Your browser doesn't support javascript.
loading
Mostrar: 20 | 50 | 100
Resultados 1 - 20 de 46
Filtrar
Mais filtros

Base de dados
País/Região como assunto
Tipo de documento
Intervalo de ano de publicação
1.
Proc Natl Acad Sci U S A ; 120(30): e2216658120, 2023 07 25.
Artigo em Inglês | MEDLINE | ID: mdl-37463203

RESUMO

There remains an urgent need for new therapies for treatment-resistant epilepsy. Sodium channel blockers are effective for seizure control in common forms of epilepsy, but loss of sodium channel function underlies some genetic forms of epilepsy. Approaches that provide bidirectional control of sodium channel expression are needed. MicroRNAs (miRNA) are small noncoding RNAs which negatively regulate gene expression. Here we show that genome-wide miRNA screening of hippocampal tissue from a rat epilepsy model, mice treated with the antiseizure medicine cannabidiol, and plasma from patients with treatment-resistant epilepsy, converge on a single target-miR-335-5p. Pathway analysis on predicted and validated miR-335-5p targets identified multiple voltage-gated sodium channels (VGSCs). Intracerebroventricular injection of antisense oligonucleotides against miR-335-5p resulted in upregulation of Scn1a, Scn2a, and Scn3a in the mouse brain and an increased action potential rising phase and greater excitability of hippocampal pyramidal neurons in brain slice recordings, consistent with VGSCs as functional targets of miR-335-5p. Blocking miR-335-5p also increased voltage-gated sodium currents and SCN1A, SCN2A, and SCN3A expression in human induced pluripotent stem cell-derived neurons. Inhibition of miR-335-5p increased susceptibility to tonic-clonic seizures in the pentylenetetrazol seizure model, whereas adeno-associated virus 9-mediated overexpression of miR-335-5p reduced seizure severity and improved survival. These studies suggest modulation of miR-335-5p may be a means to regulate VGSCs and affect neuronal excitability and seizures. Changes to miR-335-5p may reflect compensatory mechanisms to control excitability and could provide biomarker or therapeutic strategies for different types of treatment-resistant epilepsy.


Assuntos
Epilepsia , Células-Tronco Pluripotentes Induzidas , MicroRNAs , Canais de Sódio Disparados por Voltagem , Humanos , Camundongos , Ratos , Animais , Células-Tronco Pluripotentes Induzidas/metabolismo , Convulsões/induzido quimicamente , Convulsões/genética , Convulsões/metabolismo , MicroRNAs/genética , MicroRNAs/metabolismo , Canais de Sódio Disparados por Voltagem/genética , Canal de Sódio Disparado por Voltagem NAV1.1/genética , Canal de Sódio Disparado por Voltagem NAV1.1/metabolismo , Canal de Sódio Disparado por Voltagem NAV1.3/genética
2.
J Neuroinflammation ; 20(1): 30, 2023 Feb 09.
Artigo em Inglês | MEDLINE | ID: mdl-36759861

RESUMO

Patients with COVID-19 can have a variety of neurological symptoms, but the active involvement of central nervous system (CNS) in COVID-19 remains unclear. While routine cerebrospinal fluid (CSF) analyses in patients with neurological manifestations of COVID-19 generally show no or only mild inflammation, more detailed data on inflammatory mediators in the CSF of patients with COVID-19 are scarce. We studied the inflammatory response in paired CSF and serum samples of patients with COVID-19 (n = 38). Patients with herpes simplex virus encephalitis (HSVE, n = 10) and patients with non-inflammatory, non-neurodegenerative neurological diseases (n = 28) served as controls. We used proteomics, enzyme-linked immunoassays, and semiquantitative cytokine arrays to characterize inflammatory proteins. Autoantibody screening was performed with cell-based assays and native tissue staining. RNA sequencing of long-non-coding RNA and circular RNA was done to study the transcriptome. Proteomics on single protein level and subsequent pathway analysis showed similar yet strongly attenuated inflammatory changes in the CSF of COVID-19 patients compared to HSVE patients with, e.g., downregulation of the apolipoproteins and extracellular matrix proteins. Protein upregulation of the complement system, the serpin proteins pathways, and other proteins including glycoproteins alpha-2 and alpha-1 acid. Importantly, calculation of interleukin-6, interleukin-16, and CXCL10 CSF/serum indices suggest that these inflammatory mediators reach the CSF from the systemic circulation, rather than being produced within the CNS. Antibody screening revealed no pathological levels of known neuronal autoantibodies. When stratifying COVID-19 patients into those with and without bacterial superinfection as indicated by elevated procalcitonin levels, inflammatory markers were significantly (p < 0.01) higher in those with bacterial superinfection. RNA sequencing in the CSF revealed 101 linear RNAs comprising messenger RNAs, and two circRNAs being significantly differentially expressed in COVID-19 than in non-neuroinflammatory controls and neurodegenerative patients. Our findings may explain the absence of signs of intrathecal inflammation upon routine CSF testing despite the presence of SARS-CoV2 infection-associated neurological symptoms. The relevance of blood-derived mediators of inflammation in the CSF for neurological COVID-19 and post-COVID-19 symptoms deserves further investigation.


