RESUMEN
The Extracellular RNA Communication Consortium (ERCC) was launched to accelerate progress in the new field of extracellular RNA (exRNA) biology and to establish whether exRNAs and their carriers, including extracellular vesicles (EVs), can mediate intercellular communication and be utilized for clinical applications. Phase 1 of the ERCC focused on exRNA/EV biogenesis and function, discovery of exRNA biomarkers, development of exRNA/EV-based therapeutics, and construction of a robust set of reference exRNA profiles for a variety of biofluids. Here, we present progress by ERCC investigators in these areas, and we discuss collaborative projects directed at development of robust methods for EV/exRNA isolation and analysis and tools for sharing and computational analysis of exRNA profiling data.
Asunto(s)
Ácidos Nucleicos Libres de Células/genética , Ácidos Nucleicos Libres de Células/metabolismo , Vesículas Extracelulares/genética , Biomarcadores , Humanos , Bases del Conocimiento , MicroARNs/genética , ARN/genéticaRESUMEN
To develop a map of cell-cell communication mediated by extracellular RNA (exRNA), the NIH Extracellular RNA Communication Consortium created the exRNA Atlas resource (https://exrna-atlas.org). The Atlas version 4P1 hosts 5,309 exRNA-seq and exRNA qPCR profiles from 19 studies and a suite of analysis and visualization tools. To analyze variation between profiles, we apply computational deconvolution. The analysis leads to a model with six exRNA cargo types (CT1, CT2, CT3A, CT3B, CT3C, CT4), each detectable in multiple biofluids (serum, plasma, CSF, saliva, urine). Five of the cargo types associate with known vesicular and non-vesicular (lipoprotein and ribonucleoprotein) exRNA carriers. To validate utility of this model, we re-analyze an exercise response study by deconvolution to identify physiologically relevant response pathways that were not detected previously. To enable wide application of this model, as part of the exRNA Atlas resource, we provide tools for deconvolution and analysis of user-provided case-control studies.
Asunto(s)
Comunicación Celular/fisiología , ARN/metabolismo , Adulto , Líquidos Corporales/química , Ácidos Nucleicos Libres de Células/metabolismo , MicroARN Circulante/metabolismo , Vesículas Extracelulares/metabolismo , Femenino , Humanos , Masculino , Reproducibilidad de los Resultados , Análisis de Secuencia de ARN/métodos , Programas InformáticosRESUMEN
Acute exercise elicits dynamic transcriptional changes that, when repeated, form the fundamental basis of health, resilience, and performance adaptations. While moderate-intensity endurance training combined with conventional resistance training (traditional, TRAD) is often prescribed and recommended by public health guidance, high-intensity training combining maximal-effort intervals with intensive, limited-rest resistance training is a time-efficient alternative that may be used tactically (HITT) to confer similar benefits. Mechanisms of action of these distinct stimuli are incompletely characterized and have not been directly compared. We assessed transcriptome-wide responses in skeletal muscle and circulating extracellular vesicles (EVs) to a single exercise bout in young adults randomized to TRAD (n = 21, 12 M/9 F, 22 ± 3 yr) or HITT (n = 19, 11 M/8 F, 22 ± 2 yr). Next-generation sequencing captured small, long, and circular RNA in muscle and EVs. Analysis identified differentially expressed transcripts (|log2FC|>1, FDR ≤ 0.05) immediately (h0, EVs only), h3, and h24 postexercise within and between exercise protocols. In aaddition, all apparently responsive transcripts (FDR < 0.2) underwent singular value decomposition to summarize data structures into latent variables (LVs) to deconvolve molecular expression circuits and interregulatory relationships. LVs were compared across time and exercise protocol. TRAD, a longer but less intense stimulus, generally elicited a stronger transcriptional response than HITT, but considerable overlap and key differences existed. Findings reveal shared and unique molecular responses to the exercise stimuli and lay groundwork toward establishing relationships between protein-coding genes and lesser-understood transcripts that serve regulatory roles following exercise. Future work should advance the understanding of these circuits and whether they repeat in other populations or following other types of exercise/stress.NEW & NOTEWORTHY We examined small and long transcriptomics in skeletal muscle and serum-derived extracellular vesicles before and after a single exposure to traditional combined exercise (TRAD) and high-intensity tactical training (HITT). Across 40 young adults, we found more consistent protein-coding gene responses to TRAD, whereas HITT elicited differential expression of microRNA enriched in brain regions. Follow-up analysis revealed relationships and temporal dynamics across transcript networks, highlighting potential avenues for research into mechanisms of exercise response and adaptation.
