Specificity, duplex degradation and subcellular localization of antagomirs

MicroRNAs (miRNAs) are an abundant class of 20–23-nt long regulators of gene expression. The study of miRNA function in mice and potential therapeutic approaches largely depend on modified oligonucleotides. We recently demonstrated silencing miRNA function in mice using chemically modified and cholesterol-conjugated RNAs termed ‘antagomirs’. Here, we further characterize the properties and function of antagomirs in mice. We demonstrate that antagomirs harbor optimized phosphorothioate modifications, require >19-nt length for highest efficiency and can discriminate between single nucleotide mismatches of the targeted miRNA. Degradation of different chemically protected miRNA/antagomir duplexes in mouse livers and localization of antagomirs in a cytosolic compartment that is distinct from processing (P)-bodies indicates a degradation mechanism independent of the RNA interference (RNAi) pathway. Finally, we show that antagomirs, although incapable of silencing miRNAs in the central nervous system (CNS) when injected systemically, efficiently target miRNAs when injected locally into the mouse cortex. Our data further validate the effectiveness of antagomirs in vivo and should facilitate future studies to silence miRNAs for functional analysis and in clinically relevant settings

Iron-catalyzed depolymerizations of end-of-life silicones with fatty alcohols

During the last decades, polymers became one of the major materials in our society and a future without polymers is hardly imaginable. However, as negative issue of this success enormous amount of end-of-life materials are accumulated, which are mainly treated by landfill storage, thermal recycling or down-cycling. On the other hand, feedstock recycling can be an interesting option to convert end-of-life polymers to high quality polymers, via depolymerization reactions to low-molecular weight building blocks and subsequent transformation via polymerization reactions. In this regard, we present herein the depolymerization of polysiloxanes (silicones) applying fatty alcohols as depolymerization reagents. In more detail, in the presence of catalytic amounts of simple iron salts, low-molecular weight products with the motif R(OSiMe2)mOR (R = alkyl, m = 1-2) were attained. Remarkably, the reaction of R(OSiMe2)mOR with water showed the formation of new cyclic siloxanes, which are useful starting materials for long-chain silicones, and the corresponding fatty alcohol as side product, which can be directly reused in subsequent depolymerization reactions. Importantly, a recycling of the silicones and a straightforward recycling of the depolymerization reagent are feasible. © 2015 Tomsk Polytechnic University. Production and hosting by Elsevier B.V. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/). Peer review under responsibility of Tomsk Polytechnic University

Simple discrimination training and formation of functional stimulus classes by dogs

O presente estudo verificou a formação de classes de estímulos funcionalmente equivalentes e learning set a partir de treinos de discriminações simples simultâneas e repetidas reversões nas funções dos estímulos, com uso de reforçadores diferentes e específicos para cada classe. Os sujeitos foram três cães sem raça definida. Nas tentativas de teste foram encontradas, em média, porcentagens de acertos de 93,6. Esses dados somados aos resultados das primeiras tentativas de apresentação de cada par de estímulos atestaram a reversão instantânea das funções de estímulos, ambos indicando a formação de classes funcionalmente equivalentes. Os dados de learning set não foram conclusivos a respeito da progressão na eficiência dos desempenhos nas reversões, possivelmente, por influência da preferência diferencial dos cães pelos reforçadores específicos utilizados em cada classe. O procedimento parece adequado e econômico para as investigações na área de comportamento simbólico em animais não humanos.    Palavras-chave: equivalência funcional; discriminações simples; reversões; reforço específico e diferencial; cães. This study attempted to verify the establishment of functional classes and learning set in dogs through a simple discrimination procedure with repeated reversals that included specific reinforcers for each stimulus class. Two stimuli were presented in each trial and the subject had to choose one of them by jumping in its direction. After being training in such procedure, three dogs responded correctly in more than 90% of the test trials. These performances and analysis of the first responses in the test sessions provided evidences of the establishment of functional stimulus classes. Data from the repeated reversals training did not provided evidences of the establishment of a learning set by the dogs. The discussion examines whether the non-establishment of a learning set could be influenced by preferences for specific reinforcers. The procedure seems adequate and economical in investigations on symbolic potential of nonhuman animals.   Keywords: functional equivalence; simple discrimination; reversals; differential and specific reinforcement; dogs

Preferential regulation of stably expressed genes in the human genome suggests a widespread expression buffering role of microRNAs