Assuntos
COVID-19 , Encefalite por Herpes Simples , Superinfecção , Humanos , Proteoma/metabolismo , RNA Viral/metabolismo , Superinfecção/metabolismo , SARS-CoV-2 , Encéfalo/metabolismo , Inflamação/metabolismo , Encefalite por Herpes Simples/líquido cefalorraquidiano , Mediadores da Inflamação/metabolismo
3.
Proc Natl Acad Sci U S A ; 117(27): 15977-15988, 2020 07 07.
Artigo em Inglês | MEDLINE | ID: mdl-32581127

RESUMO

Temporal lobe epilepsy is the most common drug-resistant form of epilepsy in adults. The reorganization of neural networks and the gene expression landscape underlying pathophysiologic network behavior in brain structures such as the hippocampus has been suggested to be controlled, in part, by microRNAs. To systematically assess their significance, we sequenced Argonaute-loaded microRNAs to define functionally engaged microRNAs in the hippocampus of three different animal models in two species and at six time points between the initial precipitating insult through to the establishment of chronic epilepsy. We then selected commonly up-regulated microRNAs for a functional in vivo therapeutic screen using oligonucleotide inhibitors. Argonaute sequencing generated 1.44 billion small RNA reads of which up to 82% were microRNAs, with over 400 unique microRNAs detected per model. Approximately half of the detected microRNAs were dysregulated in each epilepsy model. We prioritized commonly up-regulated microRNAs that were fully conserved in humans and designed custom antisense oligonucleotides for these candidate targets. Antiseizure phenotypes were observed upon knockdown of miR-10a-5p, miR-21a-5p, and miR-142a-5p and electrophysiological analyses indicated broad safety of this approach. Combined inhibition of these three microRNAs reduced spontaneous seizures in epileptic mice. Proteomic data, RNA sequencing, and pathway analysis on predicted and validated targets of these microRNAs implicated derepressed TGF-ß signaling as a shared seizure-modifying mechanism. Correspondingly, inhibition of TGF-ß signaling occluded the antiseizure effects of the antagomirs. Together, these results identify shared, dysregulated, and functionally active microRNAs during the pathogenesis of epilepsy which represent therapeutic antiseizure targets.


Assuntos
Epilepsia do Lobo Temporal/tratamento farmacológico , Epilepsia do Lobo Temporal/metabolismo , MicroRNAs/efeitos dos fármacos , MicroRNAs/metabolismo , Oligonucleotídeos Antissenso/farmacologia , Convulsões/tratamento farmacológico , Convulsões/metabolismo , Animais , Antagomirs/farmacologia , Proteínas Argonautas/genética , Proteínas Argonautas/metabolismo , Biomarcadores , Modelos Animais de Doenças , Epilepsia , Feminino , Hipocampo/metabolismo , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , MicroRNAs/genética , Proteômica , Ratos , Ratos Sprague-Dawley , Convulsões/genética , Análise de Sistemas , Regulação para Cima/efeitos dos fármacos
4.
Bioinformatics ; 37(23): 4424-4430, 2021 12 07.
Artigo em Inglês | MEDLINE | ID: mdl-34255836

RESUMO

MOTIVATION: tRNAs were originally considered uni-functional RNA molecules involved in the delivery of amino acids to growing peptide chains on the ribosome. More recently, the liberation of tRNA fragments from tRNAs via specific enzyme cleavage has been characterized. Detection of tRNA fragments in sequencing data is difficult due to tRNA sequence redundancy and the short length of both tRNAs and their fragments. RESULTS: Here, we introduce tsRNAsearch, a Nextflow pipeline for the identification of differentially abundant tRNA fragments and other non-coding RNAs from small RNA-sequencing data. tsRNAsearch is intended for use when comparing two groups of datasets, such as control and treatment groups. tsRNAsearch comparatively searches for tRNAs and ncRNAs with irregular read distribution profiles (a proxy for RNA cleavage) using a combined score made up of four novel methods and a differential expression analysis, and reports the top ranked results in simple PDF and TEXT files. In this study, we used publicly available small RNA-seq data to replicate the identification of tsRNAs from chronic hepatitis-infected liver tissue data. In addition, we applied tsRNAsearch to pancreatic ductal adenocarcinoma (PDAC) and matched healthy pancreatic tissue small RNA-sequencing data. Our results support the identification of miR135b from the original study as a potential biomarker of PDAC and identify other potentially stronger miRNA biomarkers of PDAC. AVAILABILITY AND IMPLEMENTATION: https://github.com/GiantSpaceRobot/tsRNAsearch. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.