Asunto(s)
Entrenamiento de Fuerza , Transcriptoma , Humanos , Adulto Joven , Transcriptoma/genética , Ejercicio Físico/fisiología , Perfilación de la Expresión Génica , Músculo Esquelético/metabolismoRESUMEN
Matrin 3 is a nuclear matrix protein that has many roles in RNA processing including splicing and transport of mRNA. Many missense mutations in the Matrin 3 gene (MATR3) have been linked to familial forms of amyotrophic lateral sclerosis (ALS) and distal myopathy. However, the exact role of MATR3 mutations in ALS and myopathy pathogenesis is not understood. To demonstrate a role of MATR3 mutations in vivo, we generated a novel CRISPR/Cas9 mediated knock-in mouse model harboring the MATR3 P154S mutation expressed under the control of the endogenous promoter. The P154S variant of the MATR3 gene has been linked to familial forms of ALS. Heterozygous and homozygous MATR3 P154S knock-in mice did not develop progressive motor deficits compared to wild-type mice. In addition, ALS-like pathology did not develop in nervous or muscle tissue in either heterozygous or homozygous mice. Our results suggest that the MATR3 P154S variant is not sufficient to produce ALS-like pathology in vivo.
Asunto(s)
Esclerosis Amiotrófica Lateral , Proteínas Asociadas a Matriz Nuclear , Animales , Ratones , Esclerosis Amiotrófica Lateral/metabolismo , Músculos/metabolismo , Enfermedades Musculares/genética , Mutación , Mutación Missense , Proteínas Asociadas a Matriz Nuclear/genética , Proteínas Asociadas a Matriz Nuclear/metabolismoRESUMEN
The C9ORF72-linked diseases amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD) are characterized by the nuclear depletion and cytoplasmic accumulation of TAR DNA-binding protein 43 (TDP-43). Recent studies have shown that the loss of TDP-43 function leads to the inclusion of cryptic exons (CE) in several RNA transcript targets of TDP-43. Here, we show for the first time the detection of CEs in a single-nuclei RNA sequencing (snRNA-seq) dataset obtained from frontal and occipital cortices of C9ORF72 patients that phenotypically span the ALS-FTD disease spectrum. We assessed each cellular cluster for detection of recently described TDP-43-induced CEs. Transcripts containing CEs in the genes STMN2 and KALRN were detected in the frontal cortex of all C9ORF72 disease groups with the highest frequency in excitatory neurons in the C9ORF72-FTD group. Within the excitatory neurons, the cluster with the highest proportion of cells containing a CE had transcriptomic similarities to von Economo neurons, which are known to be vulnerable to TDP-43 pathology and selectively lost in C9ORF72-FTD. Differential gene expression and pathway analysis of CE-containing neurons revealed multiple dysregulated metabolic processes. Our findings reveal novel insights into the transcriptomic changes of neurons vulnerable to TDP-43 pathology.
Asunto(s)
Esclerosis Amiotrófica Lateral , Demencia Frontotemporal , Enfermedad de Pick , Humanos , Esclerosis Amiotrófica Lateral/genética , Esclerosis Amiotrófica Lateral/patología , Demencia Frontotemporal/genética , Demencia Frontotemporal/patología , Proteína C9orf72/genética , Proteína C9orf72/metabolismo , Transcriptoma , Enfermedad de Pick/genética , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Exones , Análisis de Secuencia de ARNRESUMEN
RATIONALE: Previous translational studies implicate plasma extracellular microRNA-30d (miR-30d) as a biomarker in left ventricular remodeling and clinical outcome in heart failure (HF) patients, although precise mechanisms remain obscure. OBJECTIVE: To investigate the mechanism of miR-30d-mediated cardioprotection in HF. METHODS AND RESULTS: In rat and mouse models of ischemic HF, we show that miR-30d gain of function (genetic, lentivirus, or agomiR-mediated) improves cardiac function, decreases myocardial fibrosis, and attenuates cardiomyocyte (CM) apoptosis. Genetic or locked nucleic acid-based knock-down of miR-30d expression potentiates pathological left ventricular remodeling, with increased dysfunction, fibrosis, and cardiomyocyte death. RNA sequencing of in vitro miR-30d gain and loss of function, together with bioinformatic prediction and experimental validation in cardiac myocytes and fibroblasts, were used to identify and validate direct targets of miR-30d. miR-30d expression is selectively enriched in cardiomyocytes, induced by hypoxic stress and is acutely protective, targeting MAP4K4 (mitogen-associate protein kinase 4) to ameliorate apoptosis. Moreover, miR-30d is secreted primarily in extracellular vesicles by cardiomyocytes and inhibits fibroblast proliferation and activation by directly targeting integrin α5 in the acute phase via paracrine signaling to cardiac fibroblasts. In the chronic phase of ischemic remodeling, lower expression of miR-30d in the heart and plasma extracellular vesicles is associated with adverse remodeling in rodent models and human subjects and is linked to whole-blood expression of genes implicated in fibrosis and inflammation, consistent with observations in model systems. CONCLUSIONS: These findings provide the mechanistic underpinning for the cardioprotective association of miR-30d in human HF. More broadly, our findings support an emerging paradigm involving intercellular communication of extracellular vesicle-contained miRNAs (microRNAs) to transregulate distinct signaling pathways across cell types. Functionally validated RNA biomarkers and their signaling networks may warrant further investigation as novel therapeutic targets in HF.
