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1.
Nat Commun ; 12(1): 3339, 2021 06 07.
Artículo en Inglés | MEDLINE | ID: mdl-34099655

RESUMEN

The intestinal epithelium is a complex structure that integrates digestive, immunological, neuroendocrine, and regenerative functions. Epithelial homeostasis is maintained by a coordinated cross-talk of different epithelial cell types. Loss of integrity of the intestinal epithelium plays a key role in inflammatory diseases and gastrointestinal infection. Here we show that the intestine-enriched miR-802 is a central regulator of intestinal epithelial cell proliferation, Paneth cell function, and enterocyte differentiation. Genetic ablation of mir-802 in the small intestine of mice leads to decreased glucose uptake, impaired enterocyte differentiation, increased Paneth cell function and intestinal epithelial proliferation. These effects are mediated in part through derepression of the miR-802 target Tmed9, a modulator of Wnt and lysozyme/defensin secretion in Paneth cells, and the downstream Wnt signaling components Fzd5 and Tcf4. Mutant Tmed9 mice harboring mutations in miR-802 binding sites partially recapitulate the augmented Paneth cell function of mice lacking miR-802. Our study demonstrates a broad miR-802 network that is important for the integration of signaling pathways of different cell types controlling epithelial homeostasis in the small intestine.


Asunto(s)
Diferenciación Celular/fisiología , Enterocitos/metabolismo , Intestino Delgado/metabolismo , MicroARNs/genética , MicroARNs/metabolismo , Células de Paneth/metabolismo , Animales , Proliferación Celular , Femenino , Receptores Frizzled/metabolismo , Expresión Génica , Células HEK293 , Homeostasis/fisiología , Humanos , Mucosa Intestinal/metabolismo , Intestinos , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Salmonella typhimurium , Factor de Transcripción 4/metabolismo , Transcriptoma , Proteínas de Transporte Vesicular/genética , Proteínas de Transporte Vesicular/metabolismo , Vía de Señalización Wnt
2.
PLoS One ; 12(9): e0185028, 2017.
Artículo en Inglés | MEDLINE | ID: mdl-28931048

RESUMEN

Cutaneous squamous cell carcinoma (SCC) is the second most common human skin cancer with a rapidly increasing incidence among the Caucasian population. Among the many regulators, responsible for cancer progression and growth, microRNAs (miRNA) are generally accepted as key players by now. In our current study we found that microRNA-181a (miR-181a) shows low abundance in SCC compared to normal epidermal skin. In vitro, miRNA downregulation in normal primary keratinocytes induced increased proliferation, while in vivo miR-181a downregulation in HaCaT normal keratinocytes showed tumor-like growth increase up to 50%. Inversely, upregulation of these miRNAs in cancer cells lead to reduced cellular proliferation and induction of apoptosis in vitro. An in vivo therapeutic model with induced miR-181a expression in SCC13 cancer cells reduced tumor formation in mice by 80%. Modulation of miR-181a levels showed an inverse correlation with the proto-oncogene KRAS both on mRNA and protein level by direct interaction. Knockdown of KRAS mimicked the anti-proliferative effects of miR-181a overexpression in patient-derived SCC cells and abolished the enhanced viability of HaCaT cells following miR-181a knockdown. Furthermore, phospho-ERK levels correlated with KRAS levels, suggesting that the observed effects were mediated via the MAPK signaling pathway. miR-181a seemed regulated during keratinocyte differentiation probably in order to amplify the tumor suppressive character of differentiation. Taken together, miR-181a plays a crucial tumor suppressive role in SCC by targeting KRAS and could be a promising candidate for a miRNA based therapy.


Asunto(s)
Carcinoma de Células Escamosas/patología , Proliferación Celular , Regulación Neoplásica de la Expresión Génica , MicroARNs/genética , Proteínas Proto-Oncogénicas p21(ras)/metabolismo , Neoplasias Cutáneas/patología , Piel/patología , Animales , Apoptosis , Carcinoma de Células Escamosas/genética , Carcinoma de Células Escamosas/metabolismo , Diferenciación Celular , Movimiento Celular , Femenino , Humanos , Ratones , Ratones Desnudos , Proto-Oncogenes Mas , Proteínas Proto-Oncogénicas p21(ras)/genética , Transducción de Señal , Piel/metabolismo , Neoplasias Cutáneas/genética , Neoplasias Cutáneas/metabolismo
3.
Nat Commun ; 8(1): 480, 2017 09 07.
Artículo en Inglés | MEDLINE | ID: mdl-28883393

