RESUMO
Phosphaturic mesenchymal tumors (PMT) are uncommon neoplasms that cause hypophosphatemia/osteomalacia mainly by secreting fibroblast growth factor 23. We previously identified FN1::FGFR1/FGF1 fusions in nearly half of the PMTs and frequent KL (Klotho or α-Klotho) overexpression in only those with no known fusion. Here, we studied a larger cohort of PMTs for KL expression and alterations. By FN1 break-apart fluorescence in situ hybridization (FISH) and reappraisal of previous RNA sequencing data, 6 tumors previously considered "fusion-negative" (defined by negative results of FISH for FN1::FGFR1 fusion and FGF1 break-apart and/or of RNA sequencing) were reclassified as fusion-positive PMTs, including 1 containing a novel FN1::ZACN fusion. The final cohort of fusion-negative PMTs included 33 tumors from 32 patients, which occurred in the bone (n = 18), soft tissue (n = 10), sinonasal tract (n = 4), and brain (n = 1). In combination with previous work, RNA sequencing, RNA in situ hybridization, and immunohistochemistry showed largely concordant results and demonstrated KL/α-Klotho overexpression in 17 of the 28 fusion-negative and none of the 10 fusion-positive PMTs studied. Prompted by a patient in this cohort harboring germline KL upstream translocation with systemic α-Klotho overexpression and multifocal PMTs, FISH was performed and revealed KL rearrangement in 16 of the 33 fusion-negative PMTs (one also with amplification), including 14 of the 17 cases with KL/α-Klotho overexpression and none of the 11 KL/α-Klotho-low fusion-negative and 11 fusion-positive cases studied. Whole genomic sequencing confirmed translocation and inversion in 2 FISH-positive cases involving the KL upstream region, warranting further investigation into the mechanism whereby these rearrangements may lead to KL upregulation. Methylated DNA immunoprecipitation and sequencing suggested no major role of promoter methylation in KL regulation in PMT. Interestingly, KL-high/-rearranged cases seemed to form a clinicopathologically homogeneous group, showing a predilection for skeletal/sinonasal locations and typically matrix-poor, cellular solitary fibrous tumor-like morphology. Importantly, FGFR1 signaling pathways were upregulated in fusion-negative PMTs regardless of the KL status compared with non-PMT mesenchymal tumors by gene set enrichment analysis, perhaps justifying FGFR1 inhibition in treating this subset of PMTs.
Assuntos
Mesenquimoma , Seios Paranasais , Neoplasias de Tecidos Moles , Humanos , Hibridização in Situ Fluorescente , Fator 1 de Crescimento de Fibroblastos/genética , Neoplasias de Tecidos Moles/genética , Mesenquimoma/genética , Mesenquimoma/patologia , Translocação Genética , Seios Paranasais/patologiaRESUMO
BACKGROUND/AIMS: Endothelial colony-forming cells (ECFCs) have the potential to be used in regenerative medicine. Dysfunction of ECFCs is correlated with the onset of cardiovascular disorders, especially coronary artery disease (CAD). Binding of vascular endothelial growth factor A (VEGFA) to vascular endothelial growth factor receptor-2 (VEGFR2) triggers cell motility and angiogenesis of ECFCs, which are crucial to vascular repair. METHODS: To identify the miRNA-VEGFR2-dependent regulation of ECFC functions, ECFCs isolated from peripheral blood of disease-free and CAD individuals were subjected to small RNA sequencing for identification of anti-VEGFR2 miRNAs. The angiogenic activities of the miRNAs were determined in both in vitro and in vivo mice models. RESULTS: Three miRNAs, namely miR-410-3p, miR-497-5p, and miR-2355-5p, were identified to be upregulated in CAD-ECFCs, and VEGFR2 was their common target gene. Knockdown of these miRNAs not only restored the expression of VEGFR2 and increased angiogenic activities of CAD-ECFCs in vitro, but also promoted blood flow recovery in ischemic limbs in vivo. miR-410-3p, miR-497-5p, and miR-2355-5p could serve as potential biomarkers for CAD detection as they are highly expressed in the plasma of CAD patients. CONCLUSIONS: This modulation could help develop new therapeutic modalities for cardiovascular diseases and other vascular dysregulated diseases, especially tumor angiogenesis.