Asunto(s)
MicroARNs/metabolismo , Infarto del Miocardio/metabolismo , Miocardio/metabolismo , Comunicación Paracrina , Función Ventricular Izquierda , Remodelación Ventricular , Animales , Apoptosis , Células Cultivadas , Modelos Animales de Enfermedad , Vesículas Extracelulares/genética , Vesículas Extracelulares/metabolismo , Vesículas Extracelulares/patología , Fibroblastos/metabolismo , Fibroblastos/patología , Fibrosis , Regulación de la Expresión Génica , Masculino , Ratones Endogámicos C57BL , Ratones Transgénicos , MicroARNs/genética , Infarto del Miocardio/genética , Infarto del Miocardio/patología , Infarto del Miocardio/fisiopatología , Miocardio/patología , Miocitos Cardíacos/metabolismo , Miocitos Cardíacos/patología , Proteínas Serina-Treonina Quinasas/genética , Proteínas Serina-Treonina Quinasas/metabolismo , Ratas Sprague-Dawley , Ratas Transgénicas , Transducción de Señal , Quinasa de Factor Nuclear kappa BRESUMEN
The ability of individuals with end-stage osteoarthritis (OA) to functionally recover from total joint arthroplasty is highly inconsistent. The molecular mechanisms driving this heterogeneity have yet to be elucidated. Furthermore, OA disproportionately impacts females, suggesting a need for identifying female-specific therapeutic targets. We profiled the skeletal muscle transcriptome in females with end-stage OA (n = 20) undergoing total knee or hip arthroplasty using RNA-Seq. Single-gene differential expression (DE) analyses tested for DE genes between skeletal muscle overlaying the surgical (SX) joint and muscle from the contralateral (CTRL) leg. Network analyses were performed using Pathway-Level Information ExtractoR (PLIER) to summarize genes into latent variables (LVs), i.e., gene circuits, and link them to biological pathways. LV differences in SX versus CTRL muscle and across sources of muscle tissue (vastus medialis, vastus lateralis, or tensor fascia latae) were determined with ANOVA. Linear models tested for associations between LVs and muscle phenotype on the SX side (inflammation, function, and integrity). DE analysis revealed 360 DE genes (|Log2 fold-difference| ≥ 1, FDR ≤ 0.05) between the SX and CTRL limbs, many associated with inflammation and lipid metabolism. PLIER analyses revealed circuits associated with protein degradation and fibro-adipogenic cell gene expression. Muscle inflammation and function were linked to an LV associated with endothelial cell gene expression highlighting a potential regulatory role of endothelial cells within skeletal muscle. These findings may provide insight into potential therapeutic targets to improve OA rehabilitation before and/or following total joint replacement.
