RESUMEN
A better understanding of the biological factors underlying antidepressant treatment in patients with major depressive disorder (MDD) is needed. We perform gene expression analyses and explore sources of variability in peripheral blood related to antidepressant treatment and treatment response in patients suffering from recurrent MDD at baseline and after 8 weeks of treatment. The study includes 281 patients, which were randomized to 8 weeks of treatment with vortioxetine (N = 184) or placebo (N = 97). To our knowledge, this is the largest dataset including both gene expression in blood and placebo-controlled treatment response measured by a clinical scale in a randomized clinical trial. We identified three novel genes whose RNA expression levels at baseline and week 8 are significantly (FDR < 0.05) associated with treatment response after 8 weeks of treatment. Among these genes were SOCS3 (FDR = 0.0039) and PROK2 (FDR = 0.0028), which have previously both been linked to depression. Downregulation of these genes was associated with poorer treatment response. We did not identify any genes that were differentially expressed between placebo and vortioxetine groups at week 8 or between baseline and week 8 of treatment. Nor did we replicate any genes identified in previous peripheral blood gene expression studies examining treatment response. Analysis of genome-wide expression variability showed that type of treatment and treatment response explains very little of the variance, a median of <0.0001% and 0.05% in gene expression across all genes, respectively. Given the relatively large size of the study, the limited findings suggest that peripheral blood gene expression might not be the best approach to explore the biological factors underlying antidepressant treatment.
Asunto(s)
Trastorno Depresivo Mayor , Antidepresivos/uso terapéutico , Trastorno Depresivo Mayor/tratamiento farmacológico , Trastorno Depresivo Mayor/genética , Método Doble Ciego , Expresión Génica , Humanos , Ensayos Clínicos Controlados Aleatorios como Asunto , Recurrencia , Resultado del Tratamiento , VortioxetinaRESUMEN
T helper (Th) cells are CD4+ effector T cells that play a critical role in immunity by shaping the inflammatory cytokine environment in a variety of physiological and pathological situations. Using a combined chemico-genetic approach, we identify histone H3K27 demethylases KDM6A and KDM6B as central regulators of human Th subsets. The prototypic KDM6 inhibitor GSK-J4 increases genome-wide levels of the repressive H3K27me3 chromatin mark and leads to suppression of the key transcription factor RORγt during Th17 differentiation. In mature Th17 cells, GSK-J4 induces an altered transcriptional program with a profound metabolic reprogramming and concomitant suppression of IL-17 cytokine levels and reduced proliferation. Single-cell analysis reveals a specific shift from highly inflammatory cell subsets toward a resting state upon demethylase inhibition. The root cause of the observed antiinflammatory phenotype in stimulated Th17 cells is reduced expression of key metabolic transcription factors, such as PPRC1. Overall, this leads to reduced mitochondrial biogenesis, resulting in a metabolic switch with concomitant antiinflammatory effects. These data are consistent with an effect of GSK-J4 on Th17 T cell differentiation pathways directly related to proliferation and include regulation of effector cytokine profiles. This suggests that inhibiting KDM6 demethylases may be an effective, even in the short term, therapeutic target for autoimmune diseases, including ankylosing spondylitis.
