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The amygdala processes positive and negative valence and contributes to addiction, but the cell-type-specific gene regulatory programs involved are unknown. We generated an atlas of single-nucleus gene expression and chromatin accessibility in the amygdala of outbred rats with high and low cocaine addiction-like behaviors following prolonged abstinence. Differentially expressed genes between the high and low groups were enriched for energy metabolism across cell types. Rats with high addiction index (AI) showed increased relapse-like behaviors and GABAergic transmission in the amygdala. Both phenotypes were reversed by pharmacological inhibition of the glyoxalase 1 enzyme, which metabolizes methylglyoxal-a GABAA receptor agonist produced by glycolysis. Differences in chromatin accessibility between high and low AI rats implicated pioneer transcription factors in the basic helix-loop-helix, FOX, SOX and activator protein 1 families. We observed opposite regulation of chromatin accessibility across many cell types. Most notably, excitatory neurons had greater accessibility in high AI rats and inhibitory neurons had greater accessibility in low AI rats.
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Trastornos Relacionados con Cocaína , Cocaína , Humanos , Ratas , Animales , Amígdala del Cerebelo/fisiología , Neuronas , Cromatina/metabolismo , Cocaína/farmacologíaRESUMEN
During adolescence, frequent and heavy cannabis use can lead to serious adverse health effects and cannabis use disorder (CUD). Rodent models of adolescent exposure to the main psychoactive component of cannabis, delta-9-tetrahydrocannabinol (THC), mimic the behavioral alterations observed in adolescent users. However, the underlying molecular mechanisms remain largely unknown. Here, we treated female and male C57BL6/N mice with high doses of THC during early adolescence and assessed their memory and social behaviors in late adolescence. We then profiled the transcriptome of five brain regions involved in cognitive and addiction-related processes. We applied gene coexpression network analysis and identified gene coexpression modules, termed cognitive modules, that simultaneously correlated with THC treatment and memory traits reduced by THC. The cognitive modules were related to endocannabinoid signaling in the female dorsal medial striatum, inflammation in the female ventral tegmental area, and synaptic transmission in the male nucleus accumbens. Moreover, cross-brain region module-module interaction networks uncovered intra- and inter-region molecular circuitries influenced by THC. Lastly, we identified key driver genes of gene networks associated with THC in mice and genetic susceptibility to CUD in humans. This analysis revealed a common regulatory mechanism linked to CUD vulnerability in the nucleus accumbens of females and males, which shared four key drivers (Hapln4, Kcnc1, Elavl2, Zcchc12). These genes regulate transcriptional subnetworks implicated in addiction processes, synaptic transmission, brain development, and lipid metabolism. Our study provides novel insights into disease mechanisms regulated by adolescent exposure to THC in a sex- and brain region-specific manner.
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Cannabis , Expresión Génica , Alucinógenos , Factores Sexuales , Adolescente , Animales , Encéfalo , Agonistas de Receptores de Cannabinoides/farmacología , Cannabis/efectos adversos , Dronabinol/metabolismo , Endocannabinoides/metabolismo , Femenino , Redes Reguladoras de Genes , Alucinógenos/farmacología , Humanos , Masculino , Ratones , Ratones Endogámicos C57BL , Canales de Potasio Shaw/metabolismoRESUMEN
BACKGROUND: Preoperative computed tomography (CT)-guided localization has been used to guide the video-assisted thoracoscopic surgery (VATS) sublobar (wedge or segmental) resection for pulmonary nodules (PNs). We aimed to assess the relative efficacy and safety of CT-guided methylene blue (MB)- and coil-based approaches to the preoperative localization of multiple PNs (MPNs). METHODS: Between January 2015 and December 2020, 31 total cases suffering from MPNs at our hospital underwent CT-guided localization and subsequent VATS resection in our hospital, of whom 15 and 16 respectively underwent MB localization (MBL) and coil localization (CL). The clinical effectiveness and complication rates were compared between 2 groups. RESULTS: The PN- and patient-based technical success rates in the MBL group were both 100%, whereas in the CL group they were 97.2% (35/36) and 93.8% (15/16), respectively, with no substantial discrepancies between groups. Patients in the MBL group illustrated a substantially shorter CT-guided localization duration compared with the CL group (18 min vs. 29.5 min, P < 0.001). Pneumothorax rates (P = 1.000) and lung hemorrhage (P = 1.000) were comparable in both groups. In the MBL and CL groups, the median interval between localization and VATS was 1 h and 15.5 h, respectively (P < 0.001). One-stage VATS sublobar resection of the target nodules was successfully performed in all patients from both groups. CONCLUSION: Both CT-guided MBL and CL can be readily and safely utilized for preoperative localization in individuals who had MPNs, with MBL being correlated with a shorter localization duration compared with CL.
