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1.
BMC Genom Data ; 22(1): 33, 2021 09 14.
Artigo em Inglês | MEDLINE | ID: mdl-34521352

RESUMO

BACKGROUND: The lymphatic and the blood vasculature are closely related systems that collaborate to ensure the organism's physiological function. Despite their common developmental origin, they present distinct functional fates in adulthood that rely on robust lineage-specific regulatory programs. The recent technological boost in sequencing approaches unveiled long noncoding RNAs (lncRNAs) as prominent regulatory players of various gene expression levels in a cell-type-specific manner. RESULTS: To investigate the potential roles of lncRNAs in vascular biology, we performed antisense oligonucleotide (ASO) knockdowns of lncRNA candidates specifically expressed either in human lymphatic or blood vascular endothelial cells (LECs or BECs) followed by Cap Analysis of Gene Expression (CAGE-Seq). Here, we describe the quality control steps adopted in our analysis pipeline before determining the knockdown effects of three ASOs per lncRNA target on the LEC or BEC transcriptomes. In this regard, we especially observed that the choice of negative control ASOs can dramatically impact the conclusions drawn from the analysis depending on the cellular background. CONCLUSION: In conclusion, the comparison of negative control ASO effects on the targeted cell type transcriptomes highlights the essential need to select a proper control set of multiple negative control ASO based on the investigated cell types.

2.
Methods Mol Biol ; 2351: 67-90, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34382184

RESUMO

The Cap Analysis of Gene Expression (CAGE) is a powerful method to identify Transcription Start Sites (TSSs) of capped RNAs while simultaneously measuring transcripts expression level. CAGE allows mapping at single nucleotide resolution at all active promoters and enhancers. Large CAGE datasets have been produced over the years from individual laboratories and consortia, including the Encyclopedia of DNA Elements (ENCODE) and Functional Annotation of the Mammalian Genome (FANTOM) consortia. These datasets constitute open resource for TSS annotations and gene expression analysis. Here, we provide an experimental protocol for the most recent CAGE method called Low Quantity (LQ) single strand (ss) CAGE "LQ-ssCAGE", which enables cost-effective profiling of low quantity RNA samples. LQ-ssCAGE is especially useful for samples derived from cells cultured in small volumes, cellular compartments such as nuclear RNAs or for samples from developmental stages. We demonstrate the reproducibility and effectiveness of the method by constructing 240 LQ-ssCAGE libraries from 50 ng of THP-1 cell extracted RNAs and discover lowly expressed novel enhancer and promoter-derived lncRNAs.


Assuntos
Biologia Computacional/métodos , Elementos Facilitadores Genéticos , Regiões Promotoras Genéticas , Capuzes de RNA , Sítio de Iniciação de Transcrição , Bases de Dados Genéticas , Regulação da Expressão Gênica , Biblioteca Gênica , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Anotação de Sequência Molecular , Sequências Reguladoras de Ácido Nucleico , Reprodutibilidade dos Testes , Fluxo de Trabalho
4.
Nat Commun ; 12(1): 3297, 2021 06 02.
Artigo em Inglês | MEDLINE | ID: mdl-34078885

RESUMO

Using the Cap Analysis of Gene Expression (CAGE) technology, the FANTOM5 consortium provided one of the most comprehensive maps of transcription start sites (TSSs) in several species. Strikingly, ~72% of them could not be assigned to a specific gene and initiate at unconventional regions, outside promoters or enhancers. Here, we probe these unassigned TSSs and show that, in all species studied, a significant fraction of CAGE peaks initiate at microsatellites, also called short tandem repeats (STRs). To confirm this transcription, we develop Cap Trap RNA-seq, a technology which combines cap trapping and long read MinION sequencing. We train sequence-based deep learning models able to predict CAGE signal at STRs with high accuracy. These models unveil the importance of STR surrounding sequences not only to distinguish STR classes, but also to predict the level of transcription initiation. Importantly, genetic variants linked to human diseases are preferentially found at STRs with high transcription initiation level, supporting the biological and clinical relevance of transcription initiation at STRs. Together, our results extend the repertoire of non-coding transcription associated with DNA tandem repeats and complexify STR polymorphism.