Asunto(s)
Benzazepinas/farmacología , Histona Demetilasas/metabolismo , Histonas/metabolismo , Histona Demetilasas con Dominio de Jumonji/metabolismo , Pirimidinas/farmacología , Células Th17/metabolismo , Enfermedades Autoinmunes/tratamiento farmacológico , Enfermedades Autoinmunes/inmunología , Benzazepinas/uso terapéutico , Diferenciación Celular/efectos de los fármacos , Células Cultivadas , Regulación hacia Abajo/efectos de los fármacos , Regulación hacia Abajo/inmunología , Código de Histonas/efectos de los fármacos , Histona Demetilasas/antagonistas & inhibidores , Humanos , Interleucina-17/metabolismo , Histona Demetilasas con Dominio de Jumonji/antagonistas & inhibidores , Cultivo Primario de Células , Pirimidinas/uso terapéutico , RNA-Seq , Espondilitis Anquilosante/tratamiento farmacológico , Espondilitis Anquilosante/inmunología , Células Th17/efectos de los fármacos , Células Th17/inmunología , Factores de Transcripción/metabolismoRESUMEN
Well-validated strategies for discovering potent and efficacious antisense oligonucleotides are central to realize the full therapeutic potential of RNA therapy. In this study, we focus on RNA targets where the same sequence of 16-20 nt is found in several regions across the RNA, and not in any other RNA. Targeting such unique repeated regions with oligonucleotides designed as gapmers and capable of recruiting RNase H has previously been proposed as a strategy for identifying potent gapmers. By sequence analysis of the human and monkey transcriptomes, we find that such unique repeated regions in RNA are often conserved between humans and monkeys, which allow pharmacodynamic effects to be evaluated in non-human primates before testing in humans. For eight potential RNA targets chosen in an unbiased fashion, we targeted their unique repeated regions with locked nucleic acid (LNA)-modified gapmers, and for six of them we identified gapmers that were significantly more potent and efficacious in vitro than non-repeat-targeting gapmer controls. We suggest a stochastic model for repeat-targeting gapmers that explains all effects observed so far and can help guide future work. Our results support the targeting of repeated regions as an effective strategy for discovering gapmer antisense oligonucleotides suitable for therapeutic development.
RESUMEN
Antisense oligonucleotides that are dependent on RNase H for cleavage and subsequent degradation of complementary RNA are being developed as therapeutics. Besides the intended RNA target, such oligonucleotides may also cause degradation of unintended RNA off-targets by binding to partially complementary target sites. Here, we characterized the global effects on the mouse liver transcriptome of four oligonucleotides designed as gapmers, two targeting Apob and two targeting Pcsk9, all in different regions on their respective intended targets. This study design allowed separation of intended- and off-target effects on the transcriptome for each gapmer. Next, we used sequence analysis to identify possible partially complementary binding sites among the potential off-targets, and validated these by measurements of melting temperature and RNase H-cleavage rates. Generally, our observations were as expected in that fewer mismatches or bulges in the gapmer/transcript duplexes resulted in a higher chance of those duplexes being effective substrates for RNase H. Follow-up experiments in mice and cells show, that off-target effects can be mitigated by ensuring that gapmers have minimal sequence complementarity to any RNA besides the intended target, and that they do not have exaggerated binding affinity to the intended target.
Asunto(s)
Terapia Genética/métodos , Ácidos Nucleicos Heterodúplex/metabolismo , Oligonucleótidos Antisentido/metabolismo , ARN Complementario/metabolismo , ARN Mensajero/metabolismo , Ribonucleasa H/metabolismo , Animales , Apolipoproteínas B/genética , Sitios de Unión/genética , Células Cultivadas , Femenino , Hígado/metabolismo , Ratones , Ratones Endogámicos C57BL , Proproteína Convertasa 9/genéticaRESUMEN
Natural killer (NK) cells are innate lymphocytes, important in immune surveillance and elimination of stressed, transformed, or virus-infected cells. They critically shape the inflammatory cytokine environment to orchestrate interactions of cells of the innate and adaptive immune systems. Some studies have reported that NK cell activation and cytokine secretion are controlled epigenetically but have yielded only limited insight into the mechanisms. Using chemical screening with small-molecule inhibitors of chromatin methylation and acetylation, further validated by knockdown approaches, we here identified Jumonji-type histone H3K27 demethylases as key regulators of cytokine production in human NK cell subsets. The prototypic JMJD3/UTX (Jumonji domain-containing protein 3) H3K27 demethylase inhibitor GSK-J4 increased global levels of the repressive H3K27me3 mark around transcription start sites of effector cytokine genes. Moreover, GSK-J4 reduced IFN-γ, TNFα, granulocyte-macrophage colony-stimulating factor (GM-CSF), and interleukin-10 levels in cytokine-stimulated NK cells while sparing their cytotoxic killing activity against cancer cells. The anti-inflammatory effect of GSK-J4 in NK cell subsets, isolated from peripheral blood or tissue from individuals with rheumatoid arthritis (RA), coupled with an inhibitory effect on formation of bone-resorbing osteoclasts, suggested that histone demethylase inhibition has broad utility for modulating immune and inflammatory responses. Overall, our results indicate that H3K27me3 is a dynamic and important epigenetic modification during NK cell activation and that JMJD3/UTX-driven H3K27 demethylation is critical for NK cell function.