In this study, we comprehensively explored the stably expressed genes (SE genes) and fluctuant genes (FL genes) in the human genome by a meta-analysis of large scale microarray data. We found that these genes have distinct function distributions. miRNA targets are shown to be significantly enriched in SE genes by using propensity analysis of miRNA regulation, supporting the hypothesis that miRNAs can buffer whole genome expression fluctuation. The expression-buffering effect of miRNA is independent of the target site number within the 3'-untranslated region. In addition, we found that gene expression fluctuation is positively correlated with the number of transcription factor binding sites in the promoter region, which suggests that coordination between transcription factors and miRNAs leads to balanced responses to external perturbations

Genomewide microRNA down‐regulation as a negative feedback mechanism in the early phases of liver regeneration

The liver is one of the few organs that have the capacity to regenerate in response to injury. We carried out genomewide microRNA (miRNA) microarray studies during liver regeneration in rats after 70% partial hepatectomy (PH) at early and mid time points to more thoroughly understand their role. At 3, 12, and 18 hours post‐PH ∼40% of the miRNAs tested were up‐regulated. Conversely, at 24 hours post‐PH, ∼70% of miRNAs were down‐regulated. Furthermore, we established that the genomewide down‐regulation of miRNA expression at 24 hours was also correlated with decreased expression of genes, such as Rnasen , Dgcr8 , Dicer , Tarbp2 , and Prkra , associated with miRNA biogenesis. To determine whether a potential negative feedback loop between miRNAs and their regulatory genes exists, 11 candidate miRNAs predicted to target the above‐mentioned genes were examined and found to be up‐regulated at 3 hours post‐PH. Using reporter and functional assays, we determined that expression of these miRNA‐processing genes could be regulated by a subset of miRNAs and that some miRNAs could target multiple miRNA biogenesis genes simultaneously. We also demonstrated that overexpression of these miRNAs inhibited cell proliferation and modulated cell cycle in both Huh‐7 human hepatoma cells and primary rat hepatocytes. From these observations, we postulated that selective up‐regulation of miRNAs in the early phase after PH was involved in the priming and commitment to liver regeneration, whereas the subsequent genomewide down‐regulation of miRNAs was required for efficient recovery of liver cell mass. Conclusion: Our data suggest that miRNA changes are regulated by negative feedback loops between miRNAs and their regulatory genes that may play an important role in the steady‐state regulation of liver regeneration. (H EPATOLOGY 2011;)Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/86868/1/HEP_24421_sm_suppinfotable.pdfhttp://deepblue.lib.umich.edu/bitstream/2027.42/86868/2/24421_ftp.pd

Targeted knock-down of miR21 primary transcripts using snoMEN vectors induces apoptosis in human cancer cell lines

We have previously reported an antisense technology, 'snoMEN vectors', for targeted knock-down of protein coding mRNAs using human snoRNAs manipulated to contain short regions of sequence complementarity with the mRNA target. Here we characterise the use of snoMEN vectors to target the knock-down of micro RNA primary transcripts. We document the specific knock-down of miR21 in HeLa cells using plasmid vectors expressing miR21-targeted snoMEN RNAs and show this induces apoptosis. Knock-down is dependent on the presence of complementary sequences in the snoMEN vector and the induction of apoptosis can be suppressed by over-expression of miR21. Furthermore, we have also developed lentiviral vectors for delivery of snoMEN RNAs and show this increases the efficiency of vector transduction in many human cell lines that are difficult to transfect with plasmid vectors. Transduction of lentiviral vectors expressing snoMEN targeted to pri-miR21 induces apoptosis in human lung adenocarcinoma cells, which express high levels of miR21, but not in human primary cells. We show that snoMEN-mediated suppression of miRNA expression is prevented by siRNA knock-down of Ago2, but not by knock-down of Ago1 or Upf1. snoMEN RNAs colocalise with Ago2 in cell nuclei and nucleoli and can be co-immunoprecipitated from nuclear extracts by antibodies specific for Ago2

Antagonism of microRNA-122 in mice by systemically administered LNA-antimiR leads to up-regulation of a large set of predicted target mRNAs in the liver