RESUMEN

Nonalcoholic fatty liver disease is one of the most prevalent metabolic disorders and it tightly associates with obesity, type 2 diabetes, and cardiovascular disease. Reduced mitochondrial lipid oxidation contributes to hepatic fatty acid accumulation. Here, we show that the Fas cell surface death receptor (Fas/CD95/Apo-1) regulates hepatic mitochondrial metabolism. Hepatic Fas overexpression in chow-fed mice compromises fatty acid oxidation, mitochondrial respiration, and the abundance of mitochondrial respiratory complexes promoting hepatic lipid accumulation and insulin resistance. In line, hepatocyte-specific ablation of Fas improves mitochondrial function and ameliorates high-fat-diet-induced hepatic steatosis, glucose tolerance, and insulin resistance. Mechanistically, Fas impairs fatty acid oxidation via the BH3 interacting-domain death agonist (BID). Mice with genetic or pharmacological inhibition of BID are protected from Fas-mediated impairment of mitochondrial oxidation and hepatic steatosis. We suggest Fas as a potential novel therapeutic target to treat obesity-associated fatty liver and insulin resistance.Hepatic steatosis is a common disease closely associated with metabolic syndrome and insulin resistance. Here Item et al. show that Fas, a member of the TNF receptor superfamily, contributes to mitochondrial dysfunction, steatosis development, and insulin resistance under high fat diet.


Asunto(s)
Metabolismo de los Lípidos/fisiología , Hígado/metabolismo , Mitocondrias Hepáticas/metabolismo , Receptor fas/metabolismo , Animales , Dieta Alta en Grasa , Proteína Ligando Fas/genética , Proteína Ligando Fas/metabolismo , Ácidos Grasos/metabolismo , Resistencia a la Insulina , Masculino , Ratones Endogámicos C57BL , Ratones Noqueados , Ratones Obesos , Ratones Transgénicos , Enfermedad del Hígado Graso no Alcohólico/etiología , Enfermedad del Hígado Graso no Alcohólico/genética , Triglicéridos/metabolismo , Receptor fas/genética
4.
J Clin Invest ; 127(3): 1061-1074, 2017 Mar 01.
Artículo en Inglés | MEDLINE | ID: mdl-28218624

RESUMEN

MicroRNAs (miRNAs) are negative modulators of gene expression that fine-tune numerous biological processes. miRNA loss-of-function rarely results in highly penetrant phenotypes, but rather, influences cellular responses to physiologic and pathophysiologic stresses. Here, we have reported that a single member of the evolutionarily conserved miR-7 family, miR-7a2, is essential for normal pituitary development and hypothalamic-pituitary-gonadal (HPG) function in adulthood. Genetic deletion of mir-7a2 causes infertility, with low levels of gonadotropic and sex steroid hormones, small testes or ovaries, impaired spermatogenesis, and lack of ovulation in male and female mice, respectively. We found that miR-7a2 is highly expressed in the pituitary, where it suppresses golgi glycoprotein 1 (GLG1) expression and downstream bone morphogenetic protein 4 (BMP4) signaling and also reduces expression of the prostaglandin F2a receptor negative regulator (PTGFRN), an inhibitor of prostaglandin signaling and follicle-stimulating hormone (FSH) and luteinizing hormone (LH) secretion. Our results reveal that miR-7a2 critically regulates sexual maturation and reproductive function by interconnecting miR-7 genomic circuits that regulate FSH and LH synthesis and secretion through their effects on pituitary prostaglandin and BMP4 signaling.


Asunto(s)
Hipogonadismo/metabolismo , Sistema Hipotálamo-Hipofisario/metabolismo , Infertilidad/metabolismo , MicroARNs/metabolismo , Transducción de Señal , Animales , Proteína Morfogenética Ósea 4/genética , Proteína Morfogenética Ósea 4/metabolismo , Femenino , Hormona Folículo Estimulante/genética , Hormona Folículo Estimulante/metabolismo , Hormonas Esteroides Gonadales/genética , Hormonas Esteroides Gonadales/metabolismo , Hipogonadismo/genética , Infertilidad/genética , Hormona Luteinizante/genética , Hormona Luteinizante/metabolismo , Masculino , Ratones , Ratones Noqueados , MicroARNs/genética , Ovario/metabolismo , Receptores de Factores de Crecimiento de Fibroblastos/genética , Receptores de Factores de Crecimiento de Fibroblastos/metabolismo , Sialoglicoproteínas/genética , Sialoglicoproteínas/metabolismo , Testículo/metabolismo
5.
Mol Cell ; 64(3): 565-579, 2016 11 03.
Artículo en Inglés | MEDLINE | ID: mdl-27871486

