RESUMEN
Exosomes are RNA and protein-containing nanovesicles secreted by all cell types and found in abundance in body fluids, including blood, urine and cerebrospinal fluid. These vesicles seem to be a perfect source of biomarkers, as their cargo largely reflects the content of parental cells, and exosomes originating from all organs can be obtained from circulation through minimally invasive or non-invasive means. Here we describe an optimized procedure for exosome isolation and analysis using clinical samples, starting from quick and robust extraction of exosomes with Total exosome isolation reagent, then isolation of RNA followed by qRT-PCR. Effectiveness of this workflow is exemplified by analysis of the miRNA content of exosomes derived from serum samples - obtained from the patients with metastatic prostate cancer, treated prostate cancer patients who have undergone prostatectomy, and control patients without prostate cancer. Three promising exosomal microRNA biomarkers were identified, discriminating these groups: hsa-miR375, hsa-miR21, hsa-miR574.
Asunto(s)
Biomarcadores de Tumor/sangre , Exosomas/química , MicroARNs/sangre , Neoplasias de la Próstata/química , Biomarcadores de Tumor/genética , Biomarcadores de Tumor/aislamiento & purificación , Estudios de Casos y Controles , Expresión Génica , Humanos , Indicadores y Reactivos/química , Masculino , MicroARNs/genética , Antígeno Prostático Específico/sangre , Antígeno Prostático Específico/genética , Prostatectomía , Neoplasias de la Próstata/diagnóstico , Neoplasias de la Próstata/genética , Neoplasias de la Próstata/cirugía , ARN Neoplásico/sangre , ARN Neoplásico/genética , ARN Neoplásico/aislamiento & purificación , Reacción en Cadena de la Polimerasa de Transcriptasa InversaRESUMEN
BACKGROUND: Cells continuously secrete a large number of microvesicles, macromolecular complexes, and small molecules into the extracellular space. Of the secreted microvesicles, the nanoparticles called exosomes are currently undergoing intense scrutiny. These are small vesicles (30-120 nm) containing nucleic acid and protein, perceived to be carriers of this cargo between diverse locations in the body. They are distinguished in their genesis by being budded into endosomes to form multivesicular bodies (MVBs) in the cytoplasm. The exosomes are released to extracellular fluids by fusion of these multivesicular bodies with the cell surface, resulting in secretion in bursts. Exosomes are secreted by all types of cells in culture, and also found in abundance in body fluids including blood, saliva, urine, and breast milk. SCOPE OF REVIEW: In this review, we summarize strategies for exosome isolation, our understanding to date of exosome composition, functions, and pathways, and discuss their potential for diagnostic and therapeutic applications. MAJOR CONCLUSIONS: Currently, the control of exosome formation, the makeup of the "cargo", biological pathways and resulting functions are incompletely understood. One of their most intriguing roles is intercellular communication--exosomes are thought to function as the messengers, delivering various effectors or signaling macromolecules between supposedly very specific cells. GENERAL SIGNIFICANCE: Both seasoned and newer investigators of nanovesicles have presented various viewpoints on what exosomes are, with some differences but a large common area. It would be useful to develop a codified definition of exosomes in both descriptive and practical terms. We hope this in turns leads to a consistent set of practices for their isolation, characterization and manipulation.
Asunto(s)
Biomarcadores/análisis , Comunicación Celular , Exosomas/metabolismo , Cuerpos Multivesiculares/fisiología , Neoplasias/diagnóstico , Neoplasias/terapia , Animales , Transporte Biológico , Humanos , Transducción de SeñalRESUMEN
Tumors of the same histologic type often comprise clinically and molecularly distinct subgroups; however, the etiology of these subgroups is unknown. Here, we report that histologically identical, but genetically distinct, ependymomas exhibit patterns of gene expression that recapitulate those of radial glia cells in the corresponding region of the central nervous system. Cancer stem cells isolated from ependymomas displayed a radial glia phenotype and formed tumors when orthotopically transplanted in mice. These findings identify restricted populations of radial glia cells as candidate stem cells of the different subgroups of ependymoma, and they support a general hypothesis that subgroups of the same histologic tumor type are generated by different populations of progenitor cells in the tissues of origin.
