hepatocito/genética , Neoplasias Hepáticas/patología , Deficiencia de alfa 1-Antitripsina/genética , Envejecimiento/genética , Análisis de Varianza , Animales , Carcinoma Hepatocelular/patología , Modelos Animales de Enfermedad , Regulación hacia Abajo , Humanos , Neoplasias Hepáticas/genética , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Mutación , Distribución Aleatoria , Estadísticas no Paramétricas , Deficiencia de alfa 1-Antitripsina/patología
9.
Nucleic Acids Res ; 44(4): 1525-40, 2016 Feb 29.
Artículo en Inglés | MEDLINE | ID: mdl-26819412

RESUMEN

MicroRNAs play a fundamental role in retinal development and function. To characterise the miRNome of the human retina, we carried out deep sequencing analysis on sixteen individuals. We established the catalogue of retina-expressed miRNAs, determined their relative abundance and found that a small number of miRNAs accounts for almost 90% of the retina miRNome. We discovered more than 3000 miRNA variants (isomiRs), encompassing a wide range of sequence variations, which include seed modifications that are predicted to have an impact on miRNA action. We demonstrated that a seed-modifying isomiR of the retina-enriched miR-124-3p was endowed with different targeting properties with respect to the corresponding canonical form. Moreover, we identified 51 putative novel, retina-specific miRNAs and experimentally validated the expression for nine of them. Finally, a parallel analysis of the human Retinal Pigment Epithelium (RPE)/choroid, two tissues that are known to be crucial for retina homeostasis, yielded notably distinct miRNA enrichment patterns compared to the retina. The generated data are accessible through an ad hoc database. This study is the first to reveal the complexity of the human retina miRNome at nucleotide resolution and constitutes a unique resource to assess the contribution of miRNAs to the pathophysiology of the human retina.


Asunto(s)
MicroARNs/genética , Retina/metabolismo , Transcriptoma/genética , Secuenciación de Nucleótidos de Alto Rendimiento/métodos , Humanos , MicroARNs/aislamiento & purificación , Epitelio Pigmentado de la Retina/metabolismo
10.
Nucleic Acids Res ; 44(12): 5773-84, 2016 07 08.
Artículo en Inglés | MEDLINE | ID: mdl-27235414

RESUMEN

The human retina is a specialized tissue involved in light stimulus transduction. Despite its unique biology, an accurate reference transcriptome is still missing. Here, we performed gene expression analysis (RNA-seq) of 50 retinal samples from non-visually impaired post-mortem donors. We identified novel transcripts with high confidence (Observed Transcriptome (ObsT)) and quantified the expression level of known transcripts (Reference Transcriptome (RefT)). The ObsT included 77 623 transcripts (23 960 genes) covering 137 Mb (35 Mb new transcribed genome). Most of the transcripts (92%) were multi-exonic: 81% with known isoforms, 16% with new isoforms and 3% belonging to new genes. The RefT included 13 792 genes across 94 521 known transcripts. Mitochondrial genes were among the most highly expressed, accounting for about 10% of the reads. Of all the protein-coding genes in Gencode, 65% are expressed in the retina. We exploited inter-individual variability in gene expression to infer a gene co-expression network and to identify genes specifically expressed in photoreceptor cells. We experimentally validated the photoreceptors localization of three genes in human retina that had not been previously reported. RNA-seq data and the gene co-expression network are available online (http://retina.tigem.it).


Asunto(s)
Proteínas del Ojo/genética , Redes Reguladoras de Genes , Genoma Humano , Proteínas Mitocondriales/genética , Retina/metabolismo , Transcriptoma , Adulto , Anciano , Empalme Alternativo , Atlas como Asunto , Mapeo Cromosómico , Exones , Proteínas del Ojo/metabolismo , Femenino , Perfilación de la Expresión Génica , Ontología de Genes , Secuenciación de Nucleótidos de Alto Rendimiento , Humanos , Masculino , Persona de Mediana Edad , Proteínas Mitocondriales/metabolismo , Anotación de Secuencia Molecular , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Retina/citología
11.
Proc Natl Acad Sci U S A ; 112(25): E3236-45, 2015 Jun 23.
Artículo en Inglés | MEDLINE | ID: mdl-26056285

