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The 14-kilodalton human growth hormone (14 kDa hGH) N-terminal fragment derived from the proteolytic cleavage of its full-length counterpart has been shown to sustain antiangiogenic potentials. This study investigated the antitumoral and antimetastatic effects of 14 kDa hGH on B16-F10 murine melanoma cells. B16-F10 murine melanoma cells transfected with 14 kDa hGH expression vectors showed a significant reduction in cellular proliferation and migration associated with an increase in cell apoptosis in vitro. In vivo, 14 kDa hGH mitigated tumor growth and metastasis of B16-F10 cells and was associated with a significant reduction in tumor angiogenesis. Similarly, 14 kDa hGH expression reduced human brain microvascular endothelial (HBME) cell proliferation, migration, and tube formation abilities and triggered apoptosis in vitro. The antiangiogenic effects of 14 kDa hGH on HBME cells were abolished when we stably downregulated plasminogen activator inhibitor-1 (PAI-1) expression in vitro. In this study, we showed the potential anticancer role of 14 kDa hGH, its ability to inhibit primary tumor growth and metastasis establishment, and the possible involvement of PAI-1 in promoting its antiangiogenic effects. Therefore, these results suggest that the 14 kDa hGH fragment can be used as a therapeutic molecule to inhibit angiogenesis and cancer progression.
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Hormônio do Crescimento Humano , Melanoma , Camundongos , Humanos , Animais , Hormônio do Crescimento Humano/metabolismo , Inibidor 1 de Ativador de Plasminogênio , Proliferação de CélulasRESUMO
BACKGROUND: Pulmonary fibrosis is an emerging complication of SARS-CoV-2 infection. In this study, we speculate that patients with COVID-19 and idiopathic pulmonary fibrosis (IPF) may share aberrant expressed microRNAs (miRNAs) associated to the progression of lung fibrosis. OBJECTIVE: To identify miRNAs presenting similar alteration in COVID-19 and IPF, and describe their impact on fibrogenesis. METHODS: A systematic review of the literature published between 2010 and January 2022 (PROSPERO, CRD42022341016) was conducted using the key words (COVID-19 OR SARS-CoV-2) AND (microRNA OR miRNA) or (idiopathic pulmonary fibrosis OR IPF) AND (microRNA OR miRNA) in Title/Abstract. RESULTS: Of the 1988 references considered, 70 original articles were appropriate for data extraction: 27 studies focused on miRNAs in COVID-19, and 43 on miRNAs in IPF. 34 miRNAs were overlapping in COVID-19 and IPF, 7 miRNAs presenting an upregulation (miR-19a-3p, miR-200c-3p, miR-21-5p, miR-145-5p, miR-199a-5p, miR-23b and miR-424) and 9 miRNAs a downregulation (miR-17-5p, miR-20a-5p, miR-92a-3p, miR-141-3p, miR-16-5p, miR-142-5p, miR-486-5p, miR-708-3p and miR-150-5p). CONCLUSION: Several studies reported elevated levels of profibrotic miRNAs in COVID-19 context. In addition, the balance of antifibrotic miRNAs responsible of the modulation of fibrotic processes is impaired in COVID-19. This evidence suggests that the deregulation of fibrotic-related miRNAs participates in the development of fibrotic lesions in the lung of post-COVID-19 patients.
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COVID-19 , Fibrose Pulmonar Idiopática , MicroRNAs , Humanos , MicroRNAs/genética , COVID-19/genética , COVID-19/patologia , SARS-CoV-2/genética , Fibrose Pulmonar Idiopática/genética , Fibrose Pulmonar Idiopática/patologia , Pulmão/patologiaRESUMO
BACKGROUND: The global coronavirus disease 2019 (COVID-19) has presented significant challenges and created concerns worldwide. Besides, patients who have experienced a SARS-CoV-2 infection could present post-viral complications that can ultimately lead to pulmonary fibrosis. Serum levels of Krebs von den Lungen 6 (KL-6), high molecular weight human MUC1 mucin, are increased in the most patients with various interstitial lung damage. Since its production is raised during epithelial damages, KL-6 could be a helpful non-invasive marker to monitor COVID-19 infection and predict post-infection sequelae. METHODS: We retrospectively evaluated KL-6 levels of 222 COVID-19 infected patients and 70 healthy control. Serum KL-6, fibrinogen, lactate dehydrogenase (LDH), platelet-lymphocytes ratio (PLR) levels and other biological parameters were analyzed. This retrospective study also characterized the relationships between serum KL-6 levels and pulmonary function variables. RESULTS: Our results showed that serum KL-6 levels in COVID-19 patients were increased compared to healthy subjects (470 U/ml vs 254 U/ml, P <0.00001). ROC curve analysis enabled us to identify that KL-6 > 453.5 U/ml was associated with COVID-19 (AUC = 0.8415, P < 0.0001). KL-6 level was positively correlated with other indicators of disease severity such as fibrinogen level (r = 0.1475, P = 0.0287), LDH level (r = 0,31, P = 0,004) and PLR level (r = 0.23, P = 0.0005). However, KL-6 levels were not correlated with pulmonary function tests (r = 0.04, P = 0.69). CONCLUSIONS: KL-6 expression was correlated with several disease severity indicators. However, the association between mortality and long-term follow-up outcomes needs further investigation. More extensive trials are required to prove that KL-6 could be a marker of disease severity in COVID-19 infection.
