RESUMO
Genome sequencing projects are discovering millions of genetic variants in humans, and interpretation of their functional effects is essential for understanding the genetic basis of variation in human traits. Here we report sequencing and deep analysis of messenger RNA and microRNA from lymphoblastoid cell lines of 462 individuals from the 1000 Genomes Project--the first uniformly processed high-throughput RNA-sequencing data from multiple human populations with high-quality genome sequences. We discover extremely widespread genetic variation affecting the regulation of most genes, with transcript structure and expression level variation being equally common but genetically largely independent. Our characterization of causal regulatory variation sheds light on the cellular mechanisms of regulatory and loss-of-function variation, and allows us to infer putative causal variants for dozens of disease-associated loci. Altogether, this study provides a deep understanding of the cellular mechanisms of transcriptome variation and of the landscape of functional variants in the human genome.
Assuntos
Variação Genética/genética , Genoma Humano/genética , Sequenciamento de Nucleotídeos em Larga Escala , Análise de Sequência de RNA , Transcriptoma/genética , Alelos , Linhagem Celular Transformada , Éxons/genética , Perfilação da Expressão Gênica , Humanos , Polimorfismo de Nucleotídeo Único/genética , Locos de Características Quantitativas/genética , RNA Mensageiro/análise , RNA Mensageiro/genéticaRESUMO
The release and uptake of nano-sized extracellular vesicles (EV) is a highly conserved means of intercellular communication. The molecular composition of EV, and thereby their signaling function to target cells, is regulated by cellular activation and differentiation stimuli. EV are regarded as snapshots of cells and are, therefore, in the limelight as biomarkers for disease. Although research on EV-associated RNA has predominantly focused on microRNAs, the transcriptome of EV consists of multiple classes of small non-coding RNAs with potential gene-regulatory functions. It is not known whether environmental cues imposed on cells induce specific changes in a broad range of EV-associated RNA classes. Here, we investigated whether immune-activating or -suppressing stimuli imposed on primary dendritic cells affected the release of various small non-coding RNAs via EV. The small RNA transcriptomes of highly pure EV populations free from ribonucleoprotein particles were analyzed by RNA sequencing and RT-qPCR. Immune stimulus-specific changes were found in the miRNA, snoRNA, and Y-RNA content of EV from dendritic cells, whereas tRNA and snRNA levels were much less affected. Only part of the changes in EV-RNA content reflected changes in cellular RNA, which urges caution in interpreting EV as snapshots of cells. By comprehensive analysis of RNA obtained from highly purified EV, we demonstrate that multiple RNA classes contribute to genetic messages conveyed via EV. The identification of multiple RNA classes that display cell stimulation-dependent association with EV is the prelude to unraveling the function and biomarker potential of these EV-RNAs.
Assuntos
Células Dendríticas/metabolismo , Vesículas Extracelulares/genética , Transcriptoma , Animais , Células da Medula Óssea/citologia , Células Cultivadas , Colecalciferol/farmacologia , Células Dendríticas/citologia , Células Dendríticas/efeitos dos fármacos , Vesículas Extracelulares/metabolismo , Corantes Fluorescentes/química , Lipopolissacarídeos/farmacologia , Camundongos , Camundongos Endogâmicos C57BL , MicroRNAs/metabolismo , Microscopia Eletrônica , Nanopartículas/química , RNA Nucleolar Pequeno/metabolismo , Pequeno RNA não Traduzido/química , Pequeno RNA não Traduzido/isolamento & purificação , Pequeno RNA não Traduzido/metabolismo , RNA de Transferência/metabolismo , Análise de Sequência de RNARESUMO
A genetic diagnosis of autosomal-dominant polycystic kidney disease (ADPKD) is challenging due to allelic heterogeneity, high GC content, and homology of the PKD1 gene with six pseudogenes. Short-read next-generation sequencing approaches, such as whole-genome sequencing and whole-exome sequencing, often fail at reliably characterizing complex regions such as PKD1. However, long-read single-molecule sequencing has been shown to be an alternative strategy that could overcome PKD1 complexities and discriminate between homologous regions of PKD1 and its pseudogenes. In this study, we present the increased power of resolution for complex regions using long-read sequencing to characterize a cohort of 19 patients with ADPKD. Our approach provided high sensitivity in identifying PKD1 pathogenic variants, diagnosing 94.7% of the patients. We show that reliable screening of ADPKD patients in a single test without interference of PKD1 homologous sequences, commonly introduced by residual amplification of PKD1 pseudogenes, by direct long-read sequencing is now possible. This strategy can be implemented in diagnostics and is highly suitable to sequence and resolve complex genomic regions that are of clinical relevance.