Asunto(s)
Artroplastia de Reemplazo de Cadera , Artroplastia de Reemplazo de Rodilla , Osteoartritis , Femenino , Humanos , Células Endoteliales , Articulación de la Rodilla , Osteoartritis/genética , Músculo EsqueléticoRESUMEN
With the emergence of RNA sequencing (RNA-seq) technologies, RNA-based biomolecules hold expanded promise for their diagnostic, prognostic and therapeutic applicability in various diseases, including cancers and infectious diseases. Detection of gene fusions and differential expression of known disease-causing transcripts by RNA-seq represent some of the most immediate opportunities. However, it is the diversity of RNA species detected through RNA-seq that holds new promise for the multi-faceted clinical applicability of RNA-based measures, including the potential of extracellular RNAs as non-invasive diagnostic indicators of disease. Ongoing efforts towards the establishment of benchmark standards, assay optimization for clinical conditions and demonstration of assay reproducibility are required to expand the clinical utility of RNA-seq.
Asunto(s)
Pruebas Diagnósticas de Rutina/métodos , Enfermedad/genética , Perfilación de la Expresión Génica , Secuenciación de Nucleótidos de Alto Rendimiento/métodos , ARN/genética , Análisis de Secuencia de ARN/métodos , Biología Computacional/métodos , HumanosRESUMEN
Gene set enrichment analysis has become one of the most frequently used applications in molecular biology research. Originally developed for gene sets, the same statistical principles are now available for all omics types. In 2016, we published the miRNA enrichment analysis and annotation tool (miEAA) for human precursor and mature miRNAs. Here, we present miEAA 2.0, supporting miRNA input from ten frequently investigated organisms. To facilitate inclusion of miEAA in workflow systems, we implemented an Application Programming Interface (API). Users can perform miRNA set enrichment analysis using either the web-interface, a dedicated Python package, or custom remote clients. Moreover, the number of category sets was raised by an order of magnitude. We implemented novel categories like annotation confidence level or localisation in biological compartments. In combination with the miRBase miRNA-version and miRNA-to-precursor converters, miEAA supports research settings where older releases of miRBase are in use. The web server also offers novel comprehensive visualizations such as heatmaps and running sum curves with background distributions. We demonstrate the new features with case studies for human kidney cancer, a biomarker study on Parkinson's disease from the PPMI cohort, and a mouse model for breast cancer. The tool is freely accessible at: https://www.ccb.uni-saarland.de/mieaa2.
Asunto(s)
MicroARNs/metabolismo , Programas Informáticos , Animales , Biomarcadores , Neoplasias de la Mama/genética , Carcinoma de Células Renales/genética , Progresión de la Enfermedad , Femenino , Humanos , Neoplasias Renales/genética , Ratones , Enfermedad de Parkinson/genética , Flujo de TrabajoRESUMEN
BACKGROUND: Metabolic disorders such as obesity and diabetes mellitus can cause dysfunction of endothelial cells (ECs) and vascular rarefaction in adipose tissues. However, the modulatory role of ECs in adipose tissue function is not fully understood. Other than vascular endothelial growth factor-vascular endothelial growth factor receptor-mediated angiogenic signaling, little is known about the EC-derived signals in adipose tissue regulation. We previously identified Argonaute 1 (AGO1; a key component of microRNA-induced silencing complex) as a crucial regulator in hypoxia-induced angiogenesis. In this study, we intend to determine the AGO1-mediated EC transcriptome, the functional importance of AGO1-regulated endothelial function in vivo, and the relevance to adipose tissue function and obesity. METHODS: We generated and subjected mice with EC-AGO1 deletion (EC-AGO1-knockout [KO]) and their wild-type littermates to a fast food-mimicking, high-fat high-sucrose diet and profiled the metabolic phenotypes. We used crosslinking immunoprecipitation- and RNA-sequencing to identify the AGO1-mediated mechanisms underlying the observed metabolic phenotype of EC-AGO1-KO. We further leveraged cell cultures and mouse models to validate the functional importance of the identified molecular pathway, for which the translational relevance was explored using human endothelium isolated from healthy donors and donors with obesity/type 2 diabetes mellitus. RESULTS: We identified an antiobesity phenotype of EC-AGO1-KO, evident by lower body weight and body fat, improved insulin sensitivity, and enhanced energy expenditure. At the organ level, we observed the most significant phenotype in the subcutaneous and brown adipose tissues of KO mice, with greater vascularity and enhanced browning and thermogenesis. Mechanistically, EC-AGO1 suppression results in inhibition of thrombospondin-1 (THBS1/TSP1), an antiangiogenic and proinflammatory cytokine that promotes insulin resistance. In EC-AGO1-KO mice, overexpression of TSP1 substantially attenuated the beneficial phenotype. In human endothelium isolated from donors with obesity or type 2 diabetes mellitus, AGO1 and THBS1 are expressed at higher levels than the healthy controls, supporting a pathological role of this pathway. CONCLUSIONS: Our study suggests a novel mechanism by which ECs, through the AGO1-TSP1 pathway, control vascularization and function of adipose tissues, insulin sensitivity, and whole-body metabolic state.