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Neoplasias Pulmonares , Nódulos Pulmonares Múltiples , Nódulo Pulmonar Solitario , Humanos , Neoplasias Pulmonares/diagnóstico por imagen , Neoplasias Pulmonares/cirugía , Azul de Metileno , Nódulos Pulmonares Múltiples/diagnóstico por imagen , Nódulos Pulmonares Múltiples/cirugía , Estudios Retrospectivos , Nódulo Pulmonar Solitario/diagnóstico por imagen , Nódulo Pulmonar Solitario/cirugía , Cirugía Torácica Asistida por Video/métodos , Tomografía Computarizada por Rayos X/métodosRESUMEN
Social network site appearance comparison refers to a tendency to compare the body image of one-self to others when using social network sites. It was found to be associated with negative emotions, for example, depression, among young females, and this association was mediated by body image and self-esteem. However, researches on the chain-mediating role of body image and self-esteem in anxiety had been limited. Therefore, the current study examined the chain-mediating role between social network site comparison and anxiety among 320 Chinese females, using the Social Network Site Appearance Comparison Scale (SNSACS), Multidimensional Body-Self Relations Questionnaire (MBSRQ), Self-esteem Scale (SES), and the anxiety subscale of Depression-Anxiety-Stress Scale (DASS). Results revealed that (1) SNSACS, SES, and DASS anxiety scores were significantly correlated with each other, and the scores of two MBSRQ subscale, that is, body area satisfaction and overweight preoccupation, were significantly correlated with SNSACS, SES, and DASS anxiety scores; (2) body area satisfaction and self-esteem played a chain-mediating role in the effect of social network site appearance comparison on anxiety; (3) overweight preoccupation and self-esteem played a chain-mediating role in the effect of social network site appearance comparison on anxiety. The findings may inspire new ideas for understanding how social comparison triggers anxiety and for developing methods to reduce anxiety derived from appearance comparison among Chinese females.
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The hypoxic tumor microenvironment serves as a niche for maintaining the glioma-initiating cells (GICs) that are critical for glioblastoma (GBM) occurrence and recurrence. Here, we report that hypoxia-induced miR-215 is vital for reprograming GICs to fit the hypoxic microenvironment via suppressing the expression of an epigenetic regulator KDM1B and modulating activities of multiple pathways. Interestingly, biogenesis of miR-215 and several miRNAs is accelerated post-transcriptionally by hypoxia-inducible factors (HIFs) through HIF-Drosha interaction. Moreover, miR-215 expression correlates inversely with KDM1B while correlating positively with HIF1α and GBM progression in patients. These findings reveal a direct role of HIF in regulating miRNA biogenesis and consequently activating the miR-215-KDM1B-mediated signaling required for GIC adaptation to hypoxia.