Asunto(s)
Ependimoma/patología , Neuroglía/patología , Células Madre/patología , Animales , Ependimoma/genética , Perfilación de la Expresión Génica , Humanos , Inmunohistoquímica , Hibridación Fluorescente in Situ , Ratones , Ratones Endogámicos C57BL , Ratones DesnudosRESUMEN
Medulloblastoma is the most common malignant pediatric brain tumor. Current treatment is associated with major long-term side effects; therefore, new nontoxic therapies, targeting specific molecular defects in this cancer, need to be developed. We use a mouse model of medulloblastoma to show that inhibition of the Sonic Hedgehog (Shh) pathway provides a novel therapy for medulloblastoma. A small molecule inhibitor of the Shh pathway, HhAntag, blocked the function of Smoothened in mice with medulloblastoma. This resulted in suppression of several genes highly expressed in medulloblastoma, inhibition of cell proliferation, increase in cell death and, at the highest dose, complete eradication of tumors. Long-term treatment with HhAntag prolonged medulloblastoma-free survival. These findings support the development of Shh antagonists for the treatment of medulloblastoma.
Asunto(s)
Neoplasias Encefálicas/metabolismo , Meduloblastoma/metabolismo , Transactivadores/antagonistas & inhibidores , Animales , Neoplasias Encefálicas/tratamiento farmacológico , Neoplasias Encefálicas/patología , División Celular , Supervivencia sin Enfermedad , Relación Dosis-Respuesta a Droga , Proteínas Hedgehog , Factores de Transcripción de Tipo Kruppel , Meduloblastoma/tratamiento farmacológico , Meduloblastoma/patología , Ratones , Ratones Transgénicos , Receptores Acoplados a Proteínas G/metabolismo , Transducción de Señal , Receptor Smoothened , Factores de Transcripción/metabolismo , Células Tumorales Cultivadas , Proteína con Dedos de Zinc GLI1RESUMEN
One critical step in RNA interference (RNAi) experiments is to design small interfering RNAs (siRNAs) that can greatly reduce the expression of the target transcripts, but not of other unintended targets. Although various statistical and computational approaches have been attempted, this remains a challenge facing RNAi researchers. Here, we present a new experimentally validated method for siRNA design. By analyzing public siRNA data and focusing on hyperfunctional siRNAs, we identified a set of sequence features as potency selection criteria to build an siRNA design algorithm with support vector machines. Additional bioinformatics filters were also included in the algorithm to increase RNAi specificity by reducing potential sequence cross-hybridization or microRNA-like effects. Independent validation experiments were performed, which indicated that the newly designed siRNAs have significantly improved performance, and worked effectively even at low concentrations. Furthermore, our cell-based studies demonstrated that the siRNA off-target effects were significantly reduced when the siRNAs were delivered into cells at the 3 nM concentration compared to 30 nM. Thus, the capability of our new design program to select highly potent siRNAs also renders increased RNAi specificity because these siRNAs can be used at a much lower concentration. The siRNA design web server is available at http://www5.appliedbiosystems.com/tools/siDesign/.
Asunto(s)
Algoritmos , Interferencia de ARN , ARN Interferente Pequeño/química , Inteligencia Artificial , Composición de Base , Línea Celular Tumoral , Biología Computacional , Técnicas de Silenciamiento del Gen , Humanos , Conformación de Ácido Nucleico , Análisis de Secuencia de ARNRESUMEN
The gene mutated in reeler (reelin) encodes a protein secreted by neurons in the developing brain that controls laminar positioning of migrating cells in the CNS by an unknown mechanism. To investigate Reelin function, we used the nestin promoter to express Reelin ectopically in the ventricular zone and other brain regions in transgenic mice. In the presence of the endogenous protein, ectopic Reelin did not alter cell migration in the neocortex or the cerebellum. However, in the reeler background, ectopic Reelin induced tyrosine phosphorylation of Dab-1 in the ventricular zone and rescued some, but not all, of the neuroanatomic and behavioral abnormalities characteristic of reeler. These results indicate that Reelin does not function simply as a positional signal. Rather, it appears to participate in multiple events critical for neuronal migration and cell positioning.