Asunto(s)
Artritis Reumatoide/enzimología , Histonas/inmunología , Histona Demetilasas con Dominio de Jumonji/inmunología , Células Asesinas Naturales/enzimología , Secuencias de Aminoácidos , Artritis Reumatoide/genética , Artritis Reumatoide/inmunología , Células Cultivadas , Citocinas/genética , Citocinas/inmunología , Histonas/química , Histonas/genética , Humanos , Histona Demetilasas con Dominio de Jumonji/genética , Células Asesinas Naturales/inmunología , Fenotipo , Factor de Necrosis Tumoral alfa/genética , Factor de Necrosis Tumoral alfa/inmunologíaRESUMEN
RNase H cleaves RNA in RNA-DNA duplexes. It is present in all domains of life as well as in multiple viruses and is essential for mammalian development and for human immunodeficiency virus replication. Here, we developed a sequencing-based method to measure the cleavage of thousands of different RNA-DNA duplexes and thereby comprehensively characterized the sequence preferences of HIV-1, human and Escherichia coli RNase H enzymes. We find that the catalytic domains of E. coli and human RNase H have nearly identical sequence preferences, which correlate with the efficiency of RNase H-recruiting antisense oligonucleotides. The sequences preferred by HIV-1 RNase H are distributed in the HIV genome in a way suggesting selection for efficient RNA cleavage during replication. Our findings can be used to improve the design of RNase H-recruiting antisense oligonucleotides and show that sequence preferences of HIV-1 RNase H may have shaped evolution of the viral genome and contributed to the use of tRNA-Lys3 as primer during viral replication.
Asunto(s)
Oligonucleótidos Antisentido/metabolismo , División del ARN , ARN/metabolismo , Ribonucleasa H/metabolismo , Secuencia de Bases , Dominio Catalítico , Escherichia coli/enzimología , Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Transcriptasa Inversa del VIH/metabolismo , VIH-1/enzimología , VIH-1/genética , Humanos , Modelos Moleculares , Conformación de Ácido Nucleico , Oligonucleótidos Antisentido/química , Oligonucleótidos Antisentido/genética , ARN/química , ARN/genética , Ribonucleasa H/química , Especificidad por Sustrato , Replicación ViralRESUMEN
All drugs perturb the expression of many genes in the cells that are exposed to them. These gene expression changes can be divided into effects resulting from engaging the intended target and effects resulting from engaging unintended targets. For antisense oligonucleotides, developments in bioinformatics algorithms, and the quality of sequence databases, allow oligonucleotide sequences to be analyzed computationally, in terms of the predictability of their interactions with intended and unintended RNA targets. Applying these tools enables selection of sequence-specific oligonucleotides where no- or only few unintended RNA targets are expected. To evaluate oligonucleotide sequence-specificity experimentally, we recommend a transcriptomics protocol where two or more oligonucleotides targeting the same RNA molecule, but with entirely different sequences, are evaluated together. This helps to clarify which changes in cellular RNA levels result from downstream processes of engaging the intended target, and which are likely to be related to engaging unintended targets. As required for all classes of drugs, the toxic potential of oligonucleotides must be evaluated in cell- and animal models before clinical testing. Since potential adverse effects related to unintended targeting are sequence-dependent and therefore species-specific, in vitro toxicology assays in human cells are especially relevant in oligonucleotide drug discovery.