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Regulación Neoplásica de la Expresión Génica/genética , Glioma/genética , MicroARNs/genética , Oxidorreductasas N-Desmetilantes/genética , Microambiente Tumoral/genética , Animales , Neoplasias Encefálicas/genética , Neoplasias Encefálicas/patología , Hipoxia de la Célula , Línea Celular Tumoral , Humanos , Ratones Desnudos , Recurrencia Local de Neoplasia/genéticaRESUMEN
Basic helix-loop-helix (bHLH) transcription factors recognize the canonical E-box (CANNTG) to regulate gene transcription; however, given the prevalence of E-boxes in a genome, it has been puzzling how individual bHLH proteins selectively recognize E-box sequences on their targets. TWIST is a bHLH transcription factor that promotes epithelial-mesenchymal transition (EMT) during development and tumor metastasis. High-resolution mapping of TWIST occupancy in human and Drosophila genomes reveals that TWIST, but not other bHLH proteins, recognizes a unique double E-box motif with two E-boxes spaced preferentially by 5 nucleotides. Using molecular modeling and binding kinetic analyses, we found that the strict spatial configuration in the double E-box motif aligns two TWIST-E47 dimers on the same face of DNA, thus providing a high-affinity site for a highly stable intramolecular tetramer. Biochemical analyses showed that the WR domain of TWIST dimerizes to mediate tetramer formation, which is functionally required for TWIST-induced EMT. These results uncover a novel mechanism for a bHLH transcription factor to recognize a unique spatial configuration of E-boxes to achieve target specificity. The WR-WR domain interaction uncovered here sets an example of target gene specificity of a bHLH protein being controlled allosterically by a domain outside of the bHLH region.
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Proteínas de Drosophila/química , Proteínas de Drosophila/metabolismo , Modelos Moleculares , Proteína 1 Relacionada con Twist/química , Proteína 1 Relacionada con Twist/metabolismo , Secuencia de Aminoácidos , Animales , Evolución Biológica , Secuencia Conservada , Drosophila/química , Drosophila/metabolismo , Regulación de la Expresión Génica , Humanos , Unión Proteica , Estabilidad Proteica , Estructura Terciaria de Proteína , Especificidad por SustratoRESUMEN
We summarize 12 experimental methods that have been developed for profiling gene expression by focusing on the 3'-end of poly(A+) mRNA, distilling both common and unique features. Of this family of methods, we provide detailed protocol for MAPS, a method we believe is the simplest and most cost-effective for profiling gene expression and quantifying alternative polyadenylation events by oligo-dT priming followed by random priming and deep sequencing. This method also enables library multiplexing by using a set of bar coding primers during PCR amplification. We also provide a general guideline for analysis of the data generated by MAPS by using the software package maps3end.
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Poli A/metabolismo , ARN/metabolismo , Programas Informáticos , Perfilación de la Expresión Génica , Secuenciación de Nucleótidos de Alto Rendimiento , Poli A/química , Poliadenilación/fisiología , ARN/químicaRESUMEN
Dysregulation of alternative splicing of mRNA precursors is known to contribute to numerous human diseases. In this study we carried out the first systematic search for asthma-associated changes in alternative splicing events, using a model of Aspergillus fumigatus (A. fumigatus)-sensitized mice and an exon junction microarray to detect potential changes in alternative splicing. One of the sensitization-associated changes identified in the search was a shift in alternative splicing of the mRNA encoding cFLIP, a modulator of the caspase-mediated extrinsic apoptosis pathway. Expanding these studies to human asthma patients, we discovered a significant decrease in the expression of both cFLIP isoforms in severe corticosteroid-resistant asthmatics. Although it is unclear whether these changes were due solely to differences in alternative splicing, these findings provide evidence that dysregulation of the extrinsic apoptosis pathway is part of the underlying immunopathogenesis of severe refractory asthma.
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Expanded hexanucleotide repeats in the chromosome 9 open reading frame 72 (C9orf72) gene are the most common genetic cause of ALS and frontotemporal degeneration (FTD). Here, we identify nuclear RNA foci containing the hexanucleotide expansion (GGGGCC) in patient cells, including white blood cells, fibroblasts, glia, and multiple neuronal cell types (spinal motor, cortical, hippocampal, and cerebellar neurons). RNA foci are not present in sporadic ALS, familial ALS/FTD caused by other mutations (SOD1, TDP-43, or tau), Parkinson disease, or nonneurological controls. Antisense oligonucleotides (ASOs) are identified that reduce GGGGCC-containing nuclear foci without altering overall C9orf72 RNA levels. By contrast, siRNAs fail to reduce nuclear RNA foci despite marked reduction in overall C9orf72 RNAs. Sustained ASO-mediated lowering of C9orf72 RNAs throughout the CNS of mice is demonstrated to be well tolerated, producing no behavioral or pathological features characteristic of ALS/FTD and only limited RNA expression alterations. Genome-wide RNA profiling identifies an RNA signature in fibroblasts from patients with C9orf72 expansion. ASOs targeting sense strand repeat-containing RNAs do not correct this signature, a failure that may be explained, at least in part, by discovery of abundant RNA foci with C9orf72 repeats transcribed in the antisense (GGCCCC) direction, which are not affected by sense strand-targeting ASOs. Taken together, these findings support a therapeutic approach by ASO administration to reduce hexanucleotide repeat-containing RNAs and raise the potential importance of targeting expanded RNAs transcribed in both directions.