RESUMEN

Ocular developmental disorders, including the group classified as microphthalmia, anophthalmia, and coloboma (MAC) and inherited retinal dystrophies, collectively represent leading causes of hereditary blindness. Characterized by extreme genetic and clinical heterogeneity, the separate groups share many common genetic causes, in particular relating to pathways controlling retinal and retinal pigment epithelial maintenance. To understand these shared pathways and delineate the overlap between these groups, we investigated the genetic cause of an autosomal dominantly inherited condition of retinal dystrophy and bilateral coloboma, present in varying degrees in a large, five-generation family. By linkage analysis and exome sequencing, we identified a previously undescribed heterozygous mutation, n.37 C > T, in the seed region of microRNA-204 (miR-204), which segregates with the disease in all affected individuals. We demonstrated that this mutation determines significant alterations of miR-204 targeting capabilities via in vitro assays, including transcriptome analysis. In vivo injection, in medaka fish (Oryzias latipes), of the mutated miR-204 caused a phenotype consistent with that observed in the family, including photoreceptor alterations with reduced numbers of both cones and rods as a result of increased apoptosis, thereby confirming the pathogenic effect of the n.37 C > T mutation. Finally, knockdown assays in medaka fish demonstrated that miR-204 is necessary for normal photoreceptor function. Overall, these data highlight the importance of miR-204 in the regulation of ocular development and maintenance and provide the first evidence, to our knowledge, of its contribution to eye disease, likely through a gain-of-function mechanism.


Asunto(s)
Coloboma/genética , MicroARNs/genética , Distrofias Retinianas/genética , Secuencia de Bases , Coloboma/complicaciones , Exoma , Femenino , Ligamiento Genético , Humanos , Masculino , Linaje , Distrofias Retinianas/complicaciones , Homología de Secuencia de Ácido Nucleico
12.
Hepatology ; 63(6): 1842-59, 2016 06.
Artículo en Inglés | MEDLINE | ID: mdl-26660341

RESUMEN

UNLABELLED: Wilson disease (WD) is an autosomal recessive disorder that is caused by the toxic accumulation of copper (Cu) in the liver. The ATP7B gene, which is mutated in WD, encodes a multitransmembrane domain adenosine triphosphatase that traffics from the trans-Golgi network to the canalicular area of hepatocytes, where it facilitates excretion of excess Cu into the bile. Several ATP7B mutations, including H1069Q and R778L that are two of the most frequent variants, result in protein products, which, although still functional, remain in the endoplasmic reticulum. Thus, they fail to reach Cu excretion sites, resulting in the toxic buildup of Cu in the liver of WD patients. Therefore, correcting the location of these mutants by leading them to the appropriate functional sites in the cell should restore Cu excretion and would be beneficial to help large cohorts of WD patients. However, molecular targets for correction of endoplasmic reticulum-retained ATP7B mutants remain elusive. Here, we show that expression of the most frequent ATP7B mutant, H1069Q, activates p38 and c-Jun N-terminal kinase signaling pathways, which favor the rapid degradation of the mutant. Suppression of these pathways with RNA interference or specific chemical inhibitors results in the substantial rescue of ATP7B(H1069Q) (as well as that of several other WD-causing mutants) from the endoplasmic reticulum to the trans-Golgi network compartment, in recovery of its Cu-dependent trafficking, and in reduction of intracellular Cu levels. CONCLUSION: Our findings indicate p38 and c-Jun N-terminal kinase as intriguing targets for correction of WD-causing mutants and, hence, as potential candidates, which could be evaluated for the development of novel therapeutic strategies to combat WD. (Hepatology 2016;63:1842-1859).