Asunto(s)
Ataxia/genética , Tipificación del Cuerpo/genética , Encéfalo/anomalías , Moléculas de Adhesión Celular Neuronal/deficiencia , Movimiento Celular/genética , Proteínas de la Matriz Extracelular/deficiencia , Ratones Mutantes Neurológicos/anomalías , Proteínas del Tejido Nervioso , Malformaciones del Sistema Nervioso/genética , Animales , Ataxia/metabolismo , Encéfalo/citología , Encéfalo/metabolismo , Moléculas de Adhesión Celular Neuronal/genética , Diferenciación Celular/genética , Coristoma/embriología , Coristoma/genética , Coristoma/metabolismo , Proteínas de la Matriz Extracelular/genética , Femenino , Regulación del Desarrollo de la Expresión Génica/genética , Inmunohistoquímica , Proteínas de Filamentos Intermediarios/genética , Proteínas de Filamentos Intermediarios/metabolismo , Masculino , Ratones , Ratones Mutantes Neurológicos/genética , Ratones Mutantes Neurológicos/metabolismo , Ratones Transgénicos , Mutación/genética , Malformaciones del Sistema Nervioso/metabolismo , Nestina , Regiones Promotoras Genéticas/genética , Proteína Reelina , Serina EndopeptidasasRESUMEN
CTP:phosphocholine cytidylyltransferase (CCT) catalyzes a rate-controlling step in the biosynthesis of phosphatidylcholine (PtdCho). Multiple CCT isoforms, CCTalpha, CCTbeta2, and CCTbeta3, are encoded by two genes, Pcyt1a and Pcyt1b. The importance of CCTalpha in mice was investigated by deleting exons 5 and 6 in the Pcyt1a gene using the Cre-lox system. Pcyt1a-/- zygotes failed to form blastocysts, did not develop past embryonic day 3.5 (E3.5), and failed to implant. In situ hybridization in E11.5 embryos showed that Pcyt1a is expressed ubiquitously, with the highest level in fetal liver, and CCTalpha transcripts are significantly more abundant than transcripts encoding CCTbeta or phosphatidylethanolamine (PtdEtn) N-methyl transferase, two other enzymes capable of producing PtdCho. Reduction of the CCTalpha transcripts in heterozygous E11.5 embryos was accompanied by upregulation of CCTbeta and PtdEtn N-methyltransferase transcripts. In contrast, enzymatic and real-time PCR data revealed that CCTbeta (Pcyt1b) expression is not upregulated to compensate for the reduction in CCTalpha expression in adult liver and other tissues from Pcyt1a+/- heterozygous mice. PtdCho biosynthesis measured by choline incorporation into isolated hepatocytes was not compromised in the Pcyt1a+/- mice. Liver PtdCho mass was the same in Pcyt1a+/+ and Pcyt1a+/- adult animals, but lung PtdCho mass decreased in the heterozygous mice. These data show that CCTalpha expression is required for early embryonic development, but that a 50% reduction in enzyme activity has little detectable impact on the operation of the CDP-choline metabolic pathway in adult tissues.
Asunto(s)
Citidililtransferasa de Colina-Fosfato/fisiología , Embrión de Mamíferos/metabolismo , Desarrollo Embrionario/fisiología , Genes Letales , Animales , Citidililtransferasa de Colina-Fosfato/genética , Citidililtransferasa de Colina-Fosfato/metabolismo , Embrión de Mamíferos/química , Desarrollo Embrionario/genética , Exones/genética , Femenino , Eliminación de Gen , Marcación de Gen , Hibridación in Situ , Isoenzimas/genética , Isoenzimas/fisiología , Metiltransferasas/genética , Metiltransferasas/metabolismo , Ratones , Fosfatidilcolinas/análisis , Fosfatidilcolinas/biosíntesis , Fosfatidiletanolamina N-Metiltransferasa , Embarazo , ARN Mensajero/análisis , ARN Mensajero/metabolismo , Distribución Tisular , Transcripción GenéticaRESUMEN
Mouse retina undergoes crucial changes during early postnatal development. By using Affymetrix microarrays, we analyzed gene expression profiles of wild-type 129SvEv/C57BL/6 mouse retinas at postnatal days (P) 7, 10, 14, 18, and 21 and found significantly altered expression of 355 genes. Characterization of these 355 genes provided insight into physiologic and pathologic processes of mouse retinal development during the second and third postnatal weeks, a period that corresponds to human embryogenesis between weeks 12 and 28. These genes formed 6 groups with similar change patterns. Among the genes, sixteen cause retinal diseases when mutated; most of these 16 genes were upregulated in retina during this period. Using the PathArt program, we identified the biological processes in which many of the 355 gene products function. Among the most active processes in the P7-P21 retina are those involved in neurogenesis, obesity, diabetes type II, apoptosis, growth and differentiation, and protein kinase activity. We examined the expression patterns of 58 genes in P7 and adult retinas by searching the Brain Gene Expression Map database. Although most genes were present in various cell types in retinas, many displayed high levels of expression specifically in the outer nuclear, inner nuclear, and/or ganglion cell layers. By combining our 3 analyses, we demonstrated that during this period of mouse retinal development, many genes play important roles in various cell types, multiple pathways are involved, and some genes in a pathway are expressed in coordinated patterns. Our results thus provide foundation for future detailed studies of specific genes and pathways in various genetic and environmental conditions during retinal development.