Asunto(s)
Tejido Adiposo Pardo/metabolismo , Proteínas Argonautas/metabolismo , Susceptibilidad a Enfermedades , Endotelio/metabolismo , Factores Eucarióticos de Iniciación/metabolismo , Enfermedades Metabólicas/etiología , Enfermedades Metabólicas/metabolismo , Adulto , Animales , Proteínas Argonautas/genética , Dieta Alta en Grasa , Modelos Animales de Enfermedad , Metabolismo Energético , Factores Eucarióticos de Iniciación/genética , Femenino , Perfilación de la Expresión Génica , Marcación de Gen , Sitios Genéticos , Humanos , Resistencia a la Insulina , Masculino , Enfermedades Metabólicas/diagnóstico , Ratones , Ratones Noqueados , Persona de Mediana Edad , Modelos Biológicos , Obesidad , FenotipoRESUMEN
Mesenchymal stem or stromal cells are currently under clinical investigation for multiple diseases. While their mechanism of action is still not fully elucidated, vesicles secreted by MSCs are believed to recapitulate their therapeutic potentials to some extent. Microvesicles (MVs), also called as microparticles or ectosome, are among secreted vesicles that could transfer cytoplasmic cargo, including RNA and proteins, from emitting (source) cells to recipient cells. Given the importance of MVs, we here attempted to establish a method to isolate and characterize MVs secreted from unmodified human bone marrow derived MSCs (referred to as native MSCs, and their microvesicles as Native-MVs) and IFNγ stimulated MSCs (referred to as IFNγ-MSCs, and their microvesicles as IFNγ-MVs). We first describe an ultracentrifugation technique to isolate MVs from the conditioned cell culture media of MSCs. Next, we describe characterization and quality control steps to analyze the protein and RNA content of MVs. Finally, we examined the potential of MVs to exert immunomodulatory effects through induction of regulatory T cells (Tregs). Secretory vesicles from MSCs are promising alternatives for cell therapy with applications in drug delivery, regenerative medicine, and immunotherapy.
Asunto(s)
Micropartículas Derivadas de Células/química , Sistemas de Liberación de Medicamentos/métodos , Células Madre Mesenquimatosas/química , Proteómica/métodos , Medicina Regenerativa/métodos , Animales , Células de la Médula Ósea/química , Células de la Médula Ósea/citología , Células de la Médula Ósea/efectos de los fármacos , Células de la Médula Ósea/inmunología , Separación Celular/métodos , Micropartículas Derivadas de Células/inmunología , Medios de Cultivo Condicionados/química , Humanos , Inmunoterapia/métodos , Interferón gamma/farmacología , Células Madre Mesenquimatosas/citología , Células Madre Mesenquimatosas/efectos de los fármacos , Células Madre Mesenquimatosas/inmunología , Proteínas/clasificación , Proteínas/aislamiento & purificación , ARN/clasificación , ARN/aislamiento & purificación , Linfocitos T Reguladores/efectos de los fármacos , Linfocitos T Reguladores/inmunologíaRESUMEN
The hexanucleotide repeat expansion GGGGCC (G4C2)n in the C9orf72 gene is the most common genetic abnormality associated with amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). Recent findings suggest that dysfunction of nuclear-cytoplasmic trafficking could affect the transport of RNA binding proteins in C9orf72 ALS/FTD. Here, we provide evidence that the RNA editing enzyme adenosine deaminase acting on RNA 2 (ADAR2) is mislocalized in C9orf72 repeat expansion mediated ALS/FTD. ADAR2 is responsible for adenosine (A) to inosine (I) editing of double-stranded RNA, and its function has been shown to be essential for survival. Here we show the mislocalization of ADAR2 in human induced pluripotent stem cell-derived motor neurons (hiPSC-MNs) from C9orf72 patients, in mice expressing (G4C2)149, and in C9orf72 ALS/FTD patient postmortem tissue. As a consequence of this mislocalization we observe alterations in RNA editing in our model systems and across multiple brain regions. Analysis of editing at 408,580 known RNA editing sites indicates that there are vast RNA A to I editing aberrations in C9orf72-mediated ALS/FTD. These RNA editing aberrations are found in many cellular pathways, such as the ALS pathway and the crucial EIF2 signaling pathway. Our findings suggest that the mislocalization of ADAR2 in C9orf72 mediated ALS/FTD is responsible for the alteration of RNA processing events that may impact vast cellular functions, including the integrated stress response (ISR) and protein translation.