MicroRNA-122 (miR-122) is an abundant liver-specific miRNA, implicated in fatty acid and cholesterol metabolism as well as hepatitis C viral replication. Here, we report that a systemically administered 16-nt, unconjugated LNA (locked nucleic acid)-antimiR oligonucleotide complementary to the 5′ end of miR-122 leads to specific, dose-dependent silencing of miR-122 and shows no hepatotoxicity in mice. Antagonism of miR-122 is due to formation of stable heteroduplexes between the LNA-antimiR and miR-122 as detected by northern analysis. Fluorescence in situ hybridization demonstrated uptake of the LNA-antimiR in mouse liver cells, which was accompanied by markedly reduced hybridization signals for mature miR-122 in treated mice. Functional antagonism of miR-122 was inferred from a low cholesterol phenotype and de-repression within 24 h of 199 liver mRNAs showing significant enrichment for miR-122 seed matches in their 3′ UTRs. Expression profiling extended to 3 weeks after the last LNA-antimiR dose revealed that most of the changes in liver gene expression were normalized to saline control levels coinciding with normalized miR-122 and plasma cholesterol levels. Combined, these data suggest that miRNA antagonists comprised of LNA are valuable tools for identifying miRNA targets in vivo and for studying the biological role of miRNAs and miRNA-associated gene-regulatory networks in a physiological context

Vectors expressing efficient RNA decoys achieve the long-term suppression of specific microRNA activity in mammalian cells

Whereas the strong and stable suppression of specific microRNA activity would be essential for the functional analysis of these molecules, and also for the development of therapeutic applications, effective inhibitory methods to achieve this have not yet been fully established. In our current study, we tested various RNA decoys which were designed to efficiently expose indigestible complementary RNAs to a specific miRNA molecule. These inhibitory RNAs were at the same time designed to be expressed in lentiviral vectors and to be transported into the cytoplasm after transcription by RNA polymerase III. We report the optimal conditions that we have established for the design of such RNA decoys (we term these molecules TuD RNAs; tough decoy RNAs). We finally demonstrate that TuD RNAs induce specific and strong biological effects and also show that TuD RNAs achieve the efficient and long-term-suppression of specific miRNAs for over 1 month in mammalian cells

Role of miR-10b in breast cancer metastasis

Ninety percent of cancer-related mortality is caused by metastasis. Current cancer treatments can control many primary tumors but rarely stop the metastatic spread. Accumulating evidence demonstrates that miRNAs are involved in cancer initiation and progression. Furthermore, several miRNAs have been found to regulate metastasis. In particular, recent studies provide the first functional evidence that overexpression of a specific miRNA, miR-10b, can contribute to the development of metastasis, which can be exploited therapeutically in treating breast cancer metastasis in mice. Further in-depth analysis should provide more precise evaluation of the roles, mechanisms, and therapeutic utility of this miRNA in breast cancer

MicroRNAs Induced During Adipogenesis that Accelerate Fat Cell Development Are Downregulated in Obesity

OBJECTIVE-- We investigated the regulation and involvement of microRNAs (miRNAs) in fat cell development and obesity. RESEARCH DESIGN AND METHODS- Using miRNA microarrays, we profiled the expression of >370 miRNAs during adipogenesis of preadipocyte 3T3-L1 cells and adipocytes from leptin deficient ob/ob and diet-induced obese mice. Changes in key miRNAs were validated by RT-PCR. We further assessed the contribution of the chronic inflammatory environment in obese adipose tissue to the dysregulated miRNA expression by tumor necrosis factor (TNF)-α treatment of adipocytes. We functionally characterized two adipocyte-enriched miRNAs, miR-103 and miR-143, by a gain-of-function approach. RESULTS--Similar miRNAs were differentially regulated during in vitro and in vivo adipogenesis. Importantly, miRNAs that were induced during adipogenesis were downregulated in adipocytes from both types of obese mice and vice versa. These changes are likely associated with the chronic inflammatory environment, since they were mimicked by TNF-α treatment of differentiated adipocytes. Ectopic expression of miR-103 or miR-143 in preadipocytes accelerated adipogenesis, as measured both by the upregulation of many adipogenesis markers and by an increase in triglyceride accumulation at an early stage of adipogenesis. CONCLUSIONS- Our results provide the first experimental evidence for miR-103 function in adipose biology. The remarkable inverse regulatory pattern for many miRNAs during adipogenesis and obesity has important implications for understanding adipose tissue dysfunction in obese mice and humans and the link between chronic inflammation and obesity with insulin resistance