Asunto(s)
Envejecimiento/metabolismo , Regulación del Desarrollo de la Expresión Génica/fisiología , Retina/crecimiento & desarrollo , Retina/metabolismo , Algoritmos , Animales , Interpretación Estadística de Datos , Hibridación in Situ , Proteínas Quinasas JNK Activadas por Mitógenos/biosíntesis , Ratones , Ratones Noqueados , Familia de Multigenes , Mutación/fisiología , Análisis de Secuencia por Matrices de Oligonucleótidos , Enfermedades de la Retina/genética , Retinitis Pigmentosa/genética , Transducción de Señal/fisiologíaRESUMEN
Two major signaling pathways that control neuronal positioning during brain development have been uncovered as a result of genetic and biochemical studies on neurological mouse mutants. Mice deficient in Reelin, Disabled 1 (Dab1), or both the very low-density lipoprotein receptor (VLDLR) and the apolipoprotein E receptor 2 (ApoER2) exhibit identical neuroanatomic defects in laminar structures throughout the brain. These proteins function as components of the Reelin signaling pathway. Reelin is a secreted glycoprotein that binds to VLDLR and ApoER2, inducing tyrosine phosphorylation of Dab1, an intracellular adapter protein. Neuronal migration is also regulated by cyclin-dependent kinase 5 (Cdk5) and its activating subunits p35 and p39. Mice deficient in Cdk5, p35, or both p35 and p39 exhibit lamination defects that are similar but not identical to those observed in mice with a defect in the Reelin signaling pathway. Cdk5 phosphorylates proteins that maintain cytoskeletal structures and promote cell motility. To explore the possibility that Cdk5 influences the Reelin pathway, we sought to determine whether Dab1 is a substrate for Cdk5. Here we show that Cdk5 phosphorylates Dab1 on serine 491 in vitro and in vivo, independently of Reelin signaling. We also show that ectopic neurons in Cdk5-deficient mice exhibit reduced levels of Reelin signaling during later stages of cortical development, although Cdk5 is not required for Reelin-induced tyrosine phosphorylation of Dab1. Although the functional significance of Dab1 serine phosphorylation is unclear, our results suggest that there is biochemical cross-talk between two signaling pathways that control cell positioning.
Asunto(s)
Encéfalo/metabolismo , Moléculas de Adhesión Celular Neuronal/metabolismo , Quinasas Ciclina-Dependientes/metabolismo , Proteínas de la Matriz Extracelular/metabolismo , Proteínas del Tejido Nervioso/metabolismo , Transducción de Señal , Secuencia de Aminoácidos , Animales , Anticuerpos/inmunología , Encéfalo/embriología , Encéfalo/enzimología , Encéfalo/crecimiento & desarrollo , Línea Celular , Cerebelo/metabolismo , Corteza Cerebral/metabolismo , Quinasa 5 Dependiente de la Ciclina , Quinasas Ciclina-Dependientes/genética , Humanos , Ratones , Ratones Noqueados , Datos de Secuencia Molecular , Proteínas del Tejido Nervioso/química , Neuronas/enzimología , Neuronas/metabolismo , Fosforilación , Fosfoserina/inmunología , Fosfoserina/metabolismo , Proteína Reelina , Serina EndopeptidasasRESUMEN
The Reelin signaling pathway in the brain involves the binding of Reelin to very-low-density lipoprotein receptors (VLDLR) and apolipoprotein E receptor 2 (ApoER2). After Reelin binds the lipoprotein receptors on migrating neurons, the intracellular adaptor protein Disabled-1 (Dab1) becomes phosphorylated, ultimately resulting in the proper positioning of cortical neurons. Previous work showed that Reelin also affects the positioning of sympathetic preganglionic neurons (SPN) in the spinal cord (Yip et al. [2000] Proc Natl Acad Sci USA 97:8612-8616). We asked in the present study whether components of the Reelin signaling pathway in the brain also function to control SPN migration in developing spinal cord. Results showed that Reelin and reelin mRNA are found adjacent to migrating SPN. In addition, dab1 mRNA and protein are expressed by migrating SPN, and dab1-null mice show abnormal SPN migration similar to that seen in reeler. Finally, vldlr and apoER2 are also expressed in migrating SPN, and mice lacking both vldlr and apoER2 show aberrant SPN location that is identical to that of reeler and dab1-null mice. Because molecules known to be involved in Reelin signaling in the brain are present in the developing spinal cord, it is likely that the Reelin signaling pathways in the brain and spinal cord function similarly. The relative simplicity of the organization of the spinal cord makes it a potentially useful model system with which to study the molecular and cellular function of the Reelin signaling pathway in control of neuronal migration.