Assuntos
MicroRNAs , RNA não Traduzido , RNA não Traduzido/genética , RNA de Transferência/metabolismo , MicroRNAs/genética , Análise de Sequência de RNA/métodos
5.
Methods ; 196: 23-29, 2021 12.
Artigo em Inglês | MEDLINE | ID: mdl-34571139

RESUMO

Circular RNA (circRNA) is a group of highly stable RNA molecules with suggested roles in development and disease. They derive from linear pre-mRNAs when a 5'-splice site splices back to an upstream 3'-splice site in a process termed back-splicing. Most circRNAs are multi-exonic and may contain several thousand nucleotides. The extensive sequence overlap between the linear and circular forms of an RNA means that circRNA identification depends on the detection of back-splice-junction sequence reads that are unique to the circRNA. However, the short-read length obtained using standard next-generation sequencing techniques means that the internal sequence, exon composition and alternative splicing of circRNAs are unknown in many cases. Recently, several labs, including ours, have reported protocols for sequencing of circRNAs using long-read nanopore sequencing and thereby expanded our understanding of circRNA size distribution and internal splicing patterns. Here, we review these protocols and discuss the different approaches taken to study the full length composition of circRNAs.


Assuntos
Sequenciamento por Nanoporos , Nanoporos , RNA/genética , RNA/metabolismo , Sítios de Splice de RNA , Splicing de RNA/genética , RNA Circular/genética
6.
EMBO J ; 36(12): 1770-1787, 2017 06 14.
Artigo em Inglês | MEDLINE | ID: mdl-28487411

RESUMO

Synaptic downscaling is a homeostatic mechanism that allows neurons to reduce firing rates during chronically elevated network activity. Although synaptic downscaling is important in neural circuit development and epilepsy, the underlying mechanisms are poorly described. We performed small RNA profiling in picrotoxin (PTX)-treated hippocampal neurons, a model of synaptic downscaling. Thereby, we identified eight microRNAs (miRNAs) that were increased in response to PTX, including miR-129-5p, whose inhibition blocked synaptic downscaling in vitro and reduced epileptic seizure severity in vivo Using transcriptome, proteome, and bioinformatic analysis, we identified the calcium pump Atp2b4 and doublecortin (Dcx) as miR-129-5p targets. Restoring Atp2b4 and Dcx expression was sufficient to prevent synaptic downscaling in PTX-treated neurons. Furthermore, we characterized a functional crosstalk between miR-129-5p and the RNA-binding protein (RBP) Rbfox1. In the absence of PTX, Rbfox1 promoted the expression of Atp2b4 and Dcx. Upon PTX treatment, Rbfox1 expression was downregulated by miR-129-5p, thereby allowing the repression of Atp2b4 and Dcx. We therefore identified a novel activity-dependent miRNA/RBP crosstalk during synaptic scaling, with potential implications for neural network homeostasis and epileptogenesis.


Assuntos
Regulação da Expressão Gênica , MicroRNAs/metabolismo , Fatores de Processamento de RNA/metabolismo , Sinapses/fisiologia , Animais , Biologia Computacional , Proteínas do Domínio Duplacortina , Proteína Duplacortina , Perfilação da Expressão Gênica , Hipocampo/efeitos dos fármacos , Hipocampo/fisiologia , Camundongos , Proteínas Associadas aos Microtúbulos/metabolismo , Neuropeptídeos/metabolismo , Picrotoxina/metabolismo , ATPases Transportadoras de Cálcio da Membrana Plasmática/metabolismo , Proteoma/análise
7.
Nucleic Acids Res ; 47(16): e93, 2019 09 19.
Artigo em Inglês | MEDLINE | ID: mdl-31216024

RESUMO

Single cell RNA sequencing methods have been increasingly used to understand cellular heterogeneity. Nevertheless, most of these methods suffer from one or more limitations, such as focusing only on polyadenylated RNA, sequencing of only the 3' end of the transcript, an exuberant fraction of reads mapping to ribosomal RNA, and the unstranded nature of the sequencing data. Here, we developed a novel single cell strand-specific total RNA library preparation method addressing all the aforementioned shortcomings. Our method was validated on a microfluidics system using three different cancer cell lines undergoing a chemical or genetic perturbation and on two other cancer cell lines sorted in microplates. We demonstrate that our total RNA-seq method detects an equal or higher number of genes compared to classic polyA[+] RNA-seq, including novel and non-polyadenylated genes. The obtained RNA expression patterns also recapitulate the expected biological signal. Inherent to total RNA-seq, our method is also able to detect circular RNAs. Taken together, SMARTer single cell total RNA sequencing is very well suited for any single cell sequencing experiment in which transcript level information is needed beyond polyadenylated genes.