Assuntos
Doença da Artéria Coronariana/metabolismo , Células Progenitoras Endoteliais/metabolismo , MicroRNAs/metabolismo , Neovascularização Fisiológica , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/metabolismo , Animais , Antagomirs/genética , Antagomirs/metabolismo , Estudos de Casos e Controles , Movimento Celular , Proliferação de Células , Células Cultivadas , Biologia Computacional , Doença da Artéria Coronariana/genética , Doença da Artéria Coronariana/patologia , Modelos Animais de Doenças , Células Progenitoras Endoteliais/patologia , Células Progenitoras Endoteliais/transplante , Perfilação da Expressão Gênica/métodos , Regulação da Expressão Gênica , Membro Posterior , Humanos , Isquemia/genética , Isquemia/metabolismo , Isquemia/fisiopatologia , Isquemia/cirurgia , Camundongos Nus , MicroRNAs/genética , Músculo Esquelético/irrigação sanguínea , Recuperação de Função Fisiológica , Fluxo Sanguíneo Regional , Fatores de Tempo , Transfecção , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/genéticaRESUMO
We previously presented YM500, which is an integrated database for miRNA quantification, isomiR identification, arm switching discovery and novel miRNA prediction from 468 human smRNA-seq datasets. Here in this updated YM500v2 database (http://ngs.ym.edu.tw/ym500/), we focus on the cancer miRNome to make the database more disease-orientated. New miRNA-related algorithms developed after YM500 were included in YM500v2, and, more significantly, more than 8000 cancer-related smRNA-seq datasets (including those of primary tumors, paired normal tissues, PBMC, recurrent tumors, and metastatic tumors) were incorporated into YM500v2. Novel miRNAs (miRNAs not included in the miRBase R21) were not only predicted by three independent algorithms but also cleaned by a new in silico filtration strategy and validated by wetlab data such as Cross-Linked ImmunoPrecipitation sequencing (CLIP-seq) to reduce the false-positive rate. A new function 'Meta-analysis' is additionally provided for allowing users to identify real-time differentially expressed miRNAs and arm-switching events according to customer-defined sample groups and dozens of clinical criteria tidying up by proficient clinicians. Cancer miRNAs identified hold the potential for both basic research and biotech applications.
Assuntos
Bases de Dados de Ácidos Nucleicos , MicroRNAs/química , MicroRNAs/metabolismo , Neoplasias/genética , Perfilação da Expressão Gênica , Humanos , Internet , Análise de Sequência de RNARESUMO
OBJECTIVE: Defects in angiogenesis/vasculogenesis or vessel repair are major complications of coronary artery disease (CAD). Endothelial progenitor cells (EPCs) play a fundamental role in postnatal vascular repair and CAD. The role of microRNAs in CAD pathogenesis and their potential as biomarkers remain to be elucidated. APPROACH AND RESULTS: MicroRNA-31 (miR-31) level in both the plasma and EPCs of patients with CAD is found lower. miR-31 regulates EPC activities by targeting FAT atypical cadherin 4 and thromboxane A2 receptor, which show increased expression in CAD EPCs. Overexpressing miR-31 in CAD EPCs rescued their angiogenic and vasculogenic abilities both in vitro and in vivo. When exploring approaches to restore endogenous miR-31, we found that far-infrared treatment enhanced the expression of not only miR-31, but also miR-720 in CAD EPCs. miR-720, which was also decreased in EPCs and the plasma of patients with CAD, stimulated EPC activity by targeting vasohibin 1. The miR720-vasohibin 1 pair was shown to be downstream of FAT atypical cadherin 4, but not of thromboxane A2 receptor. FAT atypical cadherin 4 inhibited miR-720 expression via repression of the planar cell polarity signaling gene four-jointed box 1 (FJX1), which was required for miR-720 expression through a hypoxia-inducible factor 1, α subunit-dependent mechanism. Restoring miR-720 level strengthened activity of CAD EPCs. The miR-31-miR-720 pathway is shown critical to EPC activation and that downregulation of this pathway contributes to CAD pathogenesis. Circulating levels of miR-31, miR-720, and vasohibin 1 have the potential to allow early diagnosis of CAD and to act as prognosis biomarkers for CAD and other EPC-related diseases. CONCLUSIONS: Manipulating the expression of the miR-31-miR-720 pathway in malfunction EPCs should help develop novel therapeutic modalities.
