RESUMEN
Current understanding of microRNA (miRNA) biology is limited, and antisense oligonucleotide (ASO) inhibition of miRNAs is a powerful technique for their functionalization. To uncover the role of the liver-specific miR-122 in the adult liver, we inhibited it in mice with a 2'-O-methoxyethyl phosphorothioate ASO. miR-122 inhibition in normal mice resulted in reduced plasma cholesterol levels, increased hepatic fatty-acid oxidation, and a decrease in hepatic fatty-acid and cholesterol synthesis rates. Activation of the central metabolic sensor AMPK was also increased. miR-122 inhibition in a diet-induced obesity mouse model resulted in decreased plasma cholesterol levels and a significant improvement in liver steatosis, accompanied by reductions in several lipogenic genes. These results implicate miR-122 as a key regulator of cholesterol and fatty-acid metabolism in the adult liver and suggest that miR-122 may be an attractive therapeutic target for metabolic disease.
Asunto(s)
Metabolismo de los Lípidos/fisiología , Hígado/metabolismo , MicroARNs/antagonistas & inhibidores , MicroARNs/metabolismo , Obesidad/metabolismo , Oligonucleótidos Antisentido/farmacología , Proteínas Quinasas Activadas por AMP , Animales , Análisis Químico de la Sangre , Northern Blotting , Western Blotting , Línea Celular , Colesterol/sangre , Cromatografía Líquida de Alta Presión , Cartilla de ADN , Activación Enzimática/efectos de los fármacos , Ácidos Grasos/metabolismo , Regulación de la Expresión Génica/efectos de los fármacos , Hígado/citología , Ratones , Análisis por Micromatrices , Datos de Secuencia Molecular , Complejos Multienzimáticos/metabolismo , Oligonucleótidos Antisentido/genética , Proteínas Serina-Treonina Quinasas/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa InversaRESUMEN
Chemically modified antisense oligonucleotides (ASOs) are widely used as a tool to functionalize microRNAs (miRNAs). Reduction of miRNA level after ASO inhibition is commonly reported to show efficacy. Whether this is the most relevant endpoint for measuring miRNA inhibition has not been adequately addressed in the field although it has important implications for evaluating miRNA targeting studies. Using a novel approach to quantitate miRNA levels in the presence of excess ASO, we have discovered that the outcome of miRNA inhibition can vary depending on the chemical modification of the ASO. Although some miRNA inhibitors cause a decrease in mature miRNA levels, we have identified a novel 2'-fluoro/2'-methoxyethyl modified ASO motif with dramatically improved in vivo potency which does not. These studies show there are multiple mechanisms of miRNA inhibition by ASOs and that evaluation of secondary endpoints is crucial for interpreting miRNA inhibition studies.
Asunto(s)
MicroARNs/antagonistas & inhibidores , Oligonucleótidos Antisentido/farmacología , Animales , Regulación de la Expresión Génica , Hígado/metabolismo , Masculino , Ratones , Ratones Endogámicos C57BL , MicroARNs/análisis , MicroARNs/metabolismo , Oligonucleótidos Antisentido/química , Ácidos Nucleicos de Péptidos/químicaRESUMEN
Short interfering RNAs (siRNA) guide degradation of target RNA by the RNA-induced silencing complex (RISC). The use of siRNA in animals is limited partially due to the short half-life of siRNAs in tissues. Chemically modified siRNAs are necessary that maintain mRNA degradation activity, but are more stable to nucleases. In this study, we utilized alternating 2'-O-methyl and 2'-deoxy-2'-fluoro (OMe/F) chemically modified siRNA targeting PTEN and Eg5. OMe/F-modified siRNA consistently reduced mRNA and protein levels with equal or greater potency and efficacy than unmodified siRNA. We showed that modified siRNAs use the RISC mechanism and lead to cleavage of target mRNA at the same position as unmodified siRNA. We further demonstrated that siRNAs can compete with each other, where highly potent siRNAs can compete with less potent siRNAs, thus limiting the ability of siRNAs with lower potency to mediate mRNA degradation. In contrast, a siRNA with low potency cannot compete with a highly efficient siRNA. We established a correlation between siRNA potency and ability to compete with other siRNAs. Thus, siRNAs that are more potent inhibitors for mRNA destruction have the potential to out-compete less potent siRNAs indicating that the amount of a cellular component, perhaps RISC, limits siRNA activity.