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Descubrimiento de Drogas/métodos , Oligonucleótidos Antisentido/genética , Interferencia de ARN , ARN Interferente Pequeño/genética , Análisis de Secuencia de ARN/estadística & datos numéricos , Animales , Emparejamiento Base , Evaluación Preclínica de Medicamentos , Humanos , Terapia Molecular Dirigida , Oligonucleótidos Antisentido/química , Oligonucleótidos Antisentido/metabolismo , ARN Interferente Pequeño/química , ARN Interferente Pequeño/metabolismo , Ribonucleasa H/genética , Ribonucleasa H/metabolismo , Sensibilidad y Especificidad , TermodinámicaRESUMEN
Processing and post-transcriptional regulation of RNA often depend on binding of regulatory molecules to short motifs in RNA. The effects of such interactions are difficult to study, because most regulatory molecules recognize partially degenerate RNA motifs, embedded in a sequence context specific for each RNA. Here, we describe Library Sequencing (LibSeq), an accurate massively parallel reporter method for completely characterizing the regulatory potential of thousands of short RNA sequences in a specific context. By sequencing cDNA derived from a plasmid library expressing identical reporter genes except for a degenerate 7mer subsequence in the 3'UTR, the regulatory effects of each 7mer can be determined. We show that LibSeq identifies regulatory motifs used by RNA-binding proteins and microRNAs. We furthermore apply the method to cells transfected with RNase H recruiting oligonucleotides to obtain quantitative information for >15000 potential target sequences in parallel. These comprehensive datasets provide insights into the specificity requirements of RNase H and allow a specificity measure to be calculated for each tested oligonucleotide. Moreover, we show that inclusion of chemical modifications in the central part of an RNase H recruiting oligonucleotide can increase its sequence-specificity.
Asunto(s)
Secuenciación de Nucleótidos de Alto Rendimiento/métodos , Oligonucleótidos/química , Secuencias Reguladoras de Ácido Ribonucleico , Ribonucleasa H/metabolismo , Análisis de Secuencia de ARN/métodos , Regiones no Traducidas 3' , Regulación de la Expresión Génica , Biblioteca de Genes , Genes Reporteros , Células HeLa , Humanos , MicroARNs , Motivos de Nucleótidos , Oligonucleótidos/metabolismo , Plásmidos , Unión Proteica , Proteínas de Unión al ARN/metabolismo , TermodinámicaRESUMEN
Antisense oligonucleotides complementary to RNA targets promise generality and ease of drug design. The first systemically administered antisense drug was recently approved for treatment and others are in clinical development. Chemical modifications that increase the hybridization affinity of oligonucleotides are reasoned to confer higher potency, i.e., modified oligonucleotides can be dosed at lower concentrations to achieve the same effect. Surprisingly, shorter and less affine oligonucleotides sometimes display increased potency. To explain this apparent contradiction, increased uptake or decreased propensity to form structures have been suggested as possible mechanisms. Here, we provide an alternative explanation that invokes only the kinetics behind oligonucleotide-mediated cleavage of RNA targets. A model based on the law of mass action predicts, and experiments support, the existence of an optimal binding affinity. Exaggerated affinity, and not length per se, is detrimental to potency. This finding clarifies how to optimally apply high-affinity modifications in the discovery of potent antisense oligonucleotide drugs.
RESUMEN
Quantum mechanical (QM) methodology has been employed to study the structure activity relations of DNA and locked nucleic acid (LNA). The QM calculations provide the basis for construction of molecular structure and electrostatic surface potentials from molecular orbitals. The topologies of the electrostatic potentials were compared among model oligonucleotides, and it was observed that small structural modifications induce global changes in the molecular structure and surface potentials. Since ligand structure and electrostatic potential complementarity with a receptor is a determinant for the bonding pattern between molecules, minor chemical modifications may have profound changes in the interaction profiles of oligonucleotides, possibly leading to changes in pharmacological properties. The QM modeling data can be used to understand earlier observations of antisense oligonucleotide properties, that is, the observation that small structural changes in oligonucleotide composition may lead to dramatic shifts in phenotypes. These observations should be taken into account in future oligonucleotide drug discovery, and by focusing more on non RNA target interactions it should be possible to utilize the exhibited property diversity of oligonucleotides to produce improved antisense drugs.
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ADN/química , Oligonucleótidos Antisentido/química , Oligonucleótidos/química , Humanos , Ligandos , Modelos Moleculares , Estructura Molecular , Conformación de Ácido Nucleico , Teoría Cuántica , Electricidad Estática , TermodinámicaRESUMEN
microRNAs regulate the expression of over 60% of protein coding genes by targeting their mRNAs to AGO2-containing complexes in the cytoplasm and promoting their translational inhibition and/or degradation. There is little evidence so far for microRNA-mediated regulation of other classes of non-coding RNAs. Here we report that microRNA-9 (miR-9) regulates the expression of the Metastasis Associated Lung Adenocarcinoma Transcript 1 (MALAT-1), one of the most abundant and conserved long non-coding RNAs. Intriguingly, we find that miR-9 targets AGO2-mediated regulation of MALAT1 in the nucleus. Our findings reveal a novel direct regulatory link between two important classes of non-coding RNAs, miRs and lncRNAs, and advance our understanding of microRNA functions.