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COVID-19 , Humanos , Fibrinogênio , Testes Imunológicos , Estudos Retrospectivos , SARS-CoV-2RESUMO
Tumour-derived extracellular vesicles (EVs) participate in tumour progression by deregulating various physiological processes including angiogenesis and inflammation. Here we report that EVs released by endothelial cells in a mammary tumour environment participate in the recruitment of macrophages within the tumour, leading to an immunomodulatory phenotype permissive for tumour growth. Using RNA-Seq approaches, we identified several microRNAs (miRNAs) found in endothelial EVs sharing common targets involved in the regulation of the immune system. To further study the impact of these miRNAs in a mouse tumour model, we focused on three miRNAs that are conserved between humans and mouse, that is, miR-142-5p, miR-183-5p and miR-222-3p. These miRNAs are released from endothelial cells in a tumour microenvironment and are transferred via EVs to macrophages. In mouse mammary tumour models, treatment with EVs enriched in these miRNAs leads to a polarization of macrophages toward an M2-like phenotype, which in turn promotes tumour growth.
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Vesículas Extracelulares , MicroRNAs , Neoplasias , Animais , Modelos Animais de Doenças , Células Endoteliais , Vesículas Extracelulares/genética , Camundongos , MicroRNAs/genética , Microambiente Tumoral , Macrófagos Associados a TumorRESUMO
Pancreatic ductal adenocarcinoma (PDAC) is categorized as the leading cause of cancer mortality worldwide. However, its predictive markers for long-term survival are not well known. It is interesting to delineate individual-specific perturbed genes when comparing long-term (LT) and short-term (ST) PDAC survivors and integrate individual- and group-based transcriptome profiling. Using a discovery cohort of 19 PDAC patients from CHU-Liège (Belgium), we first performed differential gene expression analysis comparing LT to ST survivor. Second, we adopted systems biology approaches to obtain clinically relevant gene modules. Third, we created individual-specific perturbation profiles. Furthermore, we used Degree-Aware disease gene prioritizing (DADA) method to develop PDAC disease modules; Network-based Integration of Multi-omics Data (NetICS) to integrate group-based and individual-specific perturbed genes in relation to PDAC LT survival. We identified 173 differentially expressed genes (DEGs) in ST and LT survivors and five modules (including 38 DEGs) showing associations to clinical traits. Validation of DEGs in the molecular lab suggested a role of REG4 and TSPAN8 in PDAC survival. Via NetICS and DADA, we identified various known oncogenes such as CUL1 and TGFB1. Our proposed analytic workflow shows the advantages of combining clinical and omics data as well as individual- and group-level transcriptome profiling.