Assuntos
Repetições de Microssatélites , Redes Neurais de Computação , Doenças Neurodegenerativas/genética , Sítio de Iniciação de Transcrição , Iniciação da Transcrição Genética , Células A549 , Animais , Sequência de Bases , Biologia Computacional/métodos , Aprendizado Profundo , Elementos Facilitadores Genéticos , Genoma Humano , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Camundongos , Doenças Neurodegenerativas/diagnóstico , Doenças Neurodegenerativas/metabolismo , Polimorfismo Genético , Regiões Promotoras Genéticas
5.
Sci Rep ; 11(1): 9355, 2021 04 30.
Artigo em Inglês | MEDLINE | ID: mdl-33931666

RESUMO

Gene expression is controlled at the transcriptional and post-transcriptional levels. The TACC2 gene was known to be associated with tumors but the control of its expression is unclear. We have reported that activity of the intronic promoter p10 of TACC2 in primary lesion of endometrial cancer is indicative of lymph node metastasis among a low-risk patient group. Here, we analyze the intronic promoter derived isoforms in JHUEM-1 endometrial cancer cells, and primary tissues of endometrial cancers and normal endometrium. Full-length cDNA amplicons are produced by long-range PCR and subjected to nanopore sequencing followed by computational error correction. We identify 16 stable, 4 variable, and 9 rare exons including 3 novel exons validated independently. All variable and rare exons reside N-terminally of the TACC domain and contribute to isoform variety. We found 240 isoforms as high-confidence, supported by more than 20 reads. The large number of isoforms produced from one minor promoter indicates the post-transcriptional complexity coupled with transcription at the TACC2 locus in cancer and normal cells.

6.
Mol Oncol ; 15(5): 1507-1527, 2021 May.
Artigo em Inglês | MEDLINE | ID: mdl-33682233

RESUMO

Cancer-associated fibroblasts (CAFs) regulate cancer progression through the modulation of extracellular matrix (ECM) and cancer cell adhesion. While undergoing a series of phenotypic changes, CAFs control cancer-stroma interactions through integrin receptor signaling. Here, we isolated CAFs from patients with non-small-cell lung cancer (NSCLC) and examined their gene expression profiles. We identified collagen type XI α1 (COL11A1), integrin α11 (ITGA11), and the ITGA11 major ligand collagen type I α1 (COL1A1) among the 390 genes that were significantly enriched in NSCLC-associated CAFs. Increased ITGA11 expression in cancer stroma was correlated with a poor clinical outcome in patients with NSCLC. Increased expression of fibronectin and collagen type I induced ITGA11 expression in CAFs. The cellular migration of CAFs toward collagen type I and fibronectin was promoted via ERK1/2 signaling, independently of the fibronectin receptor integrin α5ß1. Additionally, ERK1/2 signaling induced ITGA11 and COL11A1 expression in cancer stroma. We, therefore, propose that targeting ITGA11 and COL11A1 expressing CAFs to block cancer-stroma interactions may serve as a novel, promising anti-tumor strategy.

7.
Stem Cells Transl Med ; 10(6): 895-909, 2021 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-33599112

RESUMO

The quality and quantity of endothelial progenitor cells (EPCs) are impaired in patients with diabetes mellitus patients, leading to reduced tissue repair during autologous EPC therapy. This study aimed to address the limitations of the previously described serum-free Quantity and Quality Control Culture System (QQc) using CD34+ cells by investigating the therapeutic potential of a novel mononuclear cell (MNC)-QQ. MNCs were isolated from 50 mL of peripheral blood of patients with diabetes mellitus and healthy volunteers (n = 13 each) and subjected to QQc for 7 days in serum-free expansion media with VEGF, Flt-3 ligand, TPO, IL-6, and SCF. The vascular regeneration capability of MNC-QQ cells pre- or post-QQc was evaluated with an EPC colony-forming assay, FACS, EPC culture, tube formation assay, and quantitative real time PCR. For in vivo assessment, 1 × 104 pre- and post-MNC-QQc cells from diabetic donors were injected into a murine wound-healing model using Balb/c nude mice. The percentage of wound closure and angio-vasculogenesis was then assessed. This study revealed vasculogenic, anti-inflammatory, and wound-healing effects of MNC-QQ therapy in both in vitro and in vivo models. This system addresses the low efficiency and efficacy of the current naïve MNC therapy for wound-healing in diabetic patients. As this technique requires a simple blood draw, isolation, and peripheral blood MNC suspension culture for only a week, it can be used as a simple and effective outpatient-based vascular and regenerative therapy for patients with diabetes mellitus.