Assuntos
Sequenciamento de Nucleotídeos em Larga Escala/métodos , RNA Circular/análise , RNA Mensageiro/análise , RNA Ribossômico/análise , Análise de Célula Única/métodos , Benchmarking , Linhagem Celular Tumoral , Biblioteca Gênica , Humanos , Técnicas Analíticas Microfluídicas , Poli A/genética , Poli A/metabolismo , RNA Circular/genética , RNA Mensageiro/genética , RNA Ribossômico/genética , Análise de Sequência de RNA/estatística & dados numéricos
8.
Mol Ther ; 26(2): 593-605, 2018 02 07.
Artigo em Inglês | MEDLINE | ID: mdl-29331291

RESUMO

Bone remodeling and regeneration are highly regulated multistep processes involving posttranscriptional regulation by microRNAs (miRNAs). Here, we performed a global profiling of differentially expressed miRNAs in bone-marrow-derived skeletal cells (BMSCs; also known as stromal or mesenchymal stem cells) during in vitro osteoblast differentiation. We functionally validated the regulatory effects of several miRNAs on osteoblast differentiation and identified 15 miRNAs, most significantly miR-222 and miR-423, as regulators of osteoblastogenesis. In addition, we tested the possible targeting of miRNAs for enhancing bone tissue regeneration. Scaffolds functionalized with miRNA nano-carriers enhanced osteoblastogenesis in 3D culture and retained this ability at least 2 weeks after storage. Additionally, anti-miR-222 enhanced in vivo ectopic bone formation through targeting the cell-cycle inhibitor CDKN1B (cyclin-dependent kinase inhibitor 1B). A number of additional miRNAs exerted additive osteoinductive effects on BMSC differentiation, suggesting that pools of miRNAs delivered locally from an implanted scaffold can provide a promising approach for enhanced bone regeneration.


Assuntos
Regeneração Óssea/genética , Perfilação da Expressão Gênica , Células-Tronco Mesenquimais/metabolismo , MicroRNAs/genética , Transcriptoma , Regiões 3' não Traduzidas , Antagomirs/genética , Biomarcadores , Ciclo Celular/genética , Diferenciação Celular/genética , Linhagem Celular , Biologia Computacional/métodos , Expressão Ectópica do Gene , Regulação da Expressão Gênica , Técnicas de Transferência de Genes , Humanos , Osteogênese/genética
9.
RNA Biol ; 15(2): 280-291, 2018 02 01.
Artigo em Inglês | MEDLINE | ID: mdl-29283313

RESUMO

The expression patterns of endogenous circular RNA (circRNA) molecules during epidermal stem cell (EpSC) differentiation have not previously been explored. Here, we show that circRNAs are abundantly expressed in EpSCs and that their expression change dramatically during differentiation in a coordinated manner. Overall, circRNAs are expressed at higher levels in the differentiated cells, and many upregulated circRNAs are derived from developmental genes, including four different circRNAs from DLG1. The observed changes in circRNA expression were largely independent of host gene expression, and circRNAs independently upregulated upon differentiation are more prone to AGO2 binding and have more predicted miRNA binding sites compared to stably expressed circRNAs. In particular, upregulated circRNAs from the HECTD1 and ZNF91 genes have exceptionally high numbers of AGO2 binding sites and predicted miRNA target sites, and circZNF91 contains 24 target sites for miR-23b-3p, which is known to play important roles in keratinocyte differentiation. We also observed that upregulated circRNAs are less likely to be flanked by homologues inverted Alu repeats compared to stably expressed circRNAs. This coincide with DHX9 being upregulated in the differentiated keratinocytes. Finally, none of the circRNAs upregulated upon differentiation were also upregulated upon DNMT3A or DNMT3B knockdown, making it unlikely that epigenetic mechanisms are governing the observed circRNA expression changes. Together, we provide a map of circRNA expression in EpSCs and their differentiated counterparts and shed light on potential function and regulation of differentially expressed circRNAs.