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Esclerosis Amiotrófica Lateral/tratamiento farmacológico , Expansión de las Repeticiones de ADN/genética , Degeneración Lobar Frontotemporal/tratamiento farmacológico , Terapia Genética/métodos , Oligonucleótidos Antisentido/farmacología , Proteínas/genética , Esclerosis Amiotrófica Lateral/genética , Animales , Southern Blotting , Proteína C9orf72 , Sistema Nervioso Central/citología , Sistema Nervioso Central/metabolismo , Cartilla de ADN/genética , Fibroblastos/metabolismo , Degeneración Lobar Frontotemporal/genética , Genotipo , Hibridación Fluorescente in Situ , Ratones , Oligonucleótidos Antisentido/administración & dosificación , Oligonucleótidos Antisentido/genética , Oligonucleótidos Antisentido/uso terapéutico , Reacción en Cadena en Tiempo Real de la Polimerasa , Análisis de Secuencia de ARNRESUMEN
The generation of human induced pluripotent stem cells (iPSCs) holds great promise for the development of regenerative medicine therapies to treat a wide range of human diseases. However, the generation of iPSCs in the absence of integrative DNA vectors remains problematic. Here, we report a simple, highly reproducible RNA-based iPSC generation approach that utilizes a single, synthetic self-replicating VEE-RF RNA replicon that expresses four reprogramming factors (OCT4, KLF4, and SOX2, with c-MYC or GLIS1) at consistent high levels prior to regulated RNA degradation. A single VEE-RF RNA transfection into newborn or adult human fibroblasts resulted in efficient generation of iPSCs with all the hallmarks of stem cells, including cell surface markers, global gene expression profiles, and in vivo pluripotency, to differentiate into all three germ layers. The VEE-RF RNA-based approach has broad applicability for the generation of iPSCs for ultimate use in human stem cell therapies in regenerative medicine.
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Células Madre Pluripotentes Inducidas/citología , ARN/metabolismo , Replicón/genética , Adulto , Animales , Diferenciación Celular/genética , Línea Celular , Reprogramación Celular , Células Clonales , Fibroblastos/metabolismo , Humanos , Células Madre Pluripotentes Inducidas/metabolismo , Factor 4 Similar a Kruppel , Masculino , Ratones , Ratones Desnudos , TransfecciónRESUMEN
Autism is a highly heritable neurodevelopmental disorder, yet the genetic underpinnings of the disorder are largely unknown. Aberrant brain overgrowth is a well-replicated observation in the autism literature; but association, linkage, and expression studies have not identified genetic factors that explain this trajectory. Few studies have had sufficient statistical power to investigate whole-genome gene expression and genotypic variation in the autistic brain, especially in regions that display the greatest growth abnormality. Previous functional genomic studies have identified possible alterations in transcript levels of genes related to neurodevelopment and immune function. Thus, there is a need for genetic studies involving key brain regions to replicate these findings and solidify the role of particular functional pathways in autism pathogenesis. We therefore sought to identify abnormal brain gene expression patterns via whole-genome analysis of mRNA levels and copy number variations (CNVs) in autistic and control postmortem brain samples. We focused on prefrontal cortex tissue where excess neuron numbers and cortical overgrowth are pronounced in the majority of autism cases. We found evidence for dysregulation in pathways governing cell number, cortical patterning, and differentiation in young autistic prefrontal cortex. In contrast, adult autistic prefrontal cortex showed dysregulation of signaling and repair pathways. Genes regulating cell cycle also exhibited autism-specific CNVs in DNA derived from prefrontal cortex, and these genes were significantly associated with autism in genome-wide association study datasets. Our results suggest that CNVs and age-dependent gene expression changes in autism may reflect distinct pathological processes in the developing versus the mature autistic prefrontal cortex. Our results raise the hypothesis that genetic dysregulation in the developing brain leads to abnormal regional patterning, excess prefrontal neurons, cortical overgrowth, and neural dysfunction in autism.