Assuntos
Doença da Artéria Coronariana/sangue , Células Endoteliais/metabolismo , MicroRNAs/sangue , Músculo Esquelético/irrigação sanguínea , Células-Tronco/metabolismo , Animais , Caderinas/metabolismo , Estudos de Casos e Controles , Proteínas de Ciclo Celular/metabolismo , Células Cultivadas , Doença da Artéria Coronariana/genética , Doença da Artéria Coronariana/fisiopatologia , Modelos Animais de Doenças , Regulação para Baixo , Células Endoteliais/efeitos da radiação , Células Endoteliais/transplante , Marcadores Genéticos , Membro Posterior , Humanos , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Raios Infravermelhos , Isquemia/genética , Isquemia/metabolismo , Isquemia/fisiopatologia , Isquemia/cirurgia , Camundongos , Camundongos Nus , Neovascularização Fisiológica , Oligonucleotídeos/metabolismo , Receptores de Tromboxano A2 e Prostaglandina H2/metabolismo , Recuperação de Função Fisiológica , Fluxo Sanguíneo Regional , Transdução de Sinais , Transplante de Células-Tronco , Células-Tronco/efeitos da radiação , Fatores de Tempo , Transfecção , Proteínas Supressoras de Tumor/metabolismoRESUMO
MicroRNAs (miRNAs) are small RNAs â¼22 nt in length that are involved in the regulation of a variety of physiological and pathological processes. Advances in high-throughput small RNA sequencing (smRNA-seq), one of the next-generation sequencing applications, have reshaped the miRNA research landscape. In this study, we established an integrative database, the YM500 (http://ngs.ym.edu.tw/ym500/), containing analysis pipelines and analysis results for 609 human and mice smRNA-seq results, including public data from the Gene Expression Omnibus (GEO) and some private sources. YM500 collects analysis results for miRNA quantification, for isomiR identification (incl. RNA editing), for arm switching discovery, and, more importantly, for novel miRNA predictions. Wetlab validation on >100 miRNAs confirmed high correlation between miRNA profiling and RT-qPCR results (R = 0.84). This database allows researchers to search these four different types of analysis results via our interactive web interface. YM500 allows researchers to define the criteria of isomiRs, and also integrates the information of dbSNP to help researchers distinguish isomiRs from SNPs. A user-friendly interface is provided to integrate miRNA-related information and existing evidence from hundreds of sequencing datasets. The identified novel miRNAs and isomiRs hold the potential for both basic research and biotech applications.
Assuntos
Bases de Dados de Ácidos Nucleicos , MicroRNAs/química , MicroRNAs/metabolismo , Internet , Análise de Sequência de RNA , Transcriptoma , Interface Usuário-ComputadorRESUMO
Organ regeneration therapies using multipotent mesenchymal stem cells (MSCs) are currently being investigated for a variety of common complex diseases. Understanding the molecular regulation of MSC biology will benefit regenerative medicine. MicroRNAs (miRNAs) act as regulators in MSC stemness. There are approximately 2500 currently known human miRNAs that have been recorded in the miRBase v21 database. In the present study, we identified novel microRNAs involved in MSC stemness and differentiation by obtaining the global microRNA expression profiles (miRNomes) of MSCs from two anatomical locations bone marrow (BM-MSCs) and umbilical cord Wharton's jelly (WJ-MSCs) and from osteogenically and adipogenically differentiated progenies of BM-MSCs. Small RNA sequencing (smRNA-seq) and bioinformatics analyses predicted that 49 uncharacterized miRNA candidates had high cellular expression values in MSCs. Another independent batch of Ago1/2-based RNA immunoprecipitation (RNA-IP) sequencing datasets validated the existence of 40 unreported miRNAs in cells and their associations with the RNA-induced silencing complex (RISC). Nine of these 40 new miRNAs were universally overexpressed in both MSC types; nine others were overexpressed in differentiated cells. A novel miRNA (UNI-118-3p) was specifically expressed in BM-MSCs, as verified using RT-qPCR. Taken together, this report offers comprehensive miRNome profiles for two MSC types, as well as cells differentiated from BM-MSCs. MSC transplantation has the potential to ameliorate degenerative disorders and repair damaged tissues. Interventions involving the above 40 new microRNA members in transplanted MSCs may potentially guide future clinical applications.