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Carcinoma Ductal Pancreático , Neoplasias Pancreáticas , Biomarcadores Tumorais/genética , Carcinoma Ductal Pancreático/patologia , Perfilação da Expressão Gênica/métodos , Regulação Neoplásica da Expressão Gênica , Humanos , Neoplasias Pancreáticas/patologia , Tetraspaninas/metabolismo , Transcriptoma , Neoplasias PancreáticasRESUMO
BACKGROUND: Extracellular vesicles (EVs) are released by nearly every cell type and have attracted much attention for their ability to transfer protein and diverse RNA species from donor to recipient cells. Much attention has been given so far to the features of EV short RNAs such as miRNAs. However, while the presence of mRNA and long noncoding RNA (lncRNA) transcripts in EVs has also been reported by multiple different groups, the properties and function of these longer transcripts have been less thoroughly explored than EV miRNA. Additionally, the impact of EV export on the transcriptome of exporting cells has remained almost completely unexamined. Here, we globally investigate mRNA and lncRNA transcripts in endothelial EVs in multiple different conditions. RESULTS: In basal conditions, long RNA transcripts enriched in EVs have longer than average half-lives and distinctive stability-related sequence and structure characteristics including shorter transcript length, higher exon density, and fewer 3' UTR A/U-rich elements. EV-enriched long RNA transcripts are also enriched in HNRNPA2B1 binding motifs and are impacted by HNRNPA2B1 depletion, implicating this RNA-binding protein in the sorting of long RNA to EVs. After signaling-dependent modification of the cellular transcriptome, we observed that, unexpectedly, the rate of EV enrichment relative to cells was altered for many mRNA and lncRNA transcripts. This change in EV enrichment was negatively correlated with intracellular abundance, with transcripts whose export to EVs increased showing decreased abundance in cells and vice versa. Correspondingly, after treatment with inhibitors of EV secretion, levels of mRNA and lncRNA transcripts that are normally highly exported to EVs increased in cells, indicating a measurable impact of EV export on the long RNA transcriptome of the exporting cells. Compounds with different mechanisms of inhibition of EV secretion affected the cellular transcriptome differently, suggesting the existence of multiple EV subtypes with different long RNA profiles. CONCLUSIONS: We present evidence for an impact of EV physiology on the characteristics of EV-producing cell transcriptomes. Our work suggests a new paradigm in which the sorting and packaging of transcripts into EVs participate, together with transcription and RNA decay, in controlling RNA homeostasis and shape the cellular long RNA abundance profile.
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Vesículas Extracelulares , MicroRNAs , RNA Longo não Codificante , Movimento Celular , Vesículas Extracelulares/genética , Vesículas Extracelulares/metabolismo , MicroRNAs/metabolismo , RNA Longo não Codificante/genética , RNA Longo não Codificante/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismoRESUMO
BACKGROUND: Mitochondrial fusion and fission proteins have been nominated as druggable targets in cancer. Whether their inhibition is efficacious in triple negative breast cancer (TNBC) that almost invariably develops chemoresistance is unknown. METHODS: We used a combination of bioinformatics analyses of cancer genomic databases, genetic and pharmacological Optic Atrophy 1 (OPA1) inhibition, mitochondrial function and morphology measurements, micro-RNA (miRNA) profiling and formal epistatic analyses to address the role of OPA1 in TNBC proliferation, migration, and invasion in vitro and in vivo. RESULTS: We identified a signature of OPA1 upregulation in breast cancer that correlates with worse prognosis. Accordingly, OPA1 inhibition could reduce breast cancer cells proliferation, migration, and invasion in vitro and in vivo. Mechanistically, while OPA1 silencing did not reduce mitochondrial respiration, it increased levels of miRNAs of the 148/152 family known to inhibit tumor growth and invasiveness. Indeed, these miRNAs were epistatic to OPA1 in the regulation of TNBC cells growth and invasiveness. CONCLUSIONS: Our data show that targeted inhibition of the mitochondrial fusion protein OPA1 curtails TNBC growth and nominate OPA1 as a druggable target in TNBC.
Assuntos
GTP Fosfo-Hidrolases/metabolismo , Proteínas Mitocondriais/metabolismo , Neoplasias de Mama Triplo Negativas/genética , Animais , Apoptose , Linhagem Celular Tumoral , Proliferação de Células , Modelos Animais de Doenças , Feminino , Humanos , Camundongos Endogâmicos NOD , Transfecção , Neoplasias de Mama Triplo Negativas/patologiaRESUMO
Acute respiratory distress syndrome (ARDS) is a diffuse, acute, inflammatory lung disease characterized by a severe respiratory failure. Recognizing and promptly treating ARDS is critical to combat the high mortality associated with the disease. Despite a significant progress in the treatment of ARDS, our ability to identify early patients and predict outcomes remains limited. The development of novel biomarkers is crucial. In this study, we profiled microRNA (miRNA) expression of plasma-derived exosomes in ARDS disease by small RNA sequencing. Sequencing of 8 ARDS patients and 10 healthy subjects (HSs) allowed to identify 12 differentially expressed exosomal miRNAs (adjusted p < 0.05). Pathway analysis of their predicted targets revealed enrichment in several biological processes in agreement with ARDS pathophysiology, such as inflammation, immune cell activation, and fibrosis. By quantitative RT-PCR, we validated the alteration of nine exosomal miRNAs in an independent cohort of 15 ARDS patients and 20 HSs, among which seven present high capability in discriminating ARDS patients from HSs (area under the curve > 0.8) (miR-130a-3p, miR-221-3p, miR-24-3p, miR-98-3p, Let-7d-3p, miR-1273a, and miR-193a-5p). These findings highlight exosomal miRNA dysregulation in the plasma of ARDS patients which provide promising diagnostic biomarkers and open new perspectives for the development of therapeutics.