Assuntos
Doenças Renais Policísticas/genética , Canais de Cátion TRPP/genética , Alelos , Estudos de Coortes , Biblioteca Gênica , Testes Genéticos , Genótipo , Humanos , Perda de Heterozigosidade , Rim Policístico Autossômico Dominante/genética , Reação em Cadeia da Polimerase , Polimorfismo de Nucleotídeo Único , Pseudogenes , Análise de Sequência de DNARESUMO
Cytochrome P450 2D6 (CYP2D6) is among the most important genes involved in drug metabolism. Specific variants are associated with changes in the enzyme's amount and activity. Multiple technologies exist to determine these variants, like the AmpliChip CYP450 test, Taqman qPCR, or Second-Generation Sequencing, however, sequence homology between cytochrome P450 genes and pseudogene CYP2D7 impairs reliable CYP2D6 genotyping, and variant phasing cannot accurately be determined using these assays. To circumvent this, we sequenced CYP2D6 using the Pacific Biosciences RSII and obtained high-quality, full-length, phased CYP2D6 sequences, enabling accurate variant calling and haplotyping of the entire gene-locus including exonic, intronic, and upstream and downstream regions. Unphased diplotypes (Roche AmpliChip CYP450 test) were confirmed for 24 of the 25 samples, including gene duplications. Cases with gene deletions required additional specific assays to resolve. In total, 61 unique variants were detected, including variants that had not previously been associated with specific haplotypes. To further aid genomic analysis using standard reference sequences, we have established an LOVD-powered CYP2D6 gene-variant database, and added all reference haplotypes and data reported here. We conclude that our CYP2D6 genotyping approach produces reliable CYP2D6 diplotypes and reveals information about additional variants, including phasing and copy-number variation.
Assuntos
Citocromo P-450 CYP2D6/genética , Variação Genética , Análise de Sequência de DNA , Variações do Número de Cópias de DNA , Deleção de Genes , Duplicação Gênica , Genótipo , Humanos , Translocação GenéticaRESUMO
MOTIVATION: Advances in sequencing technologies and computational algorithms have enabled the study of genomic variants to dissect their functional consequence. Despite this unprecedented progress, current tools fail to reliably detect and characterize more complex allelic variants, such as short tandem repeats (STRs). We developed TSSV as an efficient and sensitive tool to specifically profile all allelic variants present in targeted loci. Based on its design, requiring only two short flanking sequences, TSSV can work without the use of a complete reference sequence to reliably profile highly polymorphic, repetitive or uncharacterized regions. RESULTS: We show that TSSV can accurately determine allelic STR structures in mixtures with 10% representation of minor alleles or complex mixtures in which a single STR allele is shared. Furthermore, we show the universal utility of TSSV in two other independent studies: characterizing de novo mutations introduced by transcription activator-like effector nucleases (TALENs) and profiling the noise and systematic errors in an IonTorrent sequencing experiment. TSSV complements the existing tools by aiding the study of highly polymorphic and complex regions and provides a high-resolution map that can be used in a wide range of applications, from personal genomics to forensic analysis and clinical diagnostics. AVAILABILITY AND IMPLEMENTATION: We have implemented TSSV as a Python package that can be installed through the command-line using pip install TSSV command. Its source code and documentation are available at https://pypi.python.org/pypi/tssv and http://www.lgtc.nl/tssv.