Assuntos
Células da Medula Óssea/metabolismo , Células-Tronco Mesenquimais/metabolismo , MicroRNAs/genética , Complexo de Inativação Induzido por RNA/genética , Adipócitos/citologia , Adipócitos/metabolismo , Células da Medula Óssea/citologia , Diferenciação Celular , Proliferação de Células , Regulação da Expressão Gênica , Humanos , Imunoprecipitação , Células-Tronco Mesenquimais/citologia , MicroRNAs/metabolismo , Osteoblastos/citologia , Osteoblastos/metabolismo , Complexo de Inativação Induzido por RNA/metabolismo , Análise de Sequência de RNA , Cordão Umbilical/citologia , Cordão Umbilical/metabolismoRESUMO
BACKGROUND: MicroRNAs (miRNAs) have emerged as master regulators of angiogenesis and other cancer-related events. Discovering new angiogenesis-regulating microRNAs (angiomiRs) will eventually help in developing new therapeutic strategies for tumor angiogenesis and cardiovascular diseases. Kaposi's sarcoma (KS), which is induced by the etiological infectious agent KS-associated herpesvirus (KSHV), is a peculiar neoplasm that expresses both blood and lymphatic endothelial markers and possesses extensive neovasculature. Using KSHV and its proteins as baits will be an efficient way to discover new angiomiRs in endothelial cells. Kaposin B is one of the latent viral genes and is expressed in all KSHV tumor cells. Since Kaposin B is a nuclear protein with no DNA-binding domain, it may regulate gene expression by incorporating itself into a transcription complex. RESULTS: We demonstrated that c-Myc and Kaposin B form a transcription complex and bind to the miR-221/-222 promoter, thereby affecting their expression and anti-angiogenic ability. By small RNA sequencing (smRNA-Seq), we revealed that 72.1% (173/240) of Kaposin B up-regulated and 46.5% (113/243) of Kaposin B down-regulated known miRNAs were regulated by c-Myc. We also found that 77 novel miRNA were up-regulated and 28 novel miRNAs were down-regulated in cells expressing both c-Myc and Kaposin B compared with cells expressing Kaposin B only. The result was confirmed by RNA-IP-seq data. CONCLUSIONS: Our study identifies known and novel c-Myc-regulated microRNAs and reveals that a c-Myc-oriented program is coordinated by Kaposin B in KSHV-infected cells.
Assuntos
Regulação da Expressão Gênica , MicroRNAs/genética , Neovascularização Patológica/genética , Proteínas Proto-Oncogênicas c-myc/fisiologia , Proteínas Virais/fisiologia , Células Endoteliais/metabolismo , Técnicas de Silenciamento de Genes , Células Endoteliais da Veia Umbilical Humana , Humanos , Regiões Promotoras Genéticas , Proteínas Proto-Oncogênicas c-myc/genética , Proteínas Proto-Oncogênicas c-myc/metabolismo , Proteínas Virais/genética , Proteínas Virais/metabolismoRESUMO
Pediatric high-grade gliomas (pHGGs) are aggressive brain tumors affecting children, and outcomes have remained dismal, even with access to new multimodal therapies. In this study, we compared the miRNomes and transcriptomes of pediatric low- (pLGGs) and high-grade gliomas (pHGGs) using small RNA sequencing (smRNA-Seq) and gene expression microarray, respectively. Through integrated bioinformatics analyses and experimental validation, we identified miR-137 and miR-6500-3p as significantly downregulated in pHGGs. miR-137 or miR-6500-3p overexpression reduced cell proliferation in two pHGG cell lines, SF188 and UW479. CENPE, KIF14 and NCAPG levels were significantly higher in pHGGs than pLGGs, and were direct targets of miR-137 or miR-6500-3p. Furthermore, knockdown of CENPE, KIF14 or NCAPG combined with temozolomide treatment resulted in a combined suppressive effect on pHGG cell proliferation. In summary, our results identify novel mRNA/miRNA interactions that contribute to pediatric glioma malignancy and represent potential targets for the development of new therapeutic strategies.