RESUMEN

Expression changes of competing endogenous RNAs (ceRNAs) have been proposed to influence microRNA (miRNA) activity and thereby regulate other transcripts containing miRNA-binding sites. Here, we find that although miRNA levels define the extent of repression, they have little effect on the magnitude of the ceRNA expression change required to observe derepression. Canonical 6-nt sites, which typically mediate modest repression, can nonetheless compete for miRNA binding, with potency ∼20% of that observed for canonical 8-nt sites. In aggregate, low-affinity/background sites also contribute to competition. Sites with extensive additional complementarity can appear as more potent, but only because they induce miRNA degradation. Cooperative binding of proximal sites for the same or different miRNAs does increase potency. These results provide quantitative insights into the stoichiometric relationship between miRNAs and target abundance, target-site spacing, and affinity requirements for ceRNA-mediated gene regulation, and the unusual circumstances in which ceRNA-mediated gene regulation might be observed.


Asunto(s)
Regulación de la Expresión Génica , Redes Reguladoras de Genes , Hepatocitos/metabolismo , MicroARNs/genética , ARN Mensajero/genética , Adenoviridae/genética , Adenoviridae/metabolismo , Animales , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Sitios de Unión , Unión Competitiva , Genes Reporteros , Hepatocitos/citología , Proteínas Luminiscentes/genética , Proteínas Luminiscentes/metabolismo , Masculino , Ratones , Ratones Endogámicos C57BL , MicroARNs/metabolismo , Plásmidos/química , Plásmidos/metabolismo , Cultivo Primario de Células , ARN Mensajero/metabolismo , Transformación Genética , Proteína Fluorescente Roja
6.
Mol Cell ; 60(1): 4-6, 2015 Oct 01.
Artículo en Inglés | MEDLINE | ID: mdl-26431024

RESUMEN

In this issue, Fang and Bartel (2015) report the identification of novel sequence and structural features of human pri-miRNAs, which--together with previously identified sequence motifs--define a unifying model of mammalian pri-miRNAs and advances the de novo design of artificial pri-miRNAs.


Asunto(s)
Biología Computacional/métodos , MicroARNs/metabolismo , Precursores del ARN/química , Humanos
7.
J Biol Chem ; 290(41): 25198, 2015 Oct 09.
Artículo en Inglés | MEDLINE | ID: mdl-26462297
8.
J Biol Chem ; 290(39): 23680-91, 2015 Sep 25.
Artículo en Inglés | MEDLINE | ID: mdl-26240150

RESUMEN

MicroRNAs (miRNAs) are important regulators of cell-autonomous gene expression that influence many biological processes. They are also released from cells and are present in virtually all body fluids, including blood, urine, saliva, sweat, and milk. The functional role of nutritionally obtained extracellular miRNAs is controversial, and irrefutable demonstration of exogenous miRNA uptake by cells and canonical miRNA function is still lacking. Here we show that miRNAs are present at high levels in the milk of lactating mice. To investigate intestinal uptake of miRNAs in newborn mice, we employed genetic models in which newborn miR-375 and miR-200c/141 knockout mice received milk from wild-type foster mothers. Analysis of the intestinal epithelium, blood, liver, and spleen revealed no evidence for miRNA uptake. miR-375 levels in hepatocytes were at the limit of detection and remained orders of magnitude below the threshold for target gene regulation (between 1000 and 10,000 copies/cell). Furthermore, our study revealed rapid degradation of milk miRNAs in intestinal fluid. Together, our results indicate a nutritional rather than gene-regulatory role of miRNAs in the milk of newborn mice.