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Factores de Edad , Trastorno Autístico , Variaciones en el Número de Copia de ADN , Regulación de la Expresión Génica , Corteza Prefrontal , Adolescente , Adulto , Trastorno Autístico/genética , Trastorno Autístico/metabolismo , Trastorno Autístico/patología , Autopsia , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Diferenciación Celular/genética , Niño , Preescolar , Variaciones en el Número de Copia de ADN/genética , Femenino , Eliminación de Gen , Redes Reguladoras de Genes , Genoma Humano , Humanos , Masculino , Persona de Mediana Edad , Neuronas/metabolismo , Neuronas/patología , Corteza Prefrontal/crecimiento & desarrollo , Corteza Prefrontal/metabolismo , Corteza Prefrontal/patología , Transducción de Señal/genéticaRESUMEN
Available statistical preprocessing or quality control analysis tools for gene expression microarray datasets are known to greatly affect downstream data analysis, especially when degraded samples, unique tissue samples, or novel expression assays are used. It is therefore important to assess the validity and impact of the assumptions built in to preprocessing schemes for a dataset. We developed and assessed a data preprocessing strategy for use with the Illumina DASL-based gene expression assay with partially degraded postmortem prefrontal cortex samples. The samples were obtained from individuals with autism as part of an investigation of the pathogenic factors contributing to autism. Using statistical analysis methods and metrics such as those associated with multivariate distance matrix regression and mean inter-array correlation, we developed a DASL-based assay gene expression preprocessing pipeline to accommodate and detect problems with microarray-based gene expression values obtained with degraded brain samples. Key steps in the pipeline included outlier exclusion, data transformation and normalization, and batch effect and covariate corrections. Our goal was to produce a clean dataset for subsequent downstream differential expression analysis. We ultimately settled on available transformation and normalization algorithms in the R/Bioconductor package lumi based on an assessment of their use in various combinations. A log2-transformed, quantile-normalized, and batch and seizure-corrected procedure was likely the most appropriate for our data. We empirically tested different components of our proposed preprocessing strategy and believe that our results suggest that a preprocessing strategy that effectively identifies outliers, normalizes the data, and corrects for batch effects can be applied to all studies, even those pursued with degraded samples.
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Although the lung is a defining feature of air-breathing animals, the pathway controlling the formation of type I pneumocytes, the cells that mediate gas exchange, is poorly understood. In contrast, the glucocorticoid receptor and its cognate ligand have long been known to promote type II pneumocyte maturation; prenatal administration of glucocorticoids is commonly used to attenuate the severity of infant respiratory distress syndrome (RDS). Here we show that knock-in mutations of the nuclear co-repressor SMRT (silencing mediator of retinoid and thyroid hormone receptors) in C57BL/6 mice (SMRTmRID) produces a previously unidentified respiratory distress syndrome caused by prematurity of the type I pneumocyte. Though unresponsive to glucocorticoids, treatment with anti-thyroid hormone drugs (propylthiouracil or methimazole) completely rescues SMRT-induced RDS, suggesting an unrecognized and essential role for the thyroid hormone receptor (TR) in lung development. We show that TR and SMRT control type I pneumocyte differentiation through Klf2, which, in turn, seems to directly activate the type I pneumocyte gene program. Conversely, mice without lung Klf2 lack mature type I pneumocytes and die shortly after birth, closely recapitulating the SMRTmRID phenotype. These results identify TR as a second nuclear receptor involved in lung development, specifically type I pneumocyte differentiation, and suggest a possible new type of therapeutic option in the treatment of RDS that is unresponsive to glucocorticoids.