Assuntos
Alelos , Genômica/métodos , Repetições de Microssatélites , Software , Algoritmos , Desoxirribonucleases/metabolismo , Distrofina/genética , Feminino , Genoma Humano , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Masculino , Mutação , Análise de Sequência de DNARESUMO
Recent methodological advances have improved the ease and efficiency of generating human induced pluripotent stem cells (hiPSCs), but this now typically results in a greater number of hiPSC clones being derived than can be wholly characterized. It is therefore imperative that methods are developed which facilitate rapid selection of hiPSC clones most suited for the downstream research aims. Here we describe a combination of procedures enabling the simultaneous screening of multiple clones to determine their genomic integrity as well as their cardiac differentiation potential within two weeks of the putative reprogrammed colonies initially appearing. By coupling splinkerette-PCR with Ion Torrent sequencing, we could ascertain the number and map the proviral integration sites in lentiviral-reprogrammed hiPSCs. In parallel, we developed an effective cardiac differentiation protocol that generated functional cardiomyocytes within 10 days without requiring line-specific optimization for any of the six independent human pluripotent stem cell lines tested. Finally, to demonstrate the scalable potential of these procedures, we picked 20 nascent iPSC clones and performed these independent assays concurrently. Before the clones required passaging, we were able to identify clones with a single integrated copy of the reprogramming vector and robust cardiac differentiation potential for further analysis.
Assuntos
Diferenciação Celular , Reprogramação Celular , Células-Tronco Pluripotentes Induzidas/citologia , Miócitos Cardíacos/citologia , Provírus/genética , Integração Viral/genética , Southern Blotting , Proliferação de Células , Células Cultivadas , Polpa Dentária/citologia , Polpa Dentária/metabolismo , Derme/citologia , Derme/metabolismo , Fibroblastos/citologia , Fibroblastos/metabolismo , Citometria de Fluxo , Imunofluorescência , Ensaios de Triagem em Larga Escala , Humanos , Células-Tronco Pluripotentes Induzidas/metabolismo , Miócitos Cardíacos/metabolismoRESUMO
Current microRNA target predictions are based on sequence information and empirically derived rules but do not make use of the expression of microRNAs and their targets. This study aimed to improve microRNA target predictions in a given biological context, using in silico predictions, microRNA and mRNA expression. We used target prediction tools to produce lists of predicted targets and used a gene set test designed to detect consistent effects of microRNAs on the joint expression of multiple targets. In a single test, association between microRNA expression and target gene set expression as well as the contribution of the individual target genes on the association are determined. The strongest negatively associated mRNAs as measured by the test were prioritized. We applied our integration method to a well-defined muscle differentiation model. Validation of our predictions in C2C12 cells confirmed predicted targets of known as well as novel muscle-related microRNAs. We further studied associations between microRNA-mRNA pairs in human prostate cancer, finding some pairs that have been recently experimentally validated by others. Using the same study, we showed the advantages of the global test over Pearson correlation and lasso. We conclude that our integrated approach successfully identifies regulated microRNAs and their targets.