8.
Cancer Sci ; 112(2): 884-892, 2021 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-33280191

RESUMO

Discrimination of Philadelphia-negative myeloproliferative neoplasms (Ph-MPNs) from reactive hypercytosis and myelofibrosis requires a constellation of testing including driver mutation analysis and bone marrow biopsies. We searched for a biomarker that can more easily distinguish Ph-MPNs from reactive hypercytosis and myelofibrosis by using RNA-seq analysis utilizing platelet-rich plasma (PRP)-derived RNAs from patients with essential thrombocythemia (ET) and reactive thrombocytosis, and CREB3L1 was found to have an extremely high impact in discriminating the two disorders. To validate and further explore the result, expression levels of CREB3L1 in PRP were quantified by reverse-transcription quantitative PCR and compared among patients with ET, other Ph-MPNs, chronic myeloid leukemia (CML), and reactive hypercytosis and myelofibrosis. A CREB3L1 expression cutoff value determined based on PRP of 18 healthy volunteers accurately discriminated 150 driver mutation-positive Ph-MPNs from other entities (71 reactive hypercytosis and myelofibrosis, 6 CML, and 18 healthy volunteers) and showed both sensitivity and specificity of 1.0000. Importantly, CREB3L1 expression levels were significantly higher in ET compared with reactive thrombocytosis (P < .0001), and polycythemia vera compared with reactive erythrocytosis (P < .0001). Pathology-affirmed triple-negative ET (TN-ET) patients were divided into a high- and low-CREB3L1-expression group, and some patients in the low-expression group achieved a spontaneous remission during the clinical course. In conclusion, CREB3L1 analysis has the potential to single-handedly discriminate driver mutation-positive Ph-MPNs from reactive hypercytosis and myelofibrosis, and also may identify a subgroup within TN-ET showing distinct clinical features including spontaneous remission.


Assuntos
Biomarcadores Tumorais/sangue , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/sangue , Transtornos Mieloproliferativos/diagnóstico , Proteínas do Tecido Nervoso/sangue , Diagnóstico Diferencial , Humanos , Leucemia Mielogênica Crônica BCR-ABL Positiva/sangue , Leucemia Mielogênica Crônica BCR-ABL Positiva/diagnóstico , Transtornos Mieloproliferativos/sangue
9.
PLoS One ; 15(12): e0243319, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-33320908

RESUMO

Here, we report a rapid and ultra-sensitive detection technique for fluorescent molecules called scanning single molecular counting (SSMC). The method uses a fluorescence-based digital measurement system to count single molecules in a solution. In this technique, noise is reduced by conforming the signal shape to the intensity distribution of the excitation light via a circular scan of the confocal region. This simple technique allows the fluorescent molecules to freely diffuse into the solution through the confocal region and be counted one by one and does not require statistical analysis. Using this technique, 28 to 62 aM fluorescent dye was detected through measurement for 600 s. Furthermore, we achieved a good signal-to-noise ratio (S/N = 2326) under the condition of 100 pM target nucleic acid by only mixing a hybridization-sensitive fluorescent probe, called Eprobe, into the target oligonucleotide solution. Combination of SSMC and Eprobe provides a simple, rapid, amplification-free, and high-sensitive target nucleic acid detection system. This method is promising for future applications to detect particularly difficult to design primers for amplification as miRNAs and other short oligo nucleotide biomarkers by only hybridization with high sensitivity.