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Núcleo Celular/genética , MicroARNs/genética , Estabilidad del ARN , ARN Largo no Codificante/genética , Proteínas Argonautas/metabolismo , Emparejamiento Base , Secuencia de Bases , Línea Celular Tumoral , Núcleo Celular/metabolismo , Regulación de la Expresión Génica , Humanos , MicroARNs/química , MicroARNs/metabolismo , Conformación de Ácido Nucleico , ARN Largo no Codificante/química , ARN Largo no Codificante/metabolismoRESUMEN
Antisense oligonucleotides that recruit RNase H and thereby cleave complementary messenger RNAs are being developed as therapeutics. Dose-dependent hepatic changes associated with hepatocyte necrosis and increases in serum alanine-aminotransferase levels have been observed after treatment with certain oligonucleotides. Although general mechanisms for drug-induced hepatic injury are known, the characteristics of oligonucleotides that determine their hepatotoxic potential are not well understood. Here, we present a comprehensive analysis of the hepatotoxic potential of locked nucleic acid-modified oligonucleotides in mice. We developed a random forests classifier, in which oligonucleotides are regarded as being composed of dinucleotide units, which distinguished between 206 oligonucleotides with high and low hepatotoxic potential with 80% accuracy as estimated by out-of-bag validation. In a validation set, 17 out of 23 oligonucleotides were correctly predicted (74% accuracy). In isolation, some dinucleotide units increase, and others decrease, the hepatotoxic potential of the oligonucleotides within which they are found. However, a complex interplay between all parts of an oligonucleotide can influence the hepatotoxic potential. Using the classifier, we demonstrate how an oligonucleotide with otherwise high hepatotoxic potential can be efficiently redesigned to abate hepatotoxic potential. These insights establish analysis of sequence and modification patterns as a powerful tool in the preclinical discovery process for oligonucleotide-based medicines.
Asunto(s)
Alanina Transaminasa/sangre , Diseño de Fármacos , Hígado/efectos de los fármacos , Oligonucleótidos Antisentido/toxicidad , Oligonucleótidos/toxicidad , Oligonucleótidos Fosforotioatos/toxicidad , Algoritmos , Animales , Peso Corporal , Femenino , Hígado/patología , Ratones , Ratones Endogámicos C57BL , Conformación de Ácido Nucleico , Oligonucleótidos/administración & dosificación , Oligonucleótidos/síntesis química , Oligonucleótidos Antisentido/administración & dosificación , Oligonucleótidos Antisentido/síntesis química , Tamaño de los Órganos , Oligonucleótidos Fosforotioatos/administración & dosificación , Oligonucleótidos Fosforotioatos/síntesis química , Valor Predictivo de las Pruebas , Relación Estructura-Actividad CuantitativaRESUMEN
MicroRNAs (miRNAs) are important post-transcriptional regulators of nearly every biological process in the cell and play key roles in the pathogenesis of human disease. As a result, there are many drug discovery programs that focus on developing miRNA-based therapeutics. The most advanced of these programs targets the liver-expressed miRNA-122 using the locked nucleic acid (LNA)-modified antisense oligonucleotide miravirsen. Here, we describe the discovery of miravirsen, which is currently in phase 2 clinical trials for treatment of hepatitis C virus (HCV) infection.