Assuntos
Regulação Neoplásica da Expressão Gênica , MicroRNAs/análise , Mioblastos Esqueléticos/metabolismo , RNA Mensageiro/análise , Software , Regiões 3' não Traduzidas , Algoritmos , Animais , Diferenciação Celular , Humanos , Masculino , Camundongos , MicroRNAs/genética , Mioblastos Esqueléticos/citologia , Neoplasias da Próstata/metabolismo , Neoplasias da Próstata/patologia , RNA Mensageiro/genética , TranscriptomaRESUMO
Cells release RNA-carrying vesicles and membrane-free RNA/protein complexes into the extracellular milieu. Horizontal vesicle-mediated transfer of such shuttle RNA between cells allows dissemination of genetically encoded messages, which may modify the function of target cells. Other studies used array analysis to establish the presence of microRNAs and mRNA in cell-derived vesicles from many sources. Here, we used an unbiased approach by deep sequencing of small RNA released by immune cells. We found a large variety of small non-coding RNA species representing pervasive transcripts or RNA cleavage products overlapping with protein coding regions, repeat sequences or structural RNAs. Many of these RNAs were enriched relative to cellular RNA, indicating that cells destine specific RNAs for extracellular release. Among the most abundant small RNAs in shuttle RNA were sequences derived from vault RNA, Y-RNA and specific tRNAs. Many of the highly abundant small non-coding transcripts in shuttle RNA are evolutionary well-conserved and have previously been associated to gene regulatory functions. These findings allude to a wider range of biological effects that could be mediated by shuttle RNA than previously expected. Moreover, the data present leads for unraveling how cells modify the function of other cells via transfer of specific non-coding RNA species.
Assuntos
Pequeno RNA não Traduzido/análise , Vesículas Transportadoras/química , Células Cultivadas , Técnicas de Cocultura , Células Dendríticas/química , Células Dendríticas/imunologia , Sequenciamento de Nucleotídeos em Larga Escala , MicroRNAs/análise , MicroRNAs/química , Pequeno RNA não Traduzido/química , Pequeno RNA não Traduzido/fisiologia , RNA de Transferência/análise , RNA de Transferência/química , Sequências Repetitivas de Ácido Nucleico , Análise de Sequência de RNA , Linfócitos T/química , Linfócitos T/imunologiaAssuntos
Rearranjo Gênico do Linfócito B , Imunoglobulinas/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa/métodos , Sequência de Bases , Sítios de Ligação , Primers do DNA , Humanos , Receptores de Antígenos de Linfócitos B/genética , Sensibilidade e Especificidade , Hipermutação Somática de ImunoglobulinaRESUMO
The atrioventricular (AV) node is a recurrent source of potentially life-threatening arrhythmias. Nevertheless, limited data are available on its developmental control or molecular phenotype. We used a novel AV nodal myocardium-specific reporter mouse to gain insight into the gene programs determining the formation and phenotype of the developing AV node. In this reporter, green fluorescent protein (GFP) expression was driven by a 160-kbp bacterial artificial chromosome with Tbx3 and flanking sequences. GFP was selectively active in the AV canal of embryos and AV node of adults, whereas the Tbx3-positive AV bundle and sinus node were devoid of GFP, demonstrating that distinct regulatory sequences and pathways control expression in the components of the conduction system. Fluorescent AV nodal and complementary Nppa-positive chamber myocardial cell populations of embryonic day 10.5 embryos and of embryonic day 17.5 fetuses were purified using fluorescence-activated cell sorting, and their expression profiles were assessed by genome-wide microarray analysis, providing valuable information concerning their molecular identities. We constructed a comprehensive list of sodium, calcium, and potassium channel genes specific for developing nodal or chamber myocardium. Furthermore, the data revealed that the AV node and the chamber (working) myocardium phenotypes diverge during development but that the functional gene classes characterizing both subtypes are maintained. One of the repertoires identified in the AV node-specific gene profiles consists of multiple neurotrophic factors and semaphorins, not yet appreciated to play a role in nodal development, revealing shared characteristics between nodal and nervous system development.