Asunto(s)
Interferencia de ARN , ARN Interferente Pequeño/química , Complejo Silenciador Inducido por ARN/metabolismo , Unión Competitiva , Línea Celular Tumoral , Humanos , Cinesinas/genética , Fosfohidrolasa PTEN/genética , ARN Mensajero/metabolismo , Ribonucleasas/metabolismoRESUMEN
A library of 2'-methoxyethyl-modified antisense oligonucleotides (2'MOE ASO) targeting 1,510 different genes has been developed, validated, and used to identify cell cycle regulatory genes. The most effective molecular target identified was Eg5 (kinesin-like-1), which when inhibited gave the largest increase in 4N DNA in various tumor cells. The Eg5 ASO reduced Eg5 levels, inhibited proliferation, increased apoptosis, and altered the expression of other cell cycle proteins, including survivin and Aurora-A. To examine the therapeutic utility of the Eg5 ASO, the compound was also evaluated in xenograft models. Treatment with Eg5 ASO produced a statistically significant reduction of tumor growth, reduction in Eg5 expression in the tumors, and changes in histone phosphorylation, consistent with a loss of Eg5 protein expression. These data show, for the first time, the utility of a 2'MOE ASO library for high-throughput cell culture-based functional assays and suggest that an Eg5 ASO also has potential in a therapeutic strategy.
Asunto(s)
Cinesinas/antagonistas & inhibidores , Cinesinas/genética , Oligonucleótidos Antisentido/genética , Oligonucleótidos Antisentido/farmacología , Animales , Antineoplásicos/farmacología , Apoptosis/efectos de los fármacos , Apoptosis/genética , Neoplasias de la Mama/tratamiento farmacológico , Neoplasias de la Mama/genética , Neoplasias de la Mama/patología , Procesos de Crecimiento Celular/efectos de los fármacos , Procesos de Crecimiento Celular/genética , Línea Celular Tumoral , ADN de Neoplasias/genética , Biblioteca de Genes , Glioma/tratamiento farmacológico , Glioma/genética , Glioma/patología , Células HeLa , Humanos , Cinesinas/biosíntesis , Ratones , Ratones Desnudos , ARN Mensajero/antagonistas & inhibidores , ARN Mensajero/biosíntesis , ARN Mensajero/genética , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
A new class of small molecules that bind the HCV RNA IRES IIA subdomain with sub-micromolar affinity is reported. The benzimidazole 'hit' 1 with a KD approximately 100 microM to a 29-mer RNA model of Domain IIA was identified from a 180000-member library using mass spectrometry-based screening methods. Further MS-assisted SAR (structure-activity relationships) studies afforded benzimidazole derivatives with sub-micromolar binding affinity for the IIA RNA construct. The optimized benzimidazoles demonstrated activity in a cellular replicon assay at concentrations comparable to their KD for the RNA target.
Asunto(s)
Bencimidazoles/química , Hepacivirus/genética , Modelos Moleculares , Relación Estructura-Actividad Cuantitativa , ARN Viral/química , Ribosomas/efectos de los fármacos , Secuencia de Bases , Bencimidazoles/farmacología , Bencimidazoles/toxicidad , Sitios de Unión , Línea Celular , Bases de Datos Factuales , Hepacivirus/fisiología , Humanos , Espectrometría de Masas , Datos de Secuencia Molecular , Conformación de Ácido Nucleico , ARN Viral/metabolismo , Ribosomas/química , Ribosomas/metabolismo , Replicación ViralRESUMEN
Spinocerebellar ataxia type 7 (SCA7) is a polyglutamine (polyQ) disorder characterized by specific degeneration of cerebellar, brainstem, and retinal neurons. Although they share little sequence homology, proteins implicated in polyQ disorders have common properties beyond their characteristic polyQ tract. These include the production of proteolytic fragments, nuclear accumulation, and processing by caspases. Here we report that ataxin-7 is cleaved by caspase-7, and we map two putative caspase-7 cleavage sites to Asp residues at positions 266 and 344 of the ataxin-7 protein. Site-directed mutagenesis of these two caspase-7 cleavage sites in the polyQ-expanded form of ataxin-7 produces an ataxin-7 D266N/D344N protein that is resistant to caspase cleavage. Although ataxin-7 displays toxicity, forms nuclear aggregates, and represses transcription in human embryonic kidney 293T cells in a polyQ length-dependent manner, expression of the non-cleavable D266N/D344N form of polyQ-expanded ataxin-7 attenuated cell death, aggregate formation, and transcriptional interference. Expression of the caspase-7 truncation product of ataxin-7-69Q or -92Q, which removes the putative nuclear export signal and nuclear localization signals of ataxin-7, showed increased cellular toxicity. We also detected N-terminal polyQ-expanded ataxin-7 cleavage products in SCA7 transgenic mice similar in size to those generated by caspase-7 cleavage. In a SCA7 transgenic mouse model, recruitment of caspase-7 into the nucleus by polyQ-expanded ataxin-7 correlated with its activation. Our results, thus, suggest that proteolytic processing of ataxin-7 by caspase-7 may contribute to SCA7 disease pathogenesis.