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Células Epiteliales Alveolares/metabolismo , Co-Represor 2 de Receptor Nuclear/metabolismo , Síndrome de Dificultad Respiratoria del Recién Nacido/metabolismo , Células Epiteliales Alveolares/citología , Animales , Diferenciación Celular , Células Cultivadas , Femenino , Fibroblastos/citología , Fibroblastos/fisiología , Perfilación de la Expresión Génica , Técnicas de Sustitución del Gen , Humanos , Recién Nacido , Factores de Transcripción de Tipo Kruppel/genética , Factores de Transcripción de Tipo Kruppel/metabolismo , Pulmón/citología , Pulmón/embriología , Pulmón/crecimiento & desarrollo , Pulmón/metabolismo , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Análisis por Micromatrices , Co-Represor 2 de Receptor Nuclear/genética , Receptores de Glucocorticoides/genética , Receptores de Glucocorticoides/metabolismo , Receptores de Hormona Tiroidea/genética , Receptores de Hormona Tiroidea/metabolismoRESUMEN
BACKGROUND: Gene expression assays have been shown to yield high quality genome-wide data from partially degraded RNA samples. However, these methods have not yet been applied to postmortem human brain tissue, despite their potential to overcome poor RNA quality and other technical limitations inherent in many assays. We compared cDNA-mediated annealing, selection, and ligation (DASL)- and in vitro transcription (IVT)-based genome-wide expression profiling assays on RNA samples from artificially degraded reference pools, frozen brain tissue, and formalin-fixed brain tissue. RESULTS: The DASL-based platform produced expression results of greater reliability than the IVT-based platform in artificially degraded reference brain RNA and RNA from frozen tissue-based samples. Although data associated with a small sample of formalin-fixed RNA samples were poor when obtained from both assays, the DASL-based platform exhibited greater reliability in a subset of probes and samples. CONCLUSIONS: Our results suggest that the DASL-based gene expression-profiling platform may confer some advantages on mRNA assays of the brain over traditional IVT-based methods. We ultimately consider the implications of these results on investigations of neuropsychiatric disorders.
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Encéfalo/metabolismo , Perfilación de la Expresión Génica/métodos , Análisis de Secuencia por Matrices de Oligonucleótidos/métodos , Trastorno Autístico/genética , Femenino , Fijadores , Formaldehído , Congelación , Humanos , Masculino , ARN/genética , Estabilidad del ARN , Reproducibilidad de los ResultadosRESUMEN
Transcriptome analysis by deep sequencing, more commonly known as RNA-seq is, becoming the method of choice for gene discovery and quantitative splicing detection. We published a double-random priming RNA-seq approach capable of generating strand-specific information [Li et al., Proc Natl Acad Sci USA 105:20179-20184, 2008]. Poly(A)+ RNA from a treated and an untreated sample were utilized to generate RNA-seq libraries that were sequenced on the Illumina GA1 analyzer. Statistical analysis of approximately ten million sequence reads generated from both control and treated cells suggests that this tag density is sufficient for quantitative analysis of gene expression. We were also able to detect a large fraction of reads corresponding to annotated alternative exons, with a subset of the reads matching known and detecting new splice junctions. In this chapter, we provide a detailed, bench-ready protocol for the double-random priming method and provide user-friendly templates for the curve-fitting model described in the paper to estimate the tag density needed for optimal detection of regulated gene expression and alternative splicing.
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Empalme Alternativo , Perfilación de la Expresión Génica/métodos , Expresión Génica , Secuenciación de Nucleótidos de Alto Rendimiento/métodos , Análisis de Secuencia de ARN/métodos , ADN Complementario/metabolismo , Exones , Biblioteca de Genes , Reacción en Cadena de la Polimerasa/métodos , ARN Mensajero/análisisRESUMEN
BACKGROUND: Prostate cancer is one of the leading causes of cancer illness and death among men in the United States and world wide. There is an urgent need to discover good biomarkers for early clinical diagnosis and treatment. Previously, we developed an exon-junction microarray-based assay and profiled 1532 mRNA splice isoforms from 364 potential prostate cancer related genes in 38 prostate tissues. Here, we investigate the advantage of using splice isoforms, which couple transcriptional and splicing regulation, for cancer classification. RESULTS: As many as 464 splice isoforms from more than 200 genes are differentially regulated in tumors at a false discovery rate (FDR) of 0.05. Remarkably, about 30% of genes have isoforms that are called significant but do not exhibit differential expression at the overall mRNA level. A support vector machine (SVM) classifier trained on 128 signature isoforms can correctly predict 92% of the cases, which outperforms the classifier using overall mRNA abundance by about 5%. It is also observed that the classification performance can be improved using multivariate variable selection methods, which take correlation among variables into account. CONCLUSION: These results demonstrate that profiling of splice isoforms is able to provide unique and important information which cannot be detected by conventional microarrays.