Assuntos
Nó Atrioventricular , Perfilação da Expressão Gênica/métodos , Proteínas com Domínio T/genética , Animais , Canais de Cálcio/genética , Cromossomos Artificiais Bacterianos , Embrião de Mamíferos , Genes Reporter , Proteínas de Fluorescência Verde , Camundongos , Camundongos Transgênicos , Proteínas do Tecido Nervoso/genética , Canais de Potássio/genética , Canais de Sódio/genéticaRESUMO
INTRODUCTION: The high-sequence homology of the α-globin-gene cluster is responsible for microhomology-mediated recombination events during meiosis, resulting in a high density of deletion breakpoints within a 10 kb region. Commonly used deletion detection methods, such as multiplex ligation-dependent probe amplification (MLPA) and Southern blot, cannot exactly define the breakpoints. This typically requires long-range PCR, which is not always successful. Targeted locus amplification (TLA) is a targeted enrichment method that can be used to sequence up to 70 kb of neighboring DNA sequences without prior knowledge about the target site. METHODS: Genomic DNA (gDNA) TLA is a technique that folds isolated DNA, ensuring that adjacent loci are in a close spatial proximity. Subsequent digestion and religation form DNA circles that are amplified using fragment-specific inverse primers, creating a library that is suitable for Illumina sequencing. RESULTS: Here, we describe the characterization of a rare 16 771 bp deletion, utilizing gDNA TLA with a single inverse PCR primer set on one end of the breakpoint. Primers for breakpoint PCR were designed to confirm the deletion breakpoints and were consequently used to characterize the same deletion in 10 additional carriers sharing comparable hematologic data and similar MLPA results. CONCLUSIONS: The gDNA TLA technology was successfully used to identify deletion breakpoints within the alpha-globin cluster. The deletion was described only once in an earlier study as the --gb , but as it was not registered correctly in the available databases, it was not initially recognized as such.
Assuntos
Alelos , Pontos de Quebra do Cromossomo , Deleção de Sequência , alfa-Globinas/genética , Talassemia alfa/diagnóstico , Talassemia alfa/genética , Testes Genéticos , Biblioteca Genômica , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Técnicas de Amplificação de Ácido Nucleico , Reação em Cadeia da Polimerase , Talassemia alfa/sangueRESUMO
BACKGROUND: MicroRNAs are small non-coding RNA transcripts that regulate post-transcriptional gene expression. The millions of short sequence reads generated by next generation sequencing technologies make this technique explicitly suitable for profiling of known and novel microRNAs. A modification to the small-RNA expression kit (SREK, Ambion) library preparation method for the SOLiD sequencing platform is described to generate microRNA sequencing libraries that are compatible with the Illumina Genome Analyzer. RESULTS: High quality sequencing libraries can successfully be prepared from as little as 100 ng small RNA enriched RNA. An easy to use perl-based analysis pipeline called E-miR was developed to handle the sequencing data in several automated steps including data format conversion, 3' adapter removal, genome alignment and annotation to non-coding RNA transcripts. The sample preparation and E-miR pipeline were used to identify 37 cardiac enriched microRNAs in stage 16 chicken embryos. Isomir expression profiles between the heart and embryo were highly correlated for all miRNAs suggesting that tissue or cell specific miRNA modifications do not occur. CONCLUSIONS: In conclusion, our alternative sample preparation method can successfully be applied to generate high quality miRNA sequencing libraries for the Illumina genome analyzer.