Asunto(s)
MicroARNs/metabolismo , Leche , Animales , Animales Recién Nacidos , Femenino , Silenciador del Gen , Mucosa Intestinal/metabolismo , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , MicroARNs/genética , MicroARNs/fisiología
9.
Nat Med ; 21(6): 619-27, 2015 Jun.
Artículo en Inglés | MEDLINE | ID: mdl-25985365

RESUMEN

Pancreatic beta cell death is a hallmark of type 1 (T1D) and type 2 (T2D) diabetes, but the molecular mechanisms underlying this aspect of diabetic pathology are poorly understood. Here we report that expression of the microRNA (miR)-200 family is strongly induced in islets of diabetic mice and that beta cell-specific overexpression of miR-200 in mice is sufficient to induce beta cell apoptosis and lethal T2D. Conversely, mir-200 ablation in mice reduces beta cell apoptosis and ameliorates T2D. We show that miR-200 negatively regulates a conserved anti-apoptotic and stress-resistance network that includes the essential beta cell chaperone Dnajc3 (also known as p58IPK) and the caspase inhibitor Xiap. We also observed that mir-200 dosage positively controls activation of the tumor suppressor Trp53 and thereby creates a pro-apoptotic gene-expression signature found in islets of diabetic mice. Consequently, miR-200-induced T2D is suppressed by interfering with the signaling of Trp53 and Bax, a proapoptotic member of the B cell lymphoma 2 protein family. Our results reveal a crucial role for the miR-200 family in beta cell survival and the pathophysiology of diabetes.


Asunto(s)
Diabetes Mellitus Tipo 2/genética , Células Secretoras de Insulina/metabolismo , MicroARNs/genética , Animales , Apoptosis/genética , Supervivencia Celular/genética , Diabetes Mellitus Tipo 2/patología , Regulación de la Expresión Génica , Proteínas del Choque Térmico HSP40/biosíntesis , Humanos , Insulina/metabolismo , Células Secretoras de Insulina/patología , Ratones , Ratones Endogámicos NOD , MicroARNs/metabolismo , Transducción de Señal , Proteína Inhibidora de la Apoptosis Ligada a X/biosíntesis
10.
Mol Cell ; 54(5): 766-76, 2014 Jun 05.
Artículo en Inglés | MEDLINE | ID: mdl-24793693

RESUMEN

Recent studies have reported that competitive endogenous RNAs (ceRNAs) can act as sponges for a microRNA (miRNA) through their binding sites and that changes in ceRNA abundances from individual genes can modulate the activity of miRNAs. Consideration of this hypothesis would benefit from knowing the quantitative relationship between a miRNA and its endogenous target sites. Here, we altered intracellular target site abundance through expression of an miR-122 target in hepatocytes and livers and analyzed the effects on miR-122 target genes. Target repression was released in a threshold-like manner at high target site abundance (≥1.5 × 10(5) added target sites per cell), and this threshold was insensitive to the effective levels of the miRNA. Furthermore, in response to extreme metabolic liver disease models, global target site abundance of hepatocytes did not change sufficiently to affect miRNA-mediated repression. Thus, modulation of miRNA target abundance is unlikely to cause significant effects on gene expression and metabolism through a ceRNA effect.


Asunto(s)
MicroARNs/genética , Interferencia de ARN , Animales , Células Cultivadas , Fructosa-Bifosfato Aldolasa/genética , Fructosa-Bifosfato Aldolasa/metabolismo , Hepatocitos/metabolismo , Hígado/metabolismo , Masculino , Ratones , Ratones Endogámicos C57BL , Elementos de Respuesta , Estrés Fisiológico , Transcriptoma
11.
PLoS Pathog ; 10(1): e1003844, 2014 Jan.
Artículo en Inglés | MEDLINE | ID: mdl-24391500

RESUMEN

The host's immune system plays a key role in modulating growth of pathogens and the intestinal microbiota in the gut. In particular, inflammatory bowel disorders and pathogen infections induce shifts of the resident commensal microbiota which can result in overgrowth of Enterobacteriaceae ("inflammation-inflicted blooms"). Here, we investigated competition of the human pathogenic Salmonella enterica serovar Typhimurium strain SL1344 (S. Tm) and commensal E. coli in inflammation-inflicted blooms. S. Tm produces colicin Ib (ColIb), which is a narrow-spectrum protein toxin active against related Enterobacteriaceae. Production of ColIb conferred a competitive advantage to S. Tm over sensitive E. coli strains in the inflamed gut. In contrast, an avirulent S. Tm mutant strain defective in triggering gut inflammation did not benefit from ColIb. Expression of ColIb (cib) is regulated by iron limitation and the SOS response. CirA, the cognate outer membrane receptor of ColIb on colicin-sensitive E. coli, is induced upon iron limitation. We demonstrate that growth in inflammation-induced blooms favours expression of both S. Tm ColIb and the receptor CirA, thereby fuelling ColIb dependent competition of S. Tm and commensal E. coli in the gut. In conclusion, this study uncovers a so-far unappreciated role of inflammation-inflicted blooms as an environment favouring ColIb-dependent competition of pathogenic and commensal representatives of the Enterobacteriaceae family.