Asunto(s)
Adenosina Desaminasa/genética , Proteína C9orf72/genética , Edición de ARN/genética , Proteínas de Unión al ARN/genética , Esclerosis Amiotrófica Lateral/genética , Animales , Expansión de las Repeticiones de ADN/genética , Demencia Frontotemporal/genética , Humanos , Células Madre Pluripotentes Inducidas/metabolismo , Ratones Transgénicos , Enfermedad de Pick/genéticaRESUMEN
The original article was published erroneously without mentioning the support of the U.S.
RESUMEN
BACKGROUND: Evolving interest in comprehensively profiling the full range of small RNAs present in small tissue biopsies and in circulating biofluids, and how the profile differs with disease, has launched small RNA sequencing (RNASeq) into more frequent use. However, known biases associated with small RNASeq, compounded by low RNA inputs, have been both a significant concern and a hurdle to widespread adoption. As RNASeq is becoming a viable choice for the discovery of small RNAs in low input samples and more labs are employing it, there should be benchmark datasets to test and evaluate the performance of new sequencing protocols and operators. In a recent publication from the National Institute of Standards and Technology, Pine et al., 2018, the investigators used a commercially available set of three tissues and tested performance across labs and platforms. RESULTS: In this paper, we further tested the performance of low RNA input in three commonly used and commercially available RNASeq library preparation kits; NEB Next, NEXTFlex, and TruSeq small RNA library preparation. We evaluated the performance of the kits at two different sites, using three different tissues (brain, liver, and placenta) with high (1 µg) and low RNA (10 ng) input from tissue samples, or 5.0, 3.0, 2.0, 1.0, 0.5, and 0.2 ml starting volumes of plasma. As there has been a lack of robust validation platforms for differentially expressed miRNAs, we also compared low input RNASeq data with their expression profiles on three different platforms (Abcam Fireplex, HTG EdgeSeq, and Qiagen miRNome). CONCLUSIONS: The concordance of RNASeq results on these three platforms was dependent on the RNA expression level; the higher the expression, the better the reproducibility. The results provide an extensive analysis of small RNASeq kit performance using low RNA input, and replication of these data on three downstream technologies.
Asunto(s)
Biblioteca de Genes , ARN/metabolismo , Encéfalo/metabolismo , Femenino , Secuenciación de Nucleótidos de Alto Rendimiento , Humanos , Hígado/metabolismo , MicroARNs/análisis , MicroARNs/química , Placenta/metabolismo , Embarazo , Análisis de Componente Principal , ARN/química , Juego de Reactivos para Diagnóstico , Análisis de Secuencia de ARNRESUMEN
BACKGROUND: The potential utility of microRNA as biomarkers for early detection of cancer and other diseases is being investigated with genome-scale profiling of differentially expressed microRNA. Processes for measurement assurance are critical components of genome-scale measurements. Here, we evaluated the utility of a set of total RNA samples, designed with between-sample differences in the relative abundance of miRNAs, as process controls. RESULTS: Three pure total human RNA samples (brain, liver, and placenta) and two different mixtures of these components were evaluated as measurement assurance control samples on multiple measurement systems at multiple sites and over multiple rounds. In silico modeling of mixtures provided benchmark values for comparison with physical mixtures. Biomarker development laboratories using next-generation sequencing (NGS) or genome-scale hybridization assays participated in the study and returned data from the samples using their routine workflows. Multiplexed and single assay reverse-transcription PCR (RT-PCR) was used to confirm in silico predicted sample differences. Data visualizations and summary metrics for genome-scale miRNA profiling assessment were developed using this dataset, and a range of performance was observed. These metrics have been incorporated into an online data analysis pipeline and provide a convenient dashboard view of results from experiments following the described design. The website also serves as a repository for the accumulation of performance values providing new participants in the project an opportunity to learn what may be achievable with similar measurement processes. CONCLUSIONS: The set of reference samples used in this study provides benchmark values suitable for assessing genome-scale miRNA profiling processes. Incorporation of these metrics into an online resource allows laboratories to periodically evaluate their performance and assess any changes introduced into their measurement process.