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Hepatitis C/terapia , Hígado/metabolismo , MicroARNs/genética , Terapia Molecular Dirigida , Oligonucleótidos Antisentido/uso terapéutico , Regulación de la Expresión Génica , Hepacivirus/genética , Hepatitis C/genética , Humanos , Oligonucleótidos/administración & dosificaciónRESUMEN
RATIONALE: Viral myocarditis results from an adverse immune response to cardiotropic viruses, which causes irreversible myocyte destruction and heart failure in previously healthy people. The involvement of microRNAs and their usefulness as therapeutic targets in this process are unknown. OBJECTIVE: To identify microRNAs involved in viral myocarditis pathogenesis and susceptibility. METHODS AND RESULTS: Cardiac microRNAs were profiled in both human myocarditis and in Coxsackievirus B3-injected mice, comparing myocarditis-susceptible with nonsusceptible mouse strains longitudinally. MicroRNA responses diverged depending on the susceptibility to myocarditis after viral infection in mice. MicroRNA-155, -146b, and -21 were consistently and strongly upregulated during acute myocarditis in both humans and susceptible mice. We found that microRNA-155 expression during myocarditis was localized primarily in infiltrating macrophages and T lymphocytes. Inhibition of microRNA-155 by a systemically delivered LNA-anti-miR attenuated cardiac infiltration by monocyte-macrophages, decreased T lymphocyte activation, and reduced myocardial damage during acute myocarditis in mice. These changes were accompanied by the derepression of the direct microRNA-155 target PU.1 in cardiac inflammatory cells. Beyond the acute phase, microRNA-155 inhibition reduced mortality and improved cardiac function during 7 weeks of follow-up. CONCLUSIONS: Our data show that cardiac microRNA dysregulation is a characteristic of both human and mouse viral myocarditis. The inflammatory microRNA-155 is upregulated during acute myocarditis, contributes to the adverse inflammatory response to viral infection of the heart, and is a potential therapeutic target for viral myocarditis.
Asunto(s)
Infecciones por Coxsackievirus/genética , Perfilación de la Expresión Génica , MicroARNs/metabolismo , Miocarditis/genética , Miocardio/metabolismo , Animales , Infecciones por Coxsackievirus/inmunología , Infecciones por Coxsackievirus/patología , Infecciones por Coxsackievirus/fisiopatología , Infecciones por Coxsackievirus/terapia , Infecciones por Coxsackievirus/virología , Modelos Animales de Enfermedad , Enterovirus Humano B/patogenicidad , Femenino , Perfilación de la Expresión Génica/métodos , Humanos , Activación de Linfocitos , Macrófagos/inmunología , Macrófagos/metabolismo , Macrófagos/virología , Ratones , Ratones Endogámicos C3H , Ratones Endogámicos C57BL , Miocarditis/inmunología , Miocarditis/patología , Miocarditis/fisiopatología , Miocarditis/terapia , Miocarditis/virología , Miocardio/inmunología , Miocardio/patología , Oligonucleótidos/administración & dosificación , Linfocitos T/inmunología , Linfocitos T/metabolismo , Linfocitos T/virología , Factores de TiempoRESUMEN
MicroRNAs (miRNAs) have emerged as important post-transcriptional regulators of gene expression in many developmental and cellular processes. Moreover, there is now ample evidence that perturbations in the levels of individual or entire families of miRNAs are strongly associated with the pathogenesis of a wide range of human diseases. Indeed, disease-associated miRNAs represent a new class of targets for the development of miRNA-based therapeutic modalities, which may yield patient benefits unobtainable by other therapeutic approaches. The recent explosion in miRNA research has accelerated the development of several computational and experimental approaches for probing miRNA functions in cell culture and in vivo. In this review, we focus on the use of antisense oligonucleotides (antimiRs) in miRNA inhibition for loss-of-function studies. We provide an overview of the currently employed antisense chemistries and their utility in designing antimiR oligonucleotides. Furthermore, we describe the most commonly used in vivo delivery strategies and discuss different approaches for assessment of miRNA inhibition and potential off-target effects. Finally, we summarize recent progress in antimiR mediated pharmacological inhibition of disease-associated miRNAs, which shows great promise in the development of novel miRNA-based therapeutics.
RESUMEN
microRNAs are non-coding RNAs that regulate gene expression. A significant proportion of microRNAs is perfectly conserved across the vertebrate clade, including miR-140, which is specifically expressed in cartilage. Although it has been computationally predicted that a large majority of microRNA targets are conserved, experimental evidence for this hypothesis remains scarce. In this work we use mRNA expression profiles obtained after manipulation of miR-140 activity levels in human and chicken primary chondrocytes to explore the extent of miR-140 target conservation. Our data suggest that miR-140 has a large number of targets conserved between human and chicken and we validate one of these, BMP2. However, we also found a significant number of non-conserved targets in the two species. In addition, we found that a commercially available scrambled siRNA, which is regularly used as a negative control, regulate the accumulation of many genes.