Asunto(s)
Colicinas/metabolismo , Escherichia coli/metabolismo , Intestinos/microbiología , Infecciones por Salmonella/metabolismo , Salmonella typhimurium/metabolismo , Animales , Colicinas/genética , Humanos , Hierro/metabolismo , Ratones , Respuesta SOS en Genética/fisiología , Infecciones por Salmonella/genética , Salmonella typhimurium/genética
12.
Nucleic Acids Res ; 42(1): 609-21, 2014 Jan.
Artículo en Inglés | MEDLINE | ID: mdl-24068553

RESUMEN

MicroRNAs (miRNAs) are short noncoding RNAs, which bind to messenger RNAs and regulate protein expression. The biosynthesis of miRNAs includes two precursors, a primary miRNA transcript (pri-miRNA) and a shorter pre-miRNA, both of which carry a common stem-loop bearing the mature miRNA. MiR-122 is a liver-specific miRNA with an important role in the life cycle of hepatitis C virus (HCV). It is the target of miravirsen (SPC3649), an antimiR drug candidate currently in clinical testing for treatment of HCV infections. Miravirsen is composed of locked nucleic acid (LNAs) ribonucleotides interspaced throughout a DNA phosphorothioate sequence complementary to mature miR-122. The LNA modifications endow the drug with high affinity for its target and provide resistance to nuclease degradation. While miravirsen is thought to work mainly by hybridizing to mature miR-122 and blocking its interaction with HCV RNA, its target sequence is also present in pri- and pre-miR-122. Using new in vitro and cellular assays specifically developed to discover ligands that suppress biogenesis of miR-122, we show that miravirsen binds to the stem-loop structure of pri- and pre-miR-122 with nanomolar affinity, and inhibits both Dicer- and Drosha-mediated processing of miR-122 precursors. This inhibition may contribute to the pharmacological activity of the drug in man.


Asunto(s)
MicroARNs/biosíntesis , Oligonucleótidos/metabolismo , Animales , Línea Celular , Células Cultivadas , Humanos , Masculino , Ratones , Ratones Endogámicos C57BL , MicroARNs/química , MicroARNs/metabolismo , Conformación de Ácido Nucleico , Precursores del ARN/biosíntesis , Precursores del ARN/química
13.
Proc Natl Acad Sci U S A ; 109(4): 1269-74, 2012 Jan 24.
Artículo en Inglés | MEDLINE | ID: mdl-22232693

RESUMEN

The mammalian gut harbors a dense microbial community interacting in multiple ways, including horizontal gene transfer (HGT). Pangenome analyses established particularly high levels of genetic flux between Gram-negative Enterobacteriaceae. However, the mechanisms fostering intraenterobacterial HGT are incompletely understood. Using a mouse colitis model, we found that Salmonella-inflicted enteropathy elicits parallel blooms of the pathogen and of resident commensal Escherichia coli. These blooms boosted conjugative HGT of the colicin-plasmid p2 from Salmonella enterica serovar Typhimurium to E. coli. Transconjugation efficiencies of ~100% in vivo were attributable to high intrinsic p2-transfer rates. Plasmid-encoded fitness benefits contributed little. Under normal conditions, HGT was blocked by the commensal microbiota inhibiting contact-dependent conjugation between Enterobacteriaceae. Our data show that pathogen-driven inflammatory responses in the gut can generate transient enterobacterial blooms in which conjugative transfer occurs at unprecedented rates. These blooms may favor reassortment of plasmid-encoded genes between pathogens and commensals fostering the spread of fitness-, virulence-, and antibiotic-resistance determinants.


Asunto(s)
Evolución Biológica , Colitis/microbiología , Enterobacteriaceae/genética , Transferencia de Gen Horizontal/genética , Animales , Plásmidos de Bacteriocinas/genética , Secuencia de Bases , Biología Computacional , Cartilla de ADN/genética , Enterobacteriaceae/crecimiento & desarrollo , Escherichia coli/genética , Ratones , Datos de Secuencia Molecular , Análisis de Secuencia por Matrices de Oligonucleótidos , Filogenia , ARN Ribosómico 16S/genética , Salmonella typhimurium/genética , Alineación de Secuencia , Análisis de Secuencia de ADN
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