Asunto(s)
Adenosina Trifosfatasas/genética , Proteínas de Transporte de Catión/genética , Degeneración Hepatolenticular/genética , Sistema de Señalización de MAP Quinasas , Cobre/metabolismo , ATPasas Transportadoras de Cobre , Células HeLa , Células Hep G2 , Degeneración Hepatolenticular/metabolismo , Humanos , Hígado/metabolismo , Mutación , Vías Secretoras
13.
Carcinogenesis ; 37(1): 39-48, 2016 Jan.
Artículo en Inglés | MEDLINE | ID: mdl-26542370

RESUMEN

Multidrug resistance 2 (Mdr2), also called adenosine triphosphate-binding cassette B4 (ABCB4), is the transporter of phosphatidylcholine (PC) at the canalicular membrane of mouse hepatocytes, which plays an essential role for bile formation. Mutations in human homologue MDR3 are associated with several liver diseases. Knockout of Mdr2 results in hepatic inflammation, liver fibrosis and hepatocellular carcinoma (HCC). Whereas the pathogenesis in Mdr2 (-/-) mice has been largely attributed to the toxicity of bile acids due to the absence of PC in the bile, the question of whether Mdr2 deficiency per se perturbs biological functions in the cell has been poorly addressed. As Mdr2 is expressed in many cell types, we used mouse embryonic fibroblasts (MEF) derived from Mdr2 (-/-) embryos to show that deficiency of Mdr2 increases reactive oxygen species accumulation, lipid peroxidation and DNA damage. We found that Mdr2 (-/-) MEFs undergo spontaneous transformation and that Mdr2 (-/-) mice are more susceptible to chemical carcinogen-induced intestinal tumorigenesis. Microarray analysis in Mdr2-/- MEFs and cap analysis of gene expression in Mdr2 (-/-) HCCs revealed extensively deregulated genes involved in oxidation reduction, fatty acid metabolism and lipid biosynthesis. Our findings imply a close link between Mdr2 (-/-) -associated tumorigenesis and perturbation of these biological processes and suggest potential extrahepatic functions of Mdr2/MDR3.


Asunto(s)
Subfamilia B de Transportador de Casetes de Unión a ATP/deficiencia , Transformación Celular Neoplásica/metabolismo , Estrés Oxidativo/fisiología , Subfamilia B de Transportador de Casetes de Unión a ATP/genética , Subfamilia B de Transportador de Casetes de Unión a ATP/metabolismo , Poliposis Adenomatosa del Colon/metabolismo , Poliposis Adenomatosa del Colon/patología , Animales , Apoptosis/efectos de los fármacos , Apoptosis/fisiología , Transformación Celular Neoplásica/genética , Transformación Celular Neoplásica/patología , Células Cultivadas , Daño del ADN , Femenino , Fibroblastos/metabolismo , Fibroblastos/patología , Neoplasias Intestinales/metabolismo , Neoplasias Intestinales/patología , Peroxidación de Lípido , Hígado/metabolismo , Hígado/patología , Masculino , Ratones , Ratones Endogámicos BALB C , Ratones Noqueados , Ratones Desnudos , Especies Reactivas de Oxígeno/metabolismo , Miembro 4 de la Subfamilia B de Casete de Unión a ATP
14.
BMC Genomics ; 16: 876, 2015 Oct 28.
Artículo en Inglés | MEDLINE | ID: mdl-26510930