Assuntos
Células Epidérmicas , Perfilação da Expressão Gênica/métodos , RNA/genética , Regulação para Cima , Sítios de Ligação , Diferenciação Celular , Células Cultivadas , Redes Reguladoras de Genes , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Humanos , RNA/química , RNA/metabolismo , RNA Circular , Análise de Sequência de RNA/métodos , Células-Tronco/citologia
10.
Nucleic Acids Res ; 44(6): e58, 2016 Apr 07.
Artigo em Inglês | MEDLINE | ID: mdl-26657634

RESUMO

CircRNAs are novel members of the non-coding RNA family. For several decades circRNAs have been known to exist, however only recently the widespread abundance has become appreciated. Annotation of circRNAs depends on sequencing reads spanning the backsplice junction and therefore map as non-linear reads in the genome. Several pipelines have been developed to specifically identify these non-linear reads and consequently predict the landscape of circRNAs based on deep sequencing datasets. Here, we use common RNAseq datasets to scrutinize and compare the output from five different algorithms; circRNA_finder, find_circ, CIRCexplorer, CIRI, and MapSplice and evaluate the levels of bona fide and false positive circRNAs based on RNase R resistance. By this approach, we observe surprisingly dramatic differences between the algorithms specifically regarding the highly expressed circRNAs and the circRNAs derived from proximal splice sites. Collectively, this study emphasizes that circRNA annotation should be handled with care and that several algorithms should ideally be combined to achieve reliable predictions.


Assuntos
Algoritmos , Artefatos , Anotação de Sequência Molecular/estatística & dados numéricos , RNA/química , Software , Processamento Alternativo , Biblioteca Gênica , Genoma Humano , Humanos , Anotação de Sequência Molecular/métodos , Conformação de Ácido Nucleico , RNA/genética , RNA/metabolismo , RNA Circular , Análise de Sequência de RNA
11.
Nucleic Acids Res ; 42(16): e124, 2014.
Artigo em Inglês | MEDLINE | ID: mdl-25053842

RESUMO

During recent years, miRNAs have been shown to play important roles in the regulation of gene expression. Accordingly, much effort has been put into the discovery of novel uncharacterized miRNAs in various organisms. miRNAs are structurally defined by a hairpin-loop structure recognized by the two-step processing apparatus, Drosha and Dicer, necessary for the production of mature ∼ 22-nucleotide miRNA guide strands. With the emergence of high-throughput sequencing applications, tools have been developed to identify miRNAs and profile their expression based on sequencing reads. However, as the read depth increases, false-positive predictions increase using established algorithms, underscoring the need for more stringent approaches. Here we describe a transparent pipeline for confident miRNA identification in animals, termed miRdentify. We show that miRdentify confidently discloses more than 400 novel miRNAs in humans, including the first male-specific miRNA, which we successfully validate. Moreover, novel miRNAs are predicted in the mouse, the fruit fly and nematodes, suggesting that the pipeline applies to all animals. The entire software package is available at www.ncrnalab.dk/mirdentify.


Assuntos
Sequenciamento de Nucleotídeos em Larga Escala/métodos , MicroRNAs/química , Análise de Sequência de RNA/métodos , Software , Algoritmos , Animais , Caenorhabditis elegans/genética , Drosophila melanogaster/genética , Variação Genética , Humanos , Masculino , Camundongos , Anotação de Sequência Molecular , Termodinâmica
12.
Epilepsia ; 55(12): 2017-27, 2014 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-25410734

RESUMO

OBJECTIVE: Mesial temporal lobe epilepsy (MTLE) is one of the most common types of the intractable epilepsies and is most often associated with hippocampal sclerosis (HS), which is characterized by pronounced loss of hippocampal pyramidal neurons. microRNAs (miRNAs) have been shown to be dysregulated in epilepsy and neurodegenerative diseases, and we hypothesized that miRNAs could be involved in the pathogenesis of MTLE and HS. METHODS: miRNA expression was quantified in hippocampal specimens from human patients using miRNA microarray and quantitative real-time polymerase chain reaction RT-PCR, and by RNA-seq on fetal brain specimens from domestic pigs. In situ hybridization was used to show the spatial distribution of miRNAs in the human hippocampus. The potential effect of miRNAs on targets genes was investigated using the dual luciferase reporter gene assay. RESULTS: miRNA expression profiling showed that 25 miRNAs were up-regulated and 5 were down-regulated in hippocampus biopsies of MTLE/HS patients compared to controls. We showed that miR-204 and miR-218 were significantly down-regulated in MTLE and HS, and both were expressed in neurons in all subfields of normal hippocampus. Moreover, miR-204 and miR-218 showed strong changes in expression during fetal development of the hippocampus in pigs, and we identified four target genes, involved in axonal guidance and synaptic plasticity, ROBO1, GRM1, SLC1A2, and GNAI2, as bona fide targets of miR-218. GRM1 was also shown to be a direct target of miR-204. SIGNIFICANCE: miR-204 and miR-218 are developmentally regulated in the hippocampus and may contribute to the molecular mechanisms underlying the pathogenesis of MTLE and HS.