Asunto(s)
Moléculas de Adhesión Celular Neuronal/biosíntesis , Proteínas de la Matriz Extracelular/biosíntesis , Transducción de Señal/fisiología , Médula Espinal/metabolismo , Fibras Adrenérgicas/metabolismo , Animales , Apolipoproteína E2 , Apolipoproteínas E/biosíntesis , Apolipoproteínas E/deficiencia , Apolipoproteínas E/genética , Moléculas de Adhesión Celular Neuronal/genética , Proteínas de la Matriz Extracelular/genética , Femenino , Regulación de la Expresión Génica/fisiología , Ratones , Ratones Noqueados , Ratones Mutantes Neurológicos , Proteínas del Tejido Nervioso , Embarazo , ARN Mensajero/biosíntesis , ARN Mensajero/genética , Receptores de LDL/biosíntesis , Receptores de LDL/deficiencia , Receptores de LDL/genética , Proteína Reelina , Serina EndopeptidasasRESUMEN
Reelin, Disabled-1 (Dab1), apolipoprotein E receptor 2 (ApoER2), and very low density lipoprotein receptor (VLDLR) participate in a signaling pathway required for layer formation during mammalian brain development. Binding of Reelin to ApoER2 and VLDLR induces a rapid increase in tyrosine phosphorylation of Dab1, an adaptor protein that associates with the cytoplasmic domain of the receptors. However, Reelin has also been proposed to signal through integrin and protocadherin. Here we compare the roles of ApoER2 and VLDLR in Reelin signaling. We used layer-specific markers to identify the final positions of early- and late-born neurons in the cortices of mice lacking ApoER2, VLDLR, or both ApoER2 and VLDLR. Subtle alterations were observed in mice lacking VLDLR, whereas more severe abnormalities were detected in the absence of ApoER2, and major disruptions were obvious in mice lacking both receptors. Purified Reelin associated more readily with ApoER2 than with VLDLR and no synergy was observed in the presence of both receptors. Consistent with the binding data, the level of Reelin-induced Dab1 phosphorylation was more severely reduced in neurons lacking ApoER2 than in neurons lacking VLDLR. However, similarly low levels of Dab1 tyrosine phosphorylation were observed in ApoER2(-/-) and VLDLR(-/-) mice in vivo. Finally, there was a complete absence of Reelin-induced tyrosine phosphorylation of Dab1 in cortical neurons from mice lacking both ApoER2 and VLDLR. These findings demonstrate that ApoER2 and VLDLR are essential for Reelin signaling and that no other receptor molecules can compensate for their role in mediating tyrosine phosphorylation of Dab1.