Assuntos
Perfilação da Expressão Gênica/métodos , MicroRNAs/genética , Análise de Sequência de RNA/métodos , Animais , Sequência de Bases , Embrião de Galinha , Regulação da Expressão Gênica no Desenvolvimento , Biblioteca Gênica , MicroRNAs/metabolismo , Anotação de Sequência Molecular , Dados de Sequência Molecular , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Reprodutibilidade dos Testes , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
Human lymphoid tissues harbor, in addition to CD56bright and CD56dim natural killer (NK) cells, a third NK cell population: CD69+CXCR6+ lymphoid tissue (lt)NK cells. The function and development of ltNK cells remain poorly understood. In this study, we performed RNA sequencing on the three NK cell populations derived from bone marrow (BM) and blood. In ltNK cells, 1,353 genes were differentially expressed compared to circulating NK cells. Several molecules involved in migration were downregulated in ltNK cells: S1PR1, SELPLG and CD62L. By flow cytometry we confirmed that the expression profile of adhesion molecules (CD49e-, CD29low, CD81high, CD62L-, CD11c-) and transcription factors (Eomeshigh, Tbetlow) of ltNK cells differed from their circulating counterparts. LtNK cells were characterized by enhanced expression of inhibitory receptors TIGIT and CD96 and low expression of DNAM1 and cytolytic molecules (GZMB, GZMH, GNLY). Their proliferative capacity was reduced compared to the circulating NK cells. By performing gene set enrichment analysis, we identified DUSP6 and EGR2 as potential regulators of the ltNK cell transcriptome. Remarkably, comparison of the ltNK cell transcriptome to the published human spleen-resident memory CD8+ T (Trm) cell transcriptome revealed an overlapping gene signature. Moreover, the phenotypic profile of ltNK cells resembled that of CD8+ Trm cells in BM. Together, we provide transcriptional and phenotypic data that clearly distinguish ltNK cells from both the CD56bright and CD56dim NK cells and substantiate the view that ltNK cells are tissue-resident cells, which are functionally restrained in killing and have low proliferative activity.
Assuntos
Células da Medula Óssea/imunologia , Células da Medula Óssea/metabolismo , Linfócitos T CD8-Positivos/imunologia , Linfócitos T CD8-Positivos/metabolismo , Células Matadoras Naturais/imunologia , Células Matadoras Naturais/metabolismo , Transcriptoma , Biomarcadores , Biologia Computacional/métodos , Citotoxicidade Imunológica , Perfilação da Expressão Gênica , Humanos , Memória Imunológica , Imunofenotipagem , Especificidade de Órgãos/imunologia , FenótipoRESUMO
RNA-seq is increasingly used for quantitative profiling of small RNAs (for example, microRNAs, piRNAs and snoRNAs) in diverse sample types, including isolated cells, tissues and cell-free biofluids. The accuracy and reproducibility of the currently used small RNA-seq library preparation methods have not been systematically tested. Here we report results obtained by a consortium of nine labs that independently sequenced reference, 'ground truth' samples of synthetic small RNAs and human plasma-derived RNA. We assessed three commercially available library preparation methods that use adapters of defined sequence and six methods using adapters with degenerate bases. Both protocol- and sequence-specific biases were identified, including biases that reduced the ability of small RNA-seq to accurately measure adenosine-to-inosine editing in microRNAs. We found that these biases were mitigated by library preparation methods that incorporate adapters with degenerate bases. MicroRNA relative quantification between samples using small RNA-seq was accurate and reproducible across laboratories and methods.
Assuntos
MicroRNAs/genética , Análise de Sequência de RNA/métodos , Adenosina/genética , Humanos , Inosina/genética , MicroRNAs/sangue , MicroRNAs/normas , Edição de RNA , Padrões de Referência , Reprodutibilidade dos TestesRESUMO
Hereditary cerebral hemorrhage with amyloidosis-Dutch type (HCHWA-D) is an early onset hereditary form of cerebral amyloid angiopathy (CAA) caused by a point mutation resulting in an amino acid change (NP_000475.1:p.Glu693Gln) in the amyloid precursor protein (APP). Post-mortem frontal and occipital cortical brain tissue from nine patients and nine age-related controls was used for RNA sequencing to identify biological pathways affected in HCHWA-D. Although previous studies indicated that pathology is more severe in the occipital lobe in HCHWA-D compared to the frontal lobe, the current study showed similar changes in gene expression in frontal and occipital cortex and the two brain regions were pooled for further analysis. Significantly altered pathways were analyzed using gene set enrichment analysis (GSEA) on 2036 significantly differentially expressed genes. Main pathways over-represented by down-regulated genes were related to cellular aerobic respiration (including ATP synthesis and carbon metabolism) indicating a mitochondrial dysfunction. Principal up-regulated pathways were extracellular matrix (ECM)-receptor interaction and ECM proteoglycans in relation with an increase in the transforming growth factor beta (TGFß) signaling pathway. Comparison with the publicly available dataset from pre-symptomatic APP-E693Q transgenic mice identified overlap for the ECM-receptor interaction pathway, indicating that ECM modification is an early disease specific pathomechanism.