Assuntos
Epilepsia do Lobo Temporal/patologia , Regulação da Expressão Gênica/fisiologia , Hipocampo/metabolismo , MicroRNAs/metabolismo , Adolescente , Adulto , Animais , Estudos de Coortes , Dinamarca , Embrião de Mamíferos , Epilepsia do Lobo Temporal/complicações , Epilepsia do Lobo Temporal/metabolismo , Transportador 2 de Aminoácido Excitatório , Feminino , Perfilação da Expressão Gênica , Proteínas de Transporte de Glutamato da Membrana Plasmática/genética , Proteínas de Transporte de Glutamato da Membrana Plasmática/metabolismo , Humanos , Masculino , Pessoa de Meia-Idade , Proteínas do Tecido Nervoso/metabolismo , Países Baixos , Células Piramidais/metabolismo , Células Piramidais/patologia , Receptores de Glutamato Metabotrópico/metabolismo , Reprodutibilidade dos Testes , Esclerose/etiologia , Esclerose/patologia , Análise de Sequência de RNA , Suínos , Adulto Jovem
13.
Nucleic Acids Res ; 40(19): 9876-86, 2012 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-22848101

RESUMO

Transcripts have been found to be site selectively edited from adenosine-to-inosine (A-to-I) in the mammalian brain, mostly in genes involved in neurotransmission. While A-to-I editing occurs at double-stranded structures, other structural requirements are largely unknown. We have investigated the requirements for editing at the I/M site in the Gabra-3 transcript of the GABA(A) receptor. We identify an evolutionarily conserved intronic duplex, 150 nt downstream of the exonic hairpin where the I/M site resides, which is required for its editing. This is the first time a distant RNA structure has been shown to be important for A-to-I editing. We demonstrate that the element also can induce editing in related but normally not edited RNA sequences. In human, thousands of genes are edited in duplexes formed by inverted repeats in non-coding regions. It is likely that numerous such duplexes can induce editing of coding regions throughout the transcriptome.


Assuntos
Íntrons , Edição de RNA , Sequências Reguladoras de Ácido Ribonucleico , Animais , Encéfalo/metabolismo , Células HEK293 , Células HeLa , Humanos , Camundongos , Conformação de Ácido Nucleico , Splicing de RNA , Ratos , Receptores de GABA-A/genética , Suínos
14.
Methods Mol Biol ; 2765: 143-157, 2024.
Artigo em Inglês | MEDLINE | ID: mdl-38381338

RESUMO

Circular RNAs (circRNAs) constitute a group of RNAs defined by a covalent bond between the 5' and 3' end formed by a unique back-splicing event. Most circRNAs are composed of more than one exon, which are spliced together in a linear fashion. This protocol describes methods to sequence full-length circRNA across the back-splicing junction, allowing unambiguous characterization of circRNA-specific exon-intron structures by long-read sequencing (LRS). Two different sequencing approaches are provided: (1) Global circRNA sequencing (the circNick-LRS strategy) relying on circRNA enrichment from total RNA followed by total circRNA long-read sequencing, and (2) targeted circRNA sequencing (the circPanel-LRS strategy) where a preselected panel of circRNA are sequenced without prior circRNA enrichment. Both methods were originally described in Karim et al. (Rahimi et al., Nat Commun 12: 4825, 2021) where they were applied to characterize the exon-intron structure of >10.000 circRNAs in mouse and human brains.

15.
Sci Rep ; 14(1): 15313, 2024 07 03.
Artigo em Inglês | MEDLINE | ID: mdl-38961125

RESUMO

Epileptogenesis is the process by which a normal brain becomes hyperexcitable and capable of generating spontaneous recurrent seizures. The extensive dysregulation of gene expression associated with epileptogenesis is shaped, in part, by microRNAs (miRNAs) - short, non-coding RNAs that negatively regulate protein levels. Functional miRNA-mediated regulation can, however, be difficult to elucidate due to the complexity of miRNA-mRNA interactions. Here, we integrated miRNA and mRNA expression profiles sampled over multiple time-points during and after epileptogenesis in rats, and applied bi-clustering and Bayesian modelling to construct temporal miRNA-mRNA-mRNA interaction networks. Network analysis and enrichment of network inference with sequence- and human disease-specific information identified key regulatory miRNAs with the strongest influence on the mRNA landscape, and miRNA-mRNA interactions closely associated with epileptogenesis and subsequent epilepsy. Our findings underscore the complexity of miRNA-mRNA regulation, can be used to prioritise miRNA targets in specific systems, and offer insights into key regulatory processes in epileptogenesis with therapeutic potential for further investigation.