Assuntos
Corantes Fluorescentes/química , MicroRNAs/análise , Oligonucleotídeos/química , Fluorescência , MicroRNAs/química , Hibridização de Ácido Nucleico
10.
Insects ; 11(11)2020 Nov 11.
Artigo em Inglês | MEDLINE | ID: mdl-33187095

RESUMO

The Pv11 cell line established from an African chironomid, Polypedilum vanderplanki, is the only cell line tolerant to complete desiccation. In Pv11 cells, a constitutive expression system for Pv11 cells was previously exploited and several reporter genes were successfully expressed. Here we report the identification of an effective minimal promoter for Pv11 cells and its application to the Tet-On inducible expression system. First, using a luciferase reporter assay, we showed that a 202 bp deletion fragment derived from the constitutively active 121-promoter functions in Pv11 cells as an appropriate minimal promoter with the Tet-On inducible expression system. The AcGFP1 (Aequorea coerulescens green fluorescent protein) was also successfully expressed in Pv11 cells using the inducible system. In addition to these reporter genes, the avian myeloblastosis virus reverse transcriptase α subunit (AMV RTα), which is one of the most widely commercially available RNA-dependent DNA polymerases, was successfully expressed through the inducible expression system and its catalytic activity was verified. These results demonstrate the establishment of an inducible expression system in cells that can be preserved in the dry state and highlight a possible application to the production of large and complex proteins.

11.
Sci Rep ; 10(1): 19933, 2020 11 16.
Artigo em Inglês | MEDLINE | ID: mdl-33199820

RESUMO

Myalgic encephalomyelitis/chronic fatigue syndrome (ME/CFS) is a complex and debilitating disease with no molecular diagnostics and no treatment options. To identify potential markers of this illness, we profiled 48 patients and 52 controls for standard laboratory tests, plasma metabolomics, blood immuno-phenotyping and transcriptomics, and fecal microbiome analysis. Here, we identified a set of 26 potential molecular markers that distinguished ME/CFS patients from healthy controls. Monocyte number, microbiome abundance, and lipoprotein profiles appeared to be the most informative markers. When we correlated these molecular changes to sleep and cognitive measurements of fatigue, we found that lipoprotein and microbiome profiles most closely correlated with sleep disruption while a different set of markers correlated with a cognitive parameter. Sleep, lipoprotein, and microbiome changes occur early during the course of illness suggesting that these markers can be examined in a larger cohort for potential biomarker application. Our study points to a cluster of sleep-related molecular changes as a prominent feature of ME/CFS in our Japanese cohort.


Assuntos
Biomarcadores/análise , Síndrome de Fadiga Crônica/epidemiologia , Síndrome de Fadiga Crônica/patologia , Fezes/microbiologia , Metaboloma , Microbiota , Transcriptoma , Estudos de Casos e Controles , Estudos de Coortes , Síndrome de Fadiga Crônica/genética , Síndrome de Fadiga Crônica/metabolismo , Humanos , Japão/epidemiologia
12.
Genome Res ; 30(7): 951-961, 2020 07.
Artigo em Inglês | MEDLINE | ID: mdl-32718981

RESUMO

Gene expression profiles in homologous tissues have been observed to be different between species, which may be due to differences between species in the gene expression program in each cell type, but may also reflect differences in cell type composition of each tissue in different species. Here, we compare expression profiles in matching primary cells in human, mouse, rat, dog, and chicken using Cap Analysis Gene Expression (CAGE) and short RNA (sRNA) sequencing data from FANTOM5. While we find that expression profiles of orthologous genes in different species are highly correlated across cell types, in each cell type many genes were differentially expressed between species. Expression of genes with products involved in transcription, RNA processing, and transcriptional regulation was more likely to be conserved, while expression of genes encoding proteins involved in intercellular communication was more likely to have diverged during evolution. Conservation of expression correlated positively with the evolutionary age of genes, suggesting that divergence in expression levels of genes critical for cell function was restricted during evolution. Motif activity analysis showed that both promoters and enhancers are activated by the same transcription factors in different species. An analysis of expression levels of mature miRNAs and of primary miRNAs identified by CAGE revealed that evolutionary old miRNAs are more likely to have conserved expression patterns than young miRNAs. We conclude that key aspects of the regulatory network are conserved, while differential expression of genes involved in cell-to-cell communication may contribute greatly to phenotypic differences between species.