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Proteína Morfogenética Ósea 2 , MicroARNs/genética , MicroARNs/metabolismo , ARN Mensajero/genética , Animales , Proteína Morfogenética Ósea 2/biosíntesis , Proteína Morfogenética Ósea 2/genética , Proteína Morfogenética Ósea 2/metabolismo , Células Cultivadas , Pollos , Condrocitos/citología , Condrocitos/metabolismo , Secuencia Conservada , Perfilación de la Expresión Génica , Regulación de la Expresión Génica , Humanos , Interferencia de ARN , ARN Mensajero/metabolismo , ARN Interferente Pequeño , Alineación de SecuenciaRESUMEN
Systemic iron homeostasis is mainly controlled by the liver through synthesis of the peptide hormone hepcidin (encoded by Hamp), the key regulator of duodenal iron absorption and macrophage iron release. Here we show that the liver-specific microRNA miR-122 is important for regulating Hamp mRNA expression and tissue iron levels. Efficient and specific depletion of miR-122 by injection of a locked-nucleic-acid-modified (LNA-modified) anti-miR into WT mice caused systemic iron deficiency, characterized by reduced plasma and liver iron levels, mildly impaired hematopoiesis, and increased extramedullary erythropoiesis in the spleen. Moreover, miR-122 inhibition increased the amount of mRNA transcribed by genes that control systemic iron levels, such as hemochromatosis (Hfe), hemojuvelin (Hjv), bone morphogenetic protein receptor type 1A (Bmpr1a), and Hamp. Importantly, miR-122 directly targeted the 3' untranslated region of 2 mRNAs that encode activators of hepcidin expression, Hfe and Hjv. These data help to explain the increased Hamp mRNA levels and subsequent iron deficiency in mice with reduced miR-122 levels and establish a direct mechanistic link between miR-122 and the regulation of systemic iron metabolism.
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Hierro/metabolismo , Hígado/metabolismo , MicroARNs/genética , MicroARNs/metabolismo , Regiones no Traducidas 3' , Animales , Péptidos Catiónicos Antimicrobianos/genética , Péptidos Catiónicos Antimicrobianos/metabolismo , Sitios de Unión/genética , Receptores de Proteínas Morfogenéticas Óseas de Tipo 1/genética , Regulación hacia Abajo , Femenino , Perfilación de la Expresión Génica , Hematopoyesis Extramedular/genética , Hematopoyesis Extramedular/fisiología , Proteína de la Hemocromatosis , Hepcidinas , Antígenos de Histocompatibilidad Clase I/genética , Homeostasis , Hierro/sangre , Deficiencias de Hierro , Masculino , Proteínas de la Membrana/deficiencia , Proteínas de la Membrana/genética , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , MicroARNs/antagonistas & inhibidores , Oligonucleótidos/farmacología , ARN Mensajero/genética , ARN Mensajero/metabolismoRESUMEN
The challenge of understanding the widespread biological roles of animal microRNAs (miRNAs) has prompted the development of genetic and functional genomics technologies for miRNA loss-of-function studies. However, tools for exploring the functions of entire miRNA families are still limited. We developed a method that enables antagonism of miRNA function using seed-targeting 8-mer locked nucleic acid (LNA) oligonucleotides, termed tiny LNAs. Transfection of tiny LNAs into cells resulted in simultaneous inhibition of miRNAs within families sharing the same seed with concomitant upregulation of direct targets. In addition, systemically delivered, unconjugated tiny LNAs showed uptake in many normal tissues and in breast tumors in mice, coinciding with long-term miRNA silencing. Transcriptional and proteomic profiling suggested that tiny LNAs have negligible off-target effects, not significantly altering the output from mRNAs with perfect tiny LNA complementary sites. Considered together, these data support the utility of tiny LNAs in elucidating the functions of miRNA families in vivo.