RESUMO
Conventional mitochondrial-DNA (MT DNA) sequencing approaches use Sanger sequencing of 20-40 partially overlapping PCR fragments per individual, which is a time- and resource-consuming process. We have developed a high-throughput, accurate, fast, and cost-effective human MT DNA sequencing approach. In this setup we first generate long-range PCR products for two partially overlapping 7.7 and 9.2 kb MT DNA-specific amplicons, add sample-specific barcodes, and sequence these on the PacBio RSII system to obtain full-length MT DNA sequences for genotyping/haplotyping purposes.
Assuntos
DNA Mitocondrial/genética , Reação em Cadeia da Polimerase/métodos , Análise de Sequência de DNA/métodos , HumanosRESUMO
Myocardial right ventricular (RV) hypertrophy due to pulmonary hypertension is aimed at normalizing ventricular wall stress. Depending on the degree of pressure overload, RV hypertrophy may progress to a state of impaired contractile function and heart failure, but this cannot be discerned during the early stages of ventricular remodeling. We tested whether critical differences in gene expression profiles exist between ventricles before the ultimate development of either a compensated or decompensated hypertrophic phenotype. Both phenotypes were selectively induced in Wistar rats by a single subcutaneous injection of either a low or a high dose of the pyrrolizidine alkaloid monocrotaline (MCT). Spotted oligonucleotide microarrays were used to investigate pressure-dependent cardiac gene expression profiles at 2 wk after the MCT injections, between control rats and rats that would ultimately develop either compensated or decompensated hypertrophy. Clustering of significantly regulated genes revealed specific expression profiles for each group, although the degree of hypertrophy was still similar in both. The ventricles destined to progress to failure showed activation of pro-apoptotic pathways, particularly related to mitochondria, whereas the group developing compensated hypertrophy showed blocked pro-death effector signaling via p38-MAPK, through upregulation of MAPK phosphatase-1. In summary, we show that, already at an early time point, pivotal differences in gene expression exist between ventricles that will ultimately develop either a compensated or a decompensated phenotype, depending on the degree of pressure overload. These data reveal genes that may provide markers for the early prediction of clinical outcome as well as potential targets for early intervention.
Assuntos
Insuficiência Cardíaca/genética , Hipertrofia Ventricular Esquerda/genética , Hipertrofia Ventricular Direita/genética , RNA Mensageiro/genética , Animais , Fator Natriurético Atrial/genética , ATPases Transportadoras de Cálcio/genética , Primers do DNA , Modelos Animais de Doenças , Perfilação da Expressão Gênica , Hipertensão/genética , Hipertensão Pulmonar/etiologia , Hipertensão Pulmonar/genética , Masculino , Análise de Sequência com Séries de Oligonucleotídeos , Reação em Cadeia da Polimerase , RNA/genética , RNA/isolamento & purificação , Ratos , Ratos Wistar , ATPases Transportadoras de Cálcio do Retículo SarcoplasmáticoRESUMO
BACKGROUND: Colon cancer prognosis and treatment are currently based on a classification system still showing large heterogeneity in clinical outcome, especially in TNM stages II and III. Prognostic biomarkers for metastasis risk are warranted as development of distant recurrent disease mainly accounts for the high lethality rates of colon cancer. miRNAs have been proposed as potential biomarkers for cancer. Furthermore, a verified standard for normalization of the amount of input material in PCR-based relative quantification of miRNA expression is lacking. METHODS: A selection of frozen tumor specimens from two independent patient cohorts with TNM stage II-III microsatellite stable primary adenocarcinomas was used for laser capture microdissection. Next-generation sequencing was performed on small RNAs isolated from colorectal tumors from the Dutch cohort (N = 50). Differential expression analysis, comparing in metastasized and nonmetastasized tumors, identified prognostic miRNAs. Validation was performed on colon tumors from the German cohort (N = 43) using quantitative PCR (qPCR). RESULTS: miR25-3p and miR339-5p were identified and validated as independent prognostic markers and used to construct a multivariate nomogram for metastasis risk prediction. The nomogram showed good probability prediction in validation. In addition, we recommend combination of miR16-5p and miR26a-5p as standard for normalization in qPCR of colon cancer tissue-derived miRNA expression. CONCLUSIONS: In this international study, we identified and validated a miRNA classifier in primary cancers, and propose a nomogram capable of predicting metastasis risk in microsatellite stable TNM stage II-III colon cancer. IMPACT: In conjunction with TNM staging, by means of a nomogram, this miRNA classifier may allow for personalized treatment decisions based on individual tumor characteristics.