13.
Genome Res ; 30(7): 1060-1072, 2020 07.
Artigo em Inglês | MEDLINE | ID: mdl-32718982

RESUMO

Long noncoding RNAs (lncRNAs) constitute the majority of transcripts in the mammalian genomes, and yet, their functions remain largely unknown. As part of the FANTOM6 project, we systematically knocked down the expression of 285 lncRNAs in human dermal fibroblasts and quantified cellular growth, morphological changes, and transcriptomic responses using Capped Analysis of Gene Expression (CAGE). Antisense oligonucleotides targeting the same lncRNAs exhibited global concordance, and the molecular phenotype, measured by CAGE, recapitulated the observed cellular phenotypes while providing additional insights on the affected genes and pathways. Here, we disseminate the largest-to-date lncRNA knockdown data set with molecular phenotyping (over 1000 CAGE deep-sequencing libraries) for further exploration and highlight functional roles for ZNF213-AS1 and lnc-KHDC3L-2.

14.
Methods Mol Biol ; 2120: 277-301, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32124327

RESUMO

Cap analysis of gene expression (CAGE) is an approach to identify and monitor the activity (transcription initiation frequency) of transcription start sites (TSSs) at single base-pair resolution across the genome. It has been effectively used to identify active promoter and enhancer regions in cancer cells, with potential utility to identify key factors to immunotherapy. Here, we overview a series of CAGE protocols and describe detailed experimental steps of the latest protocol based on the Illumina sequencing platform; both experimental steps (see Subheadings 3.1-3.11) and computational processing steps (see Subheadings 3.12-3.20) are described.


Assuntos
Genômica/métodos , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Sítio de Iniciação de Transcrição , Ativação Transcricional , Animais , Expressão Gênica , Humanos , Camundongos , Regiões Promotoras Genéticas
15.
Genome Res ; 30(7): 1073-1081, 2020 07.
Artigo em Inglês | MEDLINE | ID: mdl-32079618

RESUMO

Long noncoding RNAs (lncRNAs) have emerged as key coordinators of biological and cellular processes. Characterizing lncRNA expression across cells and tissues is key to understanding their role in determining phenotypes, including human diseases. We present here FC-R2, a comprehensive expression atlas across a broadly defined human transcriptome, inclusive of over 109,000 coding and noncoding genes, as described in the FANTOM CAGE-Associated Transcriptome (FANTOM-CAT) study. This atlas greatly extends the gene annotation used in the original recount2 resource. We demonstrate the utility of the FC-R2 atlas by reproducing key findings from published large studies and by generating new results across normal and diseased human samples. In particular, we (a) identify tissue-specific transcription profiles for distinct classes of coding and noncoding genes, (b) perform differential expression analysis across thirteen cancer types, identifying novel noncoding genes potentially involved in tumor pathogenesis and progression, and (c) confirm the prognostic value for several enhancer lncRNAs expression in cancer. Our resource is instrumental for the systematic molecular characterization of lncRNA by the FANTOM6 Consortium. In conclusion, comprised of over 70,000 samples, the FC-R2 atlas will empower other researchers to investigate functions and biological roles of both known coding genes and novel lncRNAs.

16.
Nat Genet ; 51(9): 1369-1379, 2019 09.
Artigo em Inglês | MEDLINE | ID: mdl-31477927

RESUMO

Promoters and enhancers are key cis-regulatory elements, but how they operate to generate cell type-specific transcriptomes is not fully understood. We developed a simple and robust method, native elongating transcript-cap analysis of gene expression (NET-CAGE), to sensitively detect 5' ends of nascent RNAs in diverse cells and tissues, including unstable transcripts such as enhancer-derived RNAs. We studied RNA synthesis and degradation at the transcription start site level, characterizing the impact of differential promoter usage on transcript stability. We quantified transcription from cis-regulatory elements without the influence of RNA turnover, and show that enhancer-promoter pairs are generally activated simultaneously on stimulation. By integrating NET-CAGE data with chromatin interaction maps, we show that cis-regulatory elements are topologically connected according to their cell type specificity. We identified new enhancers with high sensitivity, and delineated primary locations of transcription within super-enhancers. Our NET-CAGE dataset derived from human and mouse cells expands the FANTOM5 atlas of transcribed enhancers, with broad applicability to biomedical research.