RESUMEN

BACKGROUND: Parkinson's disease (PD) is a chronic progressive neurodegenerative disorder that is clinically defined in terms of motor symptoms. These are preceded by prodromal non-motor manifestations that prove the systemic nature of the disease. Identifying genes and pathways altered in living patients provide new information on the diagnosis and pathogenesis of sporadic PD. METHODS: Changes in gene expression in the blood of 40 sporadic PD patients and 20 healthy controls ("Discovery set") were analyzed by taking advantage of the Affymetrix platform. Patients were at the onset of motor symptoms and before initiating any pharmacological treatment. Data analysis was performed by applying Ranking-Principal Component Analysis, PUMA and Significance Analysis of Microarrays. Functional annotations were assigned using GO, DAVID, GSEA to unveil significant enriched biological processes in the differentially expressed genes. The expressions of selected genes were validated using RT-qPCR and samples from an independent cohort of 12 patients and controls ("Validation set"). RESULTS: Gene expression profiling of blood samples discriminates PD patients from healthy controls and identifies differentially expressed genes in blood. The majority of these are also present in dopaminergic neurons of the Substantia Nigra, the key site of neurodegeneration. Together with neuronal apoptosis, lymphocyte activation and mitochondrial dysfunction, already found in previous analysis of PD blood and post-mortem brains, we unveiled transcriptome changes enriched in biological terms related to epigenetic modifications including chromatin remodeling and methylation. Candidate transcripts as CBX5, TCF3, MAN1C1 and DOCK10 were validated by RT-qPCR. CONCLUSIONS: Our data support the use of blood transcriptomics to study neurodegenerative diseases. It identifies changes in crucial components of chromatin remodeling and methylation machineries as early events in sporadic PD suggesting epigenetics as target for therapeutic intervention.


Asunto(s)
Enfermedad de Parkinson/genética , Transcriptoma/genética , Anciano , Homólogo de la Proteína Chromobox 5 , Femenino , Perfilación de la Expresión Génica , Humanos , Masculino , Persona de Mediana Edad , Reacción en Cadena en Tiempo Real de la Polimerasa
15.
Bioinformatics ; 30(12): 1787-8, 2014 Jun 15.
Artículo en Inglés | MEDLINE | ID: mdl-24558125

RESUMEN

SUMMARY: Elucidation of molecular targets of a compound [mode of action (MoA)] and its off-targets is a crucial step in drug development. We developed an online collaborative resource (MANTRA 2.0) that supports this process by exploiting similarities between drug-induced transcriptional profiles. Drugs are organized in a network of nodes (drugs) and edges (similarities) highlighting 'communities' of drugs sharing a similar MoA. A user can upload gene expression profiles before and after drug treatment in one or multiple cell types. An automated processing pipeline transforms the gene expression profiles into a unique drug 'node' embedded in the drug-network. Visual inspection of the neighbouring drugs and communities helps in revealing its MoA and to suggest new applications of known drugs (drug repurposing). MANTRA 2.0 allows storing and sharing user-generated network nodes, thus making MANTRA 2.0 a collaborative ever-growing resource. AVAILABILITY AND IMPLEMENTATION: The web tool is freely available for academic use at http://mantra.tigem.it.


Asunto(s)
Descubrimiento de Drogas/métodos , Reposicionamiento de Medicamentos/métodos , Perfilación de la Expresión Génica , Programas Informáticos , Conducta Cooperativa , Internet , Transcriptoma/efectos de los fármacos
16.
Front Immunol ; 15: 1444130, 2024.
Artículo en Inglés | MEDLINE | ID: mdl-39224604

RESUMEN

Introduction: Ataxia telangiectasia (AT) is a rare disorder characterized by neurodegeneration, combined immunodeficiency, a predisposition to malignancies, and high clinical variability. Profiling of microRNAs (miRNAs) may offer insights into the underlying mechanisms of complex rare human diseases, as miRNAs play a role in various biological functions including proliferation, differentiation, and DNA repair. In this study, we investigate the differential expression of miRNAs in samples from AT patients to identify miRNA patterns and analyze how these patterns are related to the disease. Methods: We enrolled 20 AT patients (mean age 17.7 ± 9.6 years old) and collected clinical and genetic data. We performed short non-coding RNA-seq analysis on peripheral blood mononuclear cells (PBMCs) and fibroblasts to compare the miRNA expression profile between AT patients and controls. Results: We observed 42 differentially expressed (DE)-miRNAs in blood samples and 26 in fibroblast samples. Among these, three DE-miRNAs, miR-342-3p, miR-30a-5p, and miR-195-5p, were further validated in additional AT samples, confirming their dysregulation. Discussion: We identified an AT-related miRNA signature in blood cells and fibroblast samples collected from a group of AT patients. We also predicted several dysregulated pathways, primarily related to cancer, immune system control, or inflammatory processes. The findings suggest that miRNAs may provide insights into the pathophysiology and tumorigenesis of AT and have the potential to serve as useful biomarkers in cancer research.