Asunto(s)
Encéfalo/metabolismo , Perfilación de la Expresión Génica/normas , Genoma Humano , Hígado/metabolismo , MicroARNs/genética , Placenta/metabolismo , Femenino , Perfilación de la Expresión Génica/métodos , Secuenciación de Nucleótidos de Alto Rendimiento/métodos , Humanos , Embarazo , Estándares de ReferenciaRESUMEN
Exercise improves cardiometabolic and vascular function, although the mechanisms remain unclear. Our objective was to demonstrate the diversity of circulating extracellular RNA (ex-RNA) release during acute exercise in humans and its relevance to exercise-mediated benefits on vascular inflammation. We performed plasma small RNA sequencing in 26 individuals undergoing symptom-limited maximal treadmill exercise, with replication of our top candidate miRNA in a separate cohort of 59 individuals undergoing bicycle ergometry. We found changes in miRNAs and other ex-RNAs with exercise (e.g., Y RNAs and tRNAs) implicated in cardiovascular disease. In two independent cohorts of acute maximal exercise, we identified miR-181b-5p as a key ex-RNA increased in plasma after exercise, with validation in a separate cohort. In a mouse model of acute exercise, we found significant increases in miR-181b-5p expression in skeletal muscle after acute exercise in young (but not older) mice. Previous work revealed a strong role for miR-181b-5p in vascular inflammation in obesity, insulin resistance, sepsis, and cardiovascular disease. We conclude that circulating ex-RNAs were altered in plasma after acute exercise target pathways involved in inflammation, including miR-181b-5p. Further investigation into the role of known (e.g., miRNA) and novel (e.g., Y RNAs) RNAs is warranted to uncover new mechanisms of vascular inflammation on exercise-mediated benefits on health.NEW & NOTEWORTHY How exercise provides benefits to cardiometabolic health remains unclear. We performed RNA sequencing in plasma during exercise to identify the landscape of small noncoding circulating transcriptional changes. Our results suggest a link between inflammation and exercise, providing rich data on circulating noncoding RNAs for future studies by the scientific community.
Asunto(s)
MicroARN Circulante/sangre , Ejercicio Físico , Inflamación/sangre , Síndrome Metabólico/sangre , ARN de Transferencia/sangre , Adulto , Anciano , Animales , Ciclismo , MicroARN Circulante/genética , Prueba de Esfuerzo/métodos , Femenino , Marcadores Genéticos , Estado de Salud , Humanos , Inflamación/diagnóstico , Inflamación/genética , Masculino , Síndrome Metabólico/diagnóstico , Síndrome Metabólico/genética , Ratones , Ratones Endogámicos C57BL , MicroARNs/sangre , MicroARNs/genética , MicroARNs/metabolismo , Persona de Mediana Edad , Músculo Esquelético/metabolismo , ARN de Transferencia/genética , Factores de TiempoRESUMEN
Cells secrete extracellular RNA (exRNA) to their surrounding environment and exRNA has been found in many body fluids such as blood, breast milk and cerebrospinal fluid. However, there are conflicting results regarding the nature of exRNA. Here, we have separated 2 distinct exRNA profiles released by mast cells, here termed high-density (HD) and low-density (LD) exRNA. The exRNA in both fractions was characterized by microarray and next-generation sequencing. Both exRNA fractions contained mRNA and miRNA, and the mRNAs in the LD exRNA correlated closely with the cellular mRNA, whereas the HD mRNA did not. Furthermore, the HD exRNA was enriched in lincRNA, antisense RNA, vault RNA, snoRNA, and snRNA with little or no evidence of full-length 18S and 28S rRNA. The LD exRNA was enriched in mitochondrial rRNA, mitochondrial tRNA, tRNA, piRNA, Y RNA, and full-length 18S and 28S rRNA. The proteomes of the HD and LD exRNA-containing fractions were determined with LC-MS/MS and analyzed with Gene Ontology term finder, which showed that both proteomes were associated with the term extracellular vesicles and electron microscopy suggests that at least a part of the exRNA is associated with exosome-like extracellular vesicles. Additionally, the proteins in the HD fractions tended to be associated with the nucleus and ribosomes, whereas the LD fraction proteome tended to be associated with the mitochondrion. We show that the 2 exRNA signatures released by a single cell type can be separated by floatation on a density gradient. These results show that cells can release multiple types of exRNA with substantial differences in RNA species content. This is important for any future studies determining the nature and function of exRNA released from different cells under different conditions.