Assuntos
Regiões 5' não Traduzidas/genética , Biologia Computacional/métodos , Elementos Facilitadores Genéticos , Regulação da Expressão Gênica , Regiões Promotoras Genéticas , RNA/genética , Transcrição Genética , Perfilação da Expressão Gênica , Células HeLa , Células Hep G2 , Humanos , Células MCF-7 , Sítio de Iniciação de Transcrição , Transcriptoma
17.
BMC Genomics ; 20(1): 718, 2019 Sep 18.
Artigo em Inglês | MEDLINE | ID: mdl-31533632

RESUMO

BACKGROUND: The work of the FANTOM5 Consortium has brought forth a new level of understanding of the regulation of gene transcription and the cellular processes involved in creating diversity of cell types. In this study, we extended the analysis of the FANTOM5 Cap Analysis of Gene Expression (CAGE) transcriptome data to focus on understanding the genetic regulators involved in mouse cerebellar development. RESULTS: We used the HeliScopeCAGE library sequencing on cerebellar samples over 8 embryonic and 4 early postnatal times. This study showcases temporal expression pattern changes during cerebellar development. Through a bioinformatics analysis that focused on transcription factors, their promoters and binding sites, we identified genes that appear as strong candidates for involvement in cerebellar development. We selected several candidate transcriptional regulators for validation experiments including qRT-PCR and shRNA transcript knockdown. We observed marked and reproducible developmental defects in Atf4, Rfx3, and Scrt2 knockdown embryos, which support the role of these genes in cerebellar development. CONCLUSIONS: The successful identification of these novel gene regulators in cerebellar development demonstrates that the FANTOM5 cerebellum time series is a high-quality transcriptome database for functional investigation of gene regulatory networks in cerebellar development.


Assuntos
Cerebelo/crescimento & desenvolvimento , Perfilação da Expressão Gênica , Motivos de Nucleotídeos/genética , Transcrição Genética/genética , Fator 4 Ativador da Transcrição/deficiência , Fator 4 Ativador da Transcrição/genética , Fator 4 Ativador da Transcrição/metabolismo , Animais , Cerebelo/embriologia , Cerebelo/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Técnicas de Silenciamento de Genes , Camundongos , Camundongos Endogâmicos C57BL , Regiões Promotoras Genéticas/genética , Fatores de Transcrição de Fator Regulador X/deficiência , Fatores de Transcrição de Fator Regulador X/genética , Fatores de Transcrição de Fator Regulador X/metabolismo , Fatores de Transcrição/deficiência , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo
18.
Sci Rep ; 9(1): 10387, 2019 07 17.
Artigo em Inglês | MEDLINE | ID: mdl-31316152

RESUMO

Intracranial aneurysm (IA) is a socially important disease as a major cause of subarachnoid hemorrhage. Recent experimental studies mainly using animal models have revealed a crucial role of macrophage-mediated chronic inflammatory responses in its pathogenesis. However, as findings from comprehensive analysis of unruptured human IAs are limited, factors regulating progression and rupture of IAs in humans remain unclear. Using surgically dissected human unruptured IA lesions and control arterial walls, gene expression profiles were obtained by RNA sequence analysis. RNA sequencing analysis was done with read count about 60~100 million which yielded 6~10 billion bases per sample. 79 over-expressed and 329 under-expressed genes in IA lesions were identified. Through Gene Ontology analysis, 'chemokine activity', 'defense response' and 'extracellular region' were picked up as over-represented terms which included CCL3 and CCL4 in common. Among these genes, quantitative RT-PCR analysis using another set of samples reproduced the above result. Finally, increase of CCL3 protein compared with that in control arterial walls was clarified in IA lesions. Findings of the present study again highlight importance of macrophage recruitment via CCL3 in the pathogenesis of IA progression.