Asunto(s)
Ataxia Telangiectasia , Leucocitos Mononucleares , MicroARNs , Humanos , Ataxia Telangiectasia/genética , MicroARNs/genética , MicroARNs/sangre , Masculino , Femenino , Adulto , Adolescente , Niño , Adulto Joven , Leucocitos Mononucleares/metabolismo , Fibroblastos/metabolismo , Perfilación de la Expresión Génica , Regulación de la Expresión Génica
17.
Cell Death Dis ; 14(11): 741, 2023 Nov 14.
Artículo en Inglés | MEDLINE | ID: mdl-37963881

RESUMEN

The mammalian nervous system is made up of an extraordinary array of diverse cells that form intricate functional connections. The programs underlying cell lineage specification, identity and function of the neuronal subtypes are managed by regulatory proteins and RNAs, which coordinate the succession of steps in a stereotyped temporal order. In the central nervous system (CNS), motor neurons (MNs) are responsible for controlling essential functions such as movement, breathing, and swallowing by integrating signal transmission from the cortex, brainstem, and spinal cord (SC) towards peripheral muscles. A prime role in guiding the progression of progenitor cells towards the MN fate has been largely attributed to protein factors. More recently, the relevance of a class of regulatory RNAs abundantly expressed in the CNS - the long noncoding RNAs (lncRNAs) - has emerged overwhelmingly. LncRNA-driven gene expression control is key to regulating any step of MN differentiation and function, and its derangement profoundly impacts neuronal pathophysiology. Here, we uncover a novel function for the neuronal isoform of HOTAIRM1 (nHOTAIRM1), a lncRNA specifically expressed in the SC. Using a model system that recapitulates spinal MN (spMN) differentiation, we show that nHOTAIRM1 intervenes in the binary cell fate decision between MNs and interneurons, acting as a pro-MN factor. Furthermore, human iPSC-derived spMNs without nHOTAIRM1 display altered neurite outgrowth, with a significant reduction of both branch and junction numbers. Finally, the expression of genes essential for synaptic connectivity and neurotransmission is also profoundly impaired when nHOTAIRM1 is absent in spMNs. Mechanistically, nHOTAIRM1 establishes both direct and indirect interactions with a number of target genes in the cytoplasm, being a novel post-transcriptional regulator of MN biology. Overall, our results indicate that the lncRNA nHOTAIRM1 is essential for the specification of MN identity and the acquisition of proper morphology and synaptic activity of post-mitotic MNs.


Asunto(s)
Células Madre Pluripotentes Inducidas , ARN Largo no Codificante , Animales , Humanos , ARN Largo no Codificante/genética , ARN Largo no Codificante/metabolismo , Neuronas Motoras/metabolismo , Diferenciación Celular/genética , Médula Espinal/metabolismo , Mamíferos/genética
18.
Nat Cell Biol ; 25(5): 643-657, 2023 05.
Artículo en Inglés | MEDLINE | ID: mdl-37106060

RESUMEN

During embryonic development, naive pluripotent epiblast cells transit to a formative state. The formative epiblast cells form a polarized epithelium, exhibit distinct transcriptional and epigenetic profiles and acquire competence to differentiate into all somatic and germline lineages. However, we have limited understanding of how the transition to a formative state is molecularly controlled. Here we used murine embryonic stem cell models to show that ESRRB is both required and sufficient to activate formative genes. Genetic inactivation of Esrrb leads to illegitimate expression of mesendoderm and extra-embryonic markers, impaired formative expression and failure to self-organize in 3D. Functionally, this results in impaired ability to generate formative stem cells and primordial germ cells in the absence of Esrrb. Computational modelling and genomic analyses revealed that ESRRB occupies key formative genes in naive cells and throughout the formative state. In so doing, ESRRB kickstarts the formative transition, leading to timely and unbiased capacity for multi-lineage differentiation.