Assuntos
Neoplasias do Colo/genética , Idoso , Neoplasias do Colo/mortalidade , Feminino , Humanos , Masculino , MicroRNAs , Pessoa de Meia-Idade , Metástase Neoplásica , Nomogramas , Prognóstico , Análise de SobrevidaRESUMO
Cervical cancer is typically well infiltrated by immune cells. Because of the intricate relationship between cancer cells and immune cells, we aimed to identify both cancer cell and immune cell expressed biomarkers. Using a novel approach, we isolated RNA from flow-sorted viable EpCAM+ tumor epithelial cells and CD45+ tumor-infiltrating immune cells obtained from squamous cell cervical cancer samples (n = 24). Total RNA was sequenced and differential gene expression analysis of the CD45+ immune cell fractions identified TCL1A as a novel marker for predicting improved survival (p = 0.007). This finding was validated using qRT-PCR (p = 0.005) and partially validated using immunohistochemistry (p = 0.083). Importantly, TCL1A was found to be expressed in a subpopulation of B cells (CD3-/CD19+/CD10+/CD34-) using multicolor immunofluorescence. A high TCL1A/CD20 (B cell) ratio, determined in total tumor samples from a separate patient cohort using qRT-PCR (n = 52), was also correlated with improved survival (p = 0.027). This is the first study demonstrating the prognostic value of separating tumor epithelial cells from tumor-infiltrating immune cells and determining their RNA expression profile for identifying putative cancer biomarkers. Our results suggest that intratumoral TCL1A+ B cells are important for controlling cervical cancer development.
Assuntos
Linfócitos B/metabolismo , Proteínas Proto-Oncogênicas/genética , Proteínas Proto-Oncogênicas/metabolismo , Neoplasias do Colo do Útero/genética , Adulto , Idoso , Idoso de 80 Anos ou mais , Feminino , Perfilação da Expressão Gênica , Humanos , Pessoa de Meia-Idade , Prognóstico , Análise de Sobrevida , Microambiente Tumoral , Neoplasias do Colo do Útero/mortalidade , Neoplasias do Colo do Útero/patologiaRESUMO
Differentiated derivatives of human pluripotent stem cells in culture are generally phenotypically immature compared to their adult counterparts. Their identity is often difficult to determine with certainty because little is known about their human fetal equivalents in vivo. Cellular identity and signaling pathways directing differentiation are usually determined by extrapolating information from either human adult tissue or model organisms, assuming conservation with humans. To resolve this, we generated a collection of human fetal transcriptional profiles at different developmental stages. Moreover, we developed an algorithm, KeyGenes, which uses this dataset to quantify the extent to which next-generation sequencing or microarray data resemble specific cell or tissue types in the human fetus. Using KeyGenes combined with the human fetal atlas, we identified multiple cell and tissue samples unambiguously on a limited set of features. We thus provide a flexible and expandable platform to monitor and evaluate the efficiency of differentiation in vitro.