Asunto(s)
Moléculas de Adhesión Celular Neuronal/metabolismo , Corteza Cerebral/anomalías , Proteínas de la Matriz Extracelular/metabolismo , Proteínas del Tejido Nervioso/metabolismo , Neuronas/metabolismo , Receptores de LDL/deficiencia , Receptores de Lipoproteína/deficiencia , Proteínas Adaptadoras Transductoras de Señales , Animales , Moléculas de Adhesión Celular Neuronal/farmacología , Diferenciación Celular/genética , Movimiento Celular/genética , Células Cultivadas , Corteza Cerebral/citología , Corteza Cerebral/metabolismo , Relación Dosis-Respuesta a Droga , Proteínas de la Matriz Extracelular/farmacología , Femenino , Feto , Proteínas Relacionadas con Receptor de LDL , Ratones , Ratones Noqueados , Neuronas/citología , Neuronas/efectos de los fármacos , Fosforilación , Unión Proteica/genética , Receptores de LDL/genética , Receptores de Lipoproteína/genética , Proteína Reelina , Serina Endopeptidasas , Tirosina/metabolismoRESUMEN
The Reelin signaling pathway controls neuronal positioning during mammalian brain development by binding to the very low density lipoprotein receptor and apolipoprotein E receptor-2, and signaling through the intracellular adapter protein Disabled-1 (Dab1). To identify new components in the Reelin signaling pathway, we used a yeast two-hybrid screen to select Dab1-interacting proteins. Here, we report the characterization of a new mouse Dab1-interacting protein that is orthologous to rat Dab2IP, a Ras-GTPase activating protein previously shown to bind to Dab2/DOC. The interaction of Dab1 and Dab2IP was confirmed in biochemical assays and by co-immunoprecipitation from brain lysates. The site of interaction between Dab1 and Dab2IP was narrowed to the Dab1-PTB domain and the NPxY motif in Dab2IP. The deduced amino acid sequence of mouse Dab2IP encompasses 1,208 residues containing several protein interaction motifs as well as a Ras-like GAP-related domain. Northern blot analysis revealed at least two isoforms of Dab2IP mRNA in the brain, both of which exhibited increased expression during development. In situ hybridization analyses indicated that Dab2IP mRNA is diffusely expressed throughout the developing and the adult brain. Using a polyclonal antiserum specific for Dab2IP, we observed protein expression in the soma and processes of neurons in a variety of brain structures, including the developing cerebral cortex. Our findings suggest that Dab2IP may function as a downstream effector in the Reelin signaling pathway that influences Ras signaling during brain development.
Asunto(s)
Encéfalo/metabolismo , Proteínas Activadoras de GTPasa/metabolismo , Proteínas del Tejido Nervioso/metabolismo , Secuencia de Aminoácidos , Animales , Animales Recién Nacidos , Elementos sin Sentido (Genética)/metabolismo , Northern Blotting , Western Blotting , Encéfalo/citología , Encéfalo/embriología , Encéfalo/crecimiento & desarrollo , Calbindinas , Embrión de Mamíferos , Proteínas Activadoras de GTPasa/clasificación , Proteínas Activadoras de GTPasa/inmunología , Regulación del Desarrollo de la Expresión Génica , Inmunohistoquímica , Hibridación in Situ , Ratones , Ratones Noqueados , Datos de Secuencia Molecular , Fragmentos de Péptidos/inmunología , Fragmentos de Péptidos/metabolismo , Pruebas de Precipitina/métodos , Ratas , Proteínas Recombinantes de Fusión/metabolismo , Proteína Reelina , Proteína G de Unión al Calcio S100/metabolismo , Técnicas del Sistema de Dos Híbridos , LevadurasRESUMEN
Secreted and transmembrane proteins provide critical functions in the signaling networks essential for neurogenesis. We used a genetic signal sequence gene trap approach to isolate 189 genes expressed during development in e16.5 whole head, e16.5 hippocampus and e14.5 cerebellum. Gene ontology programs were used to classify the genes into respective biological processes. Four major classes of biological processes known to be important during development were identified: cell communication, cell physiology processes, metabolism and morphogenesis. We used in situ hybridization to determine the temporal and spatial patterns of gene expression in the developing brain using this set of probes. The results demonstrate that gene expression patterns can highlight potential gene functions in specific brain regions. We propose that combining bioinformatics with the gene expression pattern is an effective strategy to identify genes that may play critical roles during brain development.