Assuntos
Epilepsia , Perfilação da Expressão Gênica , Redes Reguladoras de Genes , MicroRNAs , RNA Mensageiro , Convulsões , Animais , MicroRNAs/genética , MicroRNAs/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Ratos , Convulsões/genética , Convulsões/metabolismo , Epilepsia/genética , Epilepsia/metabolismo , Masculino , Regulação da Expressão Gênica , Teorema de Bayes , Modelos Animais de Doenças , Transcriptoma
16.
Brain Commun ; 6(1): fcad355, 2024.
Artigo em Inglês | MEDLINE | ID: mdl-38204971

RESUMO

MicroRNAs have emerged as important regulators of the gene expression landscape in temporal lobe epilepsy. The mechanisms that control microRNA levels and influence target choice remain, however, poorly understood. RNA editing is a post-transcriptional mechanism mediated by the adenosine acting on RNA (ADAR) family of proteins that introduces base modification that diversifies the gene expression landscape. RNA editing has been studied for the mRNA landscape but the extent to which microRNA editing occurs in human temporal lobe epilepsy is unknown. Here, we used small RNA-sequencing data to characterize the identity and extent of microRNA editing in human temporal lobe epilepsy brain samples. This detected low-to-high editing in over 40 of the identified microRNAs. Among microRNA exhibiting the highest editing was miR-376a-3p, which was edited in the seed region and this was predicted to significantly change the target pool. The edited form was expressed at lower levels in human temporal lobe epilepsy samples. We modelled the shift in editing levels of miR-376a-3p in human-induced pluripotent stem cell-derived neurons. Reducing levels of the edited form of miR-376a-3p using antisense oligonucleotides resulted in extensive gene expression changes, including upregulation of mitochondrial and metabolism-associated pathways. Together, these results show that differential editing of microRNAs may re-direct targeting and result in altered functions relevant to the pathophysiology of temporal lobe epilepsy and perhaps other disorders of neuronal hyperexcitability.

17.
Int J Surg ; 2024 Jul 23.
Artigo em Inglês | MEDLINE | ID: mdl-39041944

RESUMO

BACKGROUND: Biliary obstruction can be due to both malignant and benign pancreaticobiliary disease. Currently, there are no biomarkers that can accurately help make this distinction. MicroRNAs (miRNAs) are stable molecules in tissue and biofluids that are commonly deregulated in cancer. The MIRABILE study aimed to identify miRNAs in bile that can differentiate malignant from benign pancreaticobiliary disease. MATERIALS AND METHODS: There were 111 patients recruited prospectively at endoscopic retrograde cholangiopancreatography (ERCP) or percutaneous transhepatic cholangiography (PTC) for obstructive jaundice, and bile was aspirated for cell-free RNA (cfRNA) extraction and analysis. In a discovery cohort of 78 patients (27 with pancreatic ductal adenocarcinoma (PDAC), 14 cholangiocarcinoma (CCA), 37 benign disease), cfRNA was subjected to small-RNA sequencing. LASSO regression was used to define bile miRNA signatures, and NormFinder to identify endogenous controls. In a second cohort of 87 patients (34 PDAC, 14 CCA, 39 benign disease), RT-qPCR was used for validation. RESULTS: LASSO regression identified 14 differentially-expressed bile miRNAs of which 6 were selected for validation. When comparing malignant and benign pancreaticobiliary disease, bile miR-340 and miR-182 were validated and significantly differentially expressed (P<0.05 and P<0.001, respectively). This generated an AUC of 0.79 (95%CI 0.70-0.88, sensitivity 65%; specificity 82%) in predicting malignant disease. CONCLUSION: Bile collected during biliary drainage contains miRNAs able to differentiate benign from malignant pancreaticobiliary diseases in patients with obstructive jaundice. These bile miRNAs have the potential to increase diagnostic accuracy.

18.
J Exp Clin Cancer Res ; 43(1): 189, 2024 Jul 08.
Artigo em Inglês | MEDLINE | ID: mdl-38978141

RESUMO

BACKGROUND: Distinguishing benign from malignant pancreaticobiliary disease is challenging because of the absence of reliable biomarkers. Circulating extracellular vesicles (EVs) have emerged as functional mediators between cells. Their cargos, including microRNAs (miRNAs), are increasingly acknowledged as an important source of potential biomarkers. This multicentric, prospective study aimed to establish a diagnostic plasma EV-derived miRNA signature to discriminate pancreatic ductal adenocarcinoma (PDAC) from benign pancreaticobiliary disease. METHODS: Plasma EVs were isolated using size exclusion chromatography (SEC) and characterised using nanoparticle tracking analysis, electron microscopy and Western blotting. EV-RNAs underwent small RNA sequencing to discover differentially expressed markers for PDAC (n = 10 benign vs. 10 PDAC). Candidate EV-miRNAs were then validated in a cohort of 61 patients (n = 31 benign vs. 30 PDAC) by RT-qPCR. Logistic regression and optimal thresholds (Youden Index) were used to develop an EV-miR-200 family model to detect cancer. This model was tested in an independent cohort of 95 patients (n = 30 benign, 33 PDAC, and 32 cholangiocarcinoma). RESULTS: Small RNA sequencing and RT-qPCR showed that EV-miR-200 family members were significantly overexpressed in PDAC vs. benign disease. Combined expression of the EV-miR-200 family showed an AUC of 0.823. In an independent validation cohort, application of this model showed a sensitivity, specificity and AUC of 100%, 88%, and 0.97, respectively, for diagnosing PDAC. CONCLUSIONS: This is the first study to validate plasma EV-miR-200 members as a clinically-useful diagnostic biomarker for PDAC. Further validation in larger cohorts and clinical trials is essential. These findings also suggest the potential utility in monitoring response and/or recurrence.