Asunto(s)
Vesículas Extracelulares/metabolismo , Secuenciación de Nucleótidos de Alto Rendimiento , ARN/genética , Línea Celular , Análisis por Conglomerados , Vesículas Extracelulares/ultraestructura , Perfilación de la Expresión Génica , Humanos , MicroARNs/genética , ARN/aislamiento & purificación , ARN Mensajero/genética , ARN Ribosómico/genética , ARN no Traducido/genéticaRESUMEN
BACKGROUND & AIMS: Accumulating evidence indicates that microRNAs play a role in a number of disease processes including the pathogenesis of liver fibrosis in hepatitis C infection. Our goal is to add to the accruing information regarding microRNA deregulation in liver fibrosis to increase our understanding of the underlying mechanisms of pathology and progression. METHODS: We used next generation sequencing to profile all detectable microRNAs in liver tissue and serum from patients with hepatitis C, stages F1-F4 of fibrosis. RESULTS: We found altered expression of several microRNAs, in particular, miR-182, miR199a-5p, miR-200a-5p and miR-183 were found to be significantly upregulated in tissue from liver biopsies of hepatitis C patients with advanced fibrosis, stage F3 and F4, when compared with liver biopsies from patients with early fibrosis, stages F1 and F2. We also found miR-148-5p, miR-1260b, miR-122-3p and miR-378i among the microRNAs most significantly down-regulated from early to advanced fibrosis of the liver. We also sequenced the serum microRNAs; however, we were not able to detect significant changes in circulating microRNAs associated with fibrosis stage after adjusting for multiple tests. CONCLUSIONS: Adding measurements of tissue microRNAs acquired during routine biopsies will continue to increase our knowledge of underlying mechanisms of fibrosis. Our goal is that these data, in combination with studies from other researchers and future long-term studies, could be used to enhance the staging accuracy of liver biopsies and expand the surveillance of patients at increased risk for cancer and progression to advanced fibrosis.
Asunto(s)
Hepatitis C Crónica/genética , Cirrosis Hepática/genética , Hígado/química , MicroARNs/genética , Anciano , Biopsia , Progresión de la Enfermedad , Femenino , Perfilación de la Expresión Génica/métodos , Regulación de la Expresión Génica , Redes Reguladoras de Genes , Marcadores Genéticos , Hepatitis C Crónica/complicaciones , Hepatitis C Crónica/diagnóstico , Humanos , Hígado/patología , Hígado/virología , Cirrosis Hepática/diagnóstico , Cirrosis Hepática/virología , Masculino , Persona de Mediana Edad , Análisis de Secuencia por Matrices de Oligonucleótidos , Valor Predictivo de las Pruebas , Factores de Riesgo , Análisis de Secuencia de ARN , Factores de TiempoRESUMEN
There has been a growing interest in using next-generation sequencing (NGS) to profile extracellular small RNAs from the blood and cerebrospinal fluid (CSF) of patients with neurological diseases, CNS tumors, or traumatic brain injury for biomarker discovery. Small sample volumes and samples with low RNA abundance create challenges for downstream small RNA sequencing assays. Plasma, serum, and CSF contain low amounts of total RNA, of which small RNAs make up a fraction. The purpose of this study was to maximize RNA isolation from RNA-limited samples and apply these methods to profile the miRNA in human CSF by small RNA deep sequencing. We systematically tested RNA isolation efficiency using ten commercially available kits and compared their performance on human plasma samples. We used RiboGreen to quantify total RNA yield and custom TaqMan assays to determine the efficiency of small RNA isolation for each of the kits. We significantly increased the recovery of small RNA by repeating the aqueous extraction during the phenol-chloroform purification in the top performing kits. We subsequently used the methods with the highest small RNA yield to purify RNA from CSF and serum samples from the same individual. We then prepared small RNA sequencing libraries using Illumina's TruSeq sample preparation kit and sequenced the samples on the HiSeq 2000. Not surprisingly, we found that the miRNA expression profile of CSF is substantially different from that of serum. To our knowledge, this is the first time that the small RNA fraction from CSF has been profiled using next-generation sequencing.