Assuntos
Quimiocina CCL3/genética , Aneurisma Intracraniano/genética , Idoso , Aneurisma Roto/complicações , Quimiocina CCL3/metabolismo , Feminino , Expressão Gênica/genética , Ontologia Genética , Humanos , Inflamação/complicações , Aneurisma Intracraniano/metabolismo , Macrófagos/metabolismo , Masculino , Pessoa de Meia-Idade , Análise de Sequência de RNA/métodos , Transdução de Sinais , Hemorragia Subaracnóidea/complicações , Transcriptoma/genética
19.
Sci Rep ; 9(1): 4638, 2019 03 15.
Artigo em Inglês | MEDLINE | ID: mdl-30874579

RESUMO

Selection of human induced pluripotent stem cell (hiPSC) lines with high cardiac differentiation potential is important for regenerative therapy and drug screening. We aimed to identify biomarkers for predicting cardiac differentiation potential of hiPSC lines by comparing the gene expression profiles of six undifferentiated hiPSC lines with different cardiac differentiation capabilities. We used three platforms of gene expression analysis, namely, cap analysis of gene expression (CAGE), mRNA array, and microRNA array to efficiently screen biomarkers related to cardiac differentiation of hiPSCs. Statistical analysis revealed candidate biomarker genes with significant correlation between the gene expression levels in the undifferentiated hiPSCs and their cardiac differentiation potential. Of the candidate genes, PF4 was validated as a biomarker expressed in undifferentiated hiPSCs with high potential for cardiac differentiation in 13 additional hiPSC lines. Our observations suggest that PF4 may be a useful biomarker for selecting hiPSC lines appropriate for the generation of cardiomyocytes.


Assuntos
Células-Tronco Pluripotentes Induzidas/metabolismo , Miócitos Cardíacos/metabolismo , Fator Plaquetário 4/metabolismo , Biomarcadores/metabolismo , Diferenciação Celular/fisiologia , Células Cultivadas , Reprogramação Celular/fisiologia , Humanos , Células-Tronco Pluripotentes Induzidas/citologia , MicroRNAs/genética , Organogênese/fisiologia , Fator Plaquetário 4/genética , RNA Mensageiro/genética , Transcriptoma/genética
20.
Mol Neurobiol ; 56(8): 5392-5415, 2019 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-30610612

RESUMO

Natural antisense transcripts are common features of mammalian genes providing additional regulatory layers of gene expression. A comprehensive description of antisense transcription in loci associated to familial neurodegenerative diseases may identify key players in gene regulation and provide tools for manipulating gene expression. We take advantage of the FANTOM5 sequencing datasets that represent the largest collection to date of genome-wide promoter usage in almost 2000 human samples. Transcription start sites (TSSs) are mapped at high resolution by the use of a modified protocol of cap analysis of gene expression (CAGE) for high-throughput single molecule next-generation sequencing with Helicos (hCAGE). Here we present the analysis of antisense transcription at 17 loci associated to hereditary Alzheimer's disease, Frontotemporal Dementia, Parkinson's disease, Amyotrophic Lateral Sclerosis, and Huntington's disease. We focused our analysis on libraries derived from brain tissues and primary cells. We also screened libraries from total blood and blood cell populations in the quest for peripheral biomarkers of neurodegenerative diseases. We identified 63 robust promoters in antisense orientation to genes associated to familial neurodegeneration. When applying a less stringent cutoff, this number increases to over 400. A subset of these promoters represents alternative TSSs for 24 FANTOM5 annotated long noncoding RNA (lncRNA) genes, in antisense orientation to 13 of the loci analyzed here, while the remaining contribute to the expression of additional transcript variants. Intersection with GWAS studies, sample ontology, and dynamic expression reveals association to specific genetic traits as well as cell and tissue types, not limited to neurodegenerative diseases. Antisense transcription was validated for a subset of genes, including those encoding for Microtubule-Associated Protein Tau, α-synuclein, Parkinsonism-associated deglycase DJ-1, and Leucin-Rich Repeat Kinase 2. This work provides evidence for the existence of additional regulatory mechanisms of the expression of neurodegenerative disease-causing genes by previously not-annotated and/or not-validated antisense long noncoding RNAs.


Assuntos
Loci Gênicos , Predisposição Genética para Doença , Doenças Neurodegenerativas/genética , RNA Antissenso/genética , Transcrição Genética , Linhagem Celular Tumoral , Regulação da Expressão Gênica , Pleiotropia Genética , Humanos , Anotação de Sequência Molecular , RNA Longo não Codificante/genética , RNA Longo não Codificante/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo
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