Asunto(s)
Células Madre Embrionarias , Células Madre Pluripotentes , Ratones , Animales , Diferenciación Celular/genética , Células Madre Pluripotentes/metabolismo , Estratos Germinativos/metabolismo , Células Germinativas/metabolismo , Receptores de Estrógenos/metabolismo
19.
Methods Mol Biol ; 2284: 343-365, 2021.
Artículo en Inglés | MEDLINE | ID: mdl-33835452

RESUMEN

Thanks to innovative sample-preparation and sequencing technologies, gene expression in individual cells can now be measured for thousands of cells in a single experiment. Since its introduction, single-cell RNA sequencing (scRNA-seq) approaches have revolutionized the genomics field as they created unprecedented opportunities for resolving cell heterogeneity by exploring gene expression profiles at a single-cell resolution. However, the rapidly evolving field of scRNA-seq invoked the emergence of various analytics approaches aimed to maximize the full potential of this novel strategy. Unlike population-based RNA sequencing approaches, scRNA seq necessitates comprehensive computational tools to address high data complexity and keep up with the emerging single-cell associated challenges. Despite the vast number of analytical methods, a universal standardization is lacking. While this reflects the fields' immaturity, it may also encumber a newcomer to blend in.In this review, we aim to bridge over the abovementioned hurdle and propose four ready-to-use pipelines for scRNA-seq analysis easily accessible by a newcomer, that could fit various biological data types. Here we provide an overview of the currently available single-cell technologies for cell isolation and library preparation and a step by step guide that covers the entire canonical analytic workflow to analyse scRNA-seq data including read mapping, quality controls, gene expression quantification, normalization, feature selection, dimensionality reduction, and cell clustering useful for trajectory inference and differential expression. Such workflow guidelines will escort novices as well as expert users in the analysis of complex scRNA-seq datasets, thus further expanding the research potential of single-cell approaches in basic science, and envisaging its future implementation as best practice in the field.


Asunto(s)
Algoritmos , Análisis de Secuencia de ARN/métodos , Análisis de la Célula Individual/métodos , Animales , Análisis por Conglomerados , Perfilación de la Expresión Génica/métodos , Perfilación de la Expresión Génica/estadística & datos numéricos , Genómica/métodos , Secuenciación de Nucleótidos de Alto Rendimiento/métodos , Secuenciación de Nucleótidos de Alto Rendimiento/estadística & datos numéricos , Humanos , Control de Calidad , Análisis de Secuencia de ARN/estadística & datos numéricos , Análisis de la Célula Individual/estadística & datos numéricos , Programas Informáticos , Transcriptoma
20.
Cancers (Basel) ; 13(15)2021 Jul 30.
Artículo en Inglés | MEDLINE | ID: mdl-34359754

RESUMEN

The impact of protein-coding genes on cancer onset and progression is a well-established paradigm in molecular oncology. Nevertheless, unveiling the contribution of the noncoding genes-including long noncoding RNAs (lncRNAs)-to tumorigenesis represents a great challenge for personalized medicine, since they (i) constitute the majority of the human genome, (ii) are essential and flexible regulators of gene expression and (iii) present all types of genomic alterations described for protein-coding genes. LncRNAs have been increasingly associated with cancer, their highly tissue- and cancer type-specific expression making them attractive candidates as both biomarkers and therapeutic targets. Medulloblastoma is one of the most common malignant pediatric brain tumors. Group 3 is the most aggressive subgroup, showing the highest rate of metastasis at diagnosis. Transcriptomics and reverse genetics approaches were combined to identify lncRNAs implicated in Group 3 Medulloblastoma biology. Here we present the first collection of lncRNAs dependent on the activity of the MYC oncogene, the major driver gene of Group 3 Medulloblastoma. We assessed the expression profile of selected lncRNAs in Group 3 primary tumors and functionally characterized these species. Overall, our data demonstrate the direct involvement of three lncRNAs in Medulloblastoma cancer cell phenotypes.

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