Asunto(s)
Encéfalo/embriología , Encéfalo/fisiología , Regulación del Desarrollo de la Expresión Génica , Genómica/métodos , Animales , ADN Complementario , Femenino , Hibridación in Situ , Masculino , Ratones , Ratones Endogámicos C57BL , EmbarazoAsunto(s)
Bases de Datos Genéticas , Perfilación de la Expresión Génica , Hibridación in Situ , Sistema Nervioso/embriología , Sistema Nervioso/crecimiento & desarrollo , Animales , Encéfalo/embriología , Encéfalo/crecimiento & desarrollo , Encéfalo/metabolismo , Humanos , Internet , Ratones , Ratones Endogámicos C57BL , Datos de Secuencia Molecular , Sistema Nervioso/metabolismoRESUMEN
There is an acceptance that plasmid-based delivery of interfering RNA always generates the intended targeting sequences in cells, making it as specific as its synthetic counterpart. However, recent studies have reported on cellular inefficiencies of the former, especially in light of emerging gene discordance at inter-screen level and across formats. Focusing primarily on the TRC plasmid-based shRNA hairpins, we reasoned that alleged specificities were perhaps compromised due to altered processing; resulting in a multitude of random interfering sequences. For this purpose, we opted to study the processing of hairpin TRCN#40273 targeting CTTN; which showed activity in a miRNA-21 gain-of-function shRNA screen, but inactive when used as an siRNA duplex. Using a previously described walk-through method, we identified 36 theoretical cleavage variants resulting in 78 potential siRNA duplexes targeting 53 genes. We synthesized and tested all of them. Surprisingly, six duplexes targeting ASH1L, DROSHA, GNG7, PRKCH, THEM4, and WDR92 scored as active. QRT-PCR analysis on hairpin transduced reporter cells confirmed knockdown of all six genes, besides CTTN; revealing a surprising 7 gene-signature perturbation by this one single hairpin. We expanded our qRT-PCR studies to 26 additional cell lines and observed unique knockdown profiles associated with each cell line tested; even for those lacking functional DICER1 gene suggesting no obvious dependence on dicer for shRNA hairpin processing; contrary to published models. Taken together, we report on a novel dicer independent, cell-type dependent mechanism for non-specific RNAi gene silencing we coin Alternate Targeting Sequence Generator (ATSG). In summary, ATSG adds another dimension to the already complex interpretation of RNAi screening data, and provides for the first time strong evidence in support of arrayed screening, and questions the scientific merits of performing pooled RNAi screens, where deconvolution of up to genome-scale pools is indispensable for target identification.
Asunto(s)
ARN Helicasas DEAD-box/metabolismo , Marcación de Gen/métodos , Interferencia de ARN , ARN Interferente Pequeño , Ribonucleasa III/metabolismo , Análisis de Secuencia de ARN/métodos , Células HeLa , Humanos , ARN Interferente Pequeño/biosíntesis , ARN Interferente Pequeño/genéticaRESUMEN
AIM: To develop protocols for isolation of exosomes and characterization of their RNA content. METHODS: Exosomes were extracted from HeLa cell culture media and human blood serum using the Total exosome isolation (from cell culture media) reagent, and Total exosome isolation (from serum) reagent respectively. Identity and purity of the exosomes was confirmed by Nanosight(®) analysis, electron microscopy, and Western blots for CD63 marker. Exosomal RNA cargo was recovered with the Total exosome RNA and protein isolation kit. Finally, RNA was profiled using Bioanalyzer and quantitative reverse transcription-polymerase chain reaction (qRT-PCR) methodology. RESULTS: Here we describe a novel approach for robust and scalable isolation of exosomes from cell culture media and serum, with subsequent isolation and analysis of RNA residing within these vesicles. The isolation procedure is completed in a fraction of the time, compared to the current standard protocols utilizing ultracentrifugation, and allows to recover fully intact exosomes in higher yields. Exosomes were found to contain a very diverse RNA cargo, primarily short sequences 20-200 nt (such as miRNA and fragments of mRNA), however longer RNA species were detected as well, including full-length 18S and 28S rRNA. CONCLUSION: We have successfully developed a set of reagents and a workflow allowing fast and efficient extraction of exosomes, followed by isolation of RNA and its analysis by qRT-PCR and other techniques.
RESUMEN
Exosomes are small (30-150 nm) vesicles containing unique RNA and protein cargo, secreted by all cell types in culture. They are also found in abundance in body fluids including blood, saliva, and urine. At the moment, the mechanism of exosome formation, the makeup of the cargo, biological pathways, and resulting functions are incompletely understood. One of their most intriguing roles is intercellular communication--exosomes function as the messengers, delivering various effector or signaling macromolecules between specific cells. There is an exponentially growing need to dissect structure and the function of exosomes and utilize them for development of minimally invasive diagnostics and therapeutics. Critical to further our understanding of exosomes is the development of reagents, tools, and protocols for their isolation, characterization, and analysis of their RNA and protein contents. Here we describe a complete exosome workflow solution, starting from fast and efficient extraction of exosomes from cell culture media and serum to isolation of RNA followed by characterization of exosomal RNA content using qRT-PCR and next-generation sequencing techniques. Effectiveness of this workflow is exemplified by analysis of the RNA content of exosomes derived from HeLa cell culture media and human serum, using Ion Torrent PGM as a sequencing platform.