Assuntos
Carcinoma Ductal Pancreático , Vesículas Extracelulares , MicroRNAs , Neoplasias Pancreáticas , Humanos , Carcinoma Ductal Pancreático/sangue , Carcinoma Ductal Pancreático/diagnóstico , Carcinoma Ductal Pancreático/genética , Carcinoma Ductal Pancreático/patologia , Vesículas Extracelulares/metabolismo , Vesículas Extracelulares/genética , MicroRNAs/sangue , MicroRNAs/genética , Feminino , Masculino , Pessoa de Meia-Idade , Neoplasias Pancreáticas/sangue , Neoplasias Pancreáticas/diagnóstico , Neoplasias Pancreáticas/genética , Neoplasias Pancreáticas/patologia , Idoso , Biomarcadores Tumorais/sangue , Estudos Prospectivos
19.
Cell Rep ; 43(3): 113862, 2024 Mar 26.
Artigo em Inglês | MEDLINE | ID: mdl-38446664

RESUMO

Brain functionality relies on finely tuned regulation of gene expression by networks of non-coding RNAs (ncRNAs) such as the one composed by the circular RNA ciRS-7 (also known as CDR1as), the microRNA miR-7, and the long ncRNA Cyrano. We describe ischemia-induced alterations in the ncRNA network both in vitro and in vivo and in transgenic mice lacking ciRS-7 or miR-7. Our data show that cortical neurons downregulate ciRS-7 and Cyrano and upregulate miR-7 expression during ischemia. Mice lacking ciRS-7 exhibit reduced lesion size and motor impairment, while the absence of miR-7 alone results in increased ischemia-induced neuronal death. Moreover, miR-7 levels in pyramidal excitatory neurons regulate neurite morphology and glutamatergic signaling, suggesting a potential molecular link to the in vivo phenotype. Our data reveal the role of ciRS-7 and miR-7 in modulating ischemic stroke outcome, shedding light on the pathophysiological function of intracellular ncRNA networks in the brain.


Assuntos
MicroRNAs , RNA Longo não Codificante , Camundongos , Animais , MicroRNAs/genética , MicroRNAs/metabolismo , RNA não Traduzido , RNA Circular , Transdução de Sinais , RNA Longo não Codificante/metabolismo , Isquemia
20.
Nucleic Acid Ther ; 33(1): 45-57, 2023 01.
Artigo em Inglês | MEDLINE | ID: mdl-36445751

RESUMO

Circular RNAs (circRNAs) constitute an abundant class of covalently closed noncoding RNA molecules that are formed by backsplicing from eukaryotic protein-coding genes. Recent studies have shown that circRNAs can act as microRNA or protein decoys, as well as transcriptional regulators. However, the functions of most circRNAs are still poorly understood. Because circRNA sequences overlap with their linear parent transcripts, depleting specific circRNAs without affecting host gene expression remains a challenge. In this study, we assessed the utility of LNA-modified antisense oligonucleotides (ASOs) to knock down circRNAs for loss-of-function studies. We found that, while most RNase H-dependent gapmer ASOs mediate effective knockdown of their target circRNAs, some gapmers reduce the levels of the linear parent transcript. The circRNA targeting specificity can be enhanced using design-optimized gapmer ASOs, which display potent and specific circRNA knockdown with a minimal effect on the host genes. In summary, our results demonstrate that LNA-modified ASOs complementary to backsplice-junction sequences mediate robust knockdown of circRNAs in vitro and, thus, represent a useful tool to explore the biological roles of circRNAs in loss-of-function studies in cultured cells and animal models.


Assuntos
Oligonucleotídeos Antissenso , RNA Circular , Animais , Oligonucleotídeos Antissenso/genética , Oligonucleotídeos Antissenso/metabolismo , RNA Circular/genética , Oligonucleotídeos/genética
SELEÇÃO DE REFERÊNCIAS
DETALHE DA PESQUISA