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Algoritmos , Biomarcadores de Tumor/metabolismo , Diagnóstico por Computador/métodos , Perfilación de la Expresión Génica/métodos , Proteínas de Neoplasias/metabolismo , Neoplasias de la Próstata/clasificación , Neoplasias de la Próstata/genética , Empalme Alternativo/genética , Biomarcadores de Tumor/genética , Humanos , Masculino , Proteínas de Neoplasias/genética , Neoplasias de la Próstata/diagnóstico , Neoplasias de la Próstata/metabolismo , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , ARN Mensajero/genética , Reproducibilidad de los Resultados , Sensibilidad y Especificidad , Análisis de Secuencia de ARN/métodosRESUMEN
The expression of specific mRNA isoforms may uniquely reflect the biological state of a cell because it reflects the integrated outcome of both transcriptional and posttranscriptional regulation. In this study, we constructed a splicing array to examine approximately 1,500 mRNA isoforms from a panel of genes previously implicated in prostate cancer and identified a large number of cell type-specific mRNA isoforms. We also developed a novel "two-dimensional" profiling strategy to simultaneously quantify changes in splicing and transcript abundance; the results revealed extensive covariation between transcription and splicing in prostate cancer cells. Taking advantage of the ability of our technology to analyze RNA from formalin-fixed, paraffin-embedded tissues, we derived a specific set of mRNA isoform biomarkers for prostate cancer using independent panels of tissue samples for feature selection and cross-analysis. A number of cancer-specific splicing switch events were further validated by laser capture microdissection. Quantitative changes in transcription/RNA stability and qualitative differences in splicing ratio may thus be combined to characterize tumorigenic programs and signature mRNA isoforms may serve as unique biomarkers for tumor diagnosis and prognosis.
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Empalme Alternativo , Neoplasias de la Próstata/genética , ARN Mensajero/genética , Anciano , Anciano de 80 o más Años , Biomarcadores de Tumor/biosíntesis , Biomarcadores de Tumor/genética , Línea Celular Tumoral , Perfilación de la Expresión Génica , Humanos , Masculino , Persona de Mediana Edad , Análisis de Secuencia por Matrices de Oligonucleótidos , Adhesión en Parafina , Neoplasias de la Próstata/metabolismo , Neoplasias de la Próstata/patología , ARN Mensajero/biosíntesis , Transcripción GenéticaRESUMEN
Alternative splicing is a prominent feature of higher eukaryotes. Understanding of the function of mRNA isoforms and the regulation of alternative splicing is a major challenge in the post-genomic era. The development of mRNA isoform sensitive microarrays, which requires precise splice-junction sequence information, is a promising approach. Despite the availability of a large number of mRNAs and ESTs in various databases and the efforts made to align transcript sequences to genomic sequences, existing alternative splicing databases do not offer adequate information in an appropriate format to aid in splicing array design. Here we describe our effort in constructing the Manually Annotated Alternatively Spliced Events (MAASE) database system, which is specifically designed to support splicing microarray applications. MAASE comprises two components: (1) a manual/computational annotation tool for the efficient extraction of critical sequence and functional information for alternative splicing events and (2) a user-friendly database of annotated events that allows convenient export of information to aid in microarray design and data analysis. We provide a detailed introduction and a step-by-step user guide to the MAASE database system to facilitate future large-scale annotation efforts, integration with other alternative splicing databases, and splicing array fabrication.