Asunto(s)
Exosomas/metabolismo , ARN/metabolismo , Secuencia de Bases , Transporte Biológico , Western Blotting , Medios de Cultivo , Biblioteca de Genes , Células HeLa , Humanos , MicroARNs/sangre , MicroARNs/genética , ARN/sangre , ARN/genética , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Análisis de Secuencia de ARN , Suero/metabolismo , Tetraspanina 29/metabolismo , Tetraspanina 30/metabolismo , UltracentrifugaciónRESUMEN
MicroRNAs (miRNAs) are evolutionary conserved noncoding molecules that regulate gene expression. They influence a number of diverse biological functions, such as development and differentiation. However, their dysregulation has been shown to be associated with disease states, such as cancer. Genes and pathways regulating their biogenesis remain unknown and are highly sought after. For this purpose, we have validated a multiplexed high-content assay strategy to screen for such modulators. Here, we describe its implementation that makes use of a cell-based gain-of-function reporter assay monitoring enhanced green fluorescent protein expression under the control of miRNA 21 (miR-21); combined with measures of both cell metabolic activities through the use of Alamar Blue and cell death through imaged Hoechst-stained nuclei. The strategy was validated using a panel of known genes and enabled us to successfully progress to and complete an arrayed genome-wide short interfering RNA (siRNA) screen against the Ambion Silencer Select v4.0 library containing 64,755 siRNA duplexes covering 21,565 genes. We applied a high-stringency hit analysis method, referred to as the Bhinder-Djaballah analysis method, leading to the nomination of 1,273 genes as candidate inhibitors of the miR-21 biogenesis pathway; after several iterations eliminating those genes with only one active duplex and those enriched in seed sequence mediated off-target effects. Biological classifications revealed four major control junctions among them vesicular transport via clathrin-mediated endocytosis. Altogether, our screen has uncovered a number of novel candidate regulators that are potentially good druggable targets allowing for the discovery and development of small molecules for regulating miRNA function.
Asunto(s)
MicroARNs/biosíntesis , MicroARNs/genética , Interferencia de ARN , Algoritmos , Automatización , Células Cultivadas , Colorantes , Biblioteca de Genes , Marcación de Gen , Proteínas Fluorescentes Verdes , Ensayos Analíticos de Alto Rendimiento , Humanos , Procesamiento de Imagen Asistido por Computador , MicroARNs/efectos de los fármacos , Oxazinas , Reacción en Cadena de la Polimerasa , ARN Interferente Pequeño/genética , Reproducibilidad de los Resultados , XantenosRESUMEN
RNA interference (RNAi) is a regulatory mechanism of eukaryotic cells that uses small interfering RNAs (siRNA) to direct homology-dependent control of gene activity. Applications of RNAi include functional genomics, in vivo target validation, and gene-specific medicines. A key to in vivo application of siRNA is the advancement of efficient delivery to organs, tissues, or cell types of interest. There is a need to develop reliable and easy-to-use assays to evaluate siRNA delivery efficiency and distribution, study pathways, and stability of siRNAs in cells (post-transfection) and in animals (post- injection). We have adopted the Applied Biosystems TaqMan(®) based stem-loop RT-PCR technology, originally developed for quantification of endogenous microRNAs in cells, to fulfill these needs. In this chapter, application protocols are described, which enable robust quantification of siRNA, including chemically modified molecules, in vitro and in vivo.
Asunto(s)
Bioensayo , MicroARNs/análisis , ARN Interferente Pequeño/análisis , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa/métodos , Animales , Bioensayo/instrumentación , Bioensayo/métodos , Silenciador del Gen , Células HeLa , Humanos , Ratones , Ratones Endogámicos BALB C , MicroARNs/aislamiento & purificación , Interferencia de ARN , Estabilidad del ARN , ARN Interferente Pequeño/aislamiento & purificación , TransfecciónRESUMEN
RNA interference (RNAi) is a mechanism by which the introduction of small interfering RNAs (siRNAs) into cultured cells causes degradation of the complementary mRNA. Applications of RNAi include gene function analysis, pathway analysis, and target validation. While RNAi experiments have become common practice in research labs, multiple factors can influence the extent of siRNA-induced knockdown (and thus biological outcome). A properly designed and selected siRNA sequence, siRNA modification format, choice of transfection reagent/technique, optimized protocols of siRNA in vitro delivery, and an appropriate and optimized readout are all critical for ensuring a successful experiment. In this chapter, we describe a typical in vitro siRNA experiment with optimization of transfection conditions and analysis of siRNA potency, i.e., mRNA knockdown with quantitative real-time PCR.