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Age-related macular degeneration (AMD) is a worldwide leading cause of blindness affecting individuals over 50 years old. The most aggressive form, wet AMD, is characterized by choroidal neovascularization (CNV) and inflammation involving microglia recruitment. By using a laser-induced CNV mouse model, we provide evidence for a key role played by miR-142-3p during CNV formation. MiR-142-3p was overexpressed in murine CNV lesions and its pharmacological inhibition decreased vascular and microglia densities by 46% and 30%, respectively. Consistently, miR-142-3p overexpression with mimics resulted in an increase of 136% and 126% of blood vessels and microglia recruitment. Interestingly, miR-142-3p expression was linked to the activation state of mouse microglia cells as determined by morphological analysis (cell solidity) through a computational method. In vitro, miR-142-3p overexpression in human microglia cells (HMC3) modulated microglia activation, as shown by CD68 levels. Interestingly, miR142-3p modulation also regulated the production of VEGF-A, the main pro-angiogenic factor. Together, these data strongly support the unprecedented importance of miR-142-3p-dependent vascular-inflammation axis during CNV progression, through microglia activation.
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Neovascularização de Coroide/tratamento farmacológico , Neovascularização de Coroide/genética , Lasers , MicroRNAs/farmacologia , Animais , Neovascularização de Coroide/patologia , Modelos Animais de Doenças , Injeções Intravítreas/métodos , Ativação de Macrófagos/genética , Degeneração Macular/tratamento farmacológico , Degeneração Macular/metabolismo , Camundongos , Microglia/efeitos dos fármacos , Microglia/metabolismoRESUMO
The recruitment of pericytes and vascular smooth muscle cells (SMCs) that enwrap endothelial cells (ECs) is a crucial process for vascular maturation and stabilization. Communication between these two cell types is crucial during vascular development and in maintaining vessel homeostasis. Extracellular vesicles (EVs) have emerged as a new communication tool involving the exchange of microRNAs between cells. In the present study, we searched for microRNAs that could be transferred via EVs from ECs to SMCs and vice versa. Thanks to a microRNA profiling experiment, we found that two microRNAs are more exported in each cell type in coculture experiments: while miR-539 is more secreted by ECs, miR-582 is more present in EVs from SMCs. Functional assays revealed that both microRNAs can modulate both cell-type phenotypes. We further identified miR-539 and miR-582 targets, in agreement with their respective cell functions. The results obtained in vivo in the neovascularization model suggest that miR-539 and miR-582 might cooperate to trigger the process of blood vessel coverage by smooth muscle cells in a mature plexus. Taken together, these results are the first to highlight the role of miR-539 and miR-582 in angiogenesis and communication between ECs and SMCs.
Assuntos
Comunicação Celular , Vesículas Extracelulares/metabolismo , Células Endoteliais da Veia Umbilical Humana/citologia , Músculo Liso Vascular/citologia , Miócitos de Músculo Liso/citologia , Vasos Sanguíneos/metabolismo , Técnicas de Cocultura , Vesículas Extracelulares/ultraestrutura , Regulação da Expressão Gênica , Humanos , MicroRNAs/genética , MicroRNAs/metabolismo , Modelos Biológicos , Neovascularização Fisiológica/genética , Remodelação VascularRESUMO
INTRODUCTION: Idiopathic pulmonary fibrosis (IPF) is a progressive fibrosing interstitial lung disease of unknown aetiology and cure. Recent studies have reported a dysregulation of exosomal microRNAs (miRs) in the IPF context. However, the impact of IPF-related exosomal miRs on the progression of pulmonary fibrosis is unknown. METHODS: Two independent cohorts were enrolled at the ambulatory care polyclinic of Liège University. Exosomes from sputum were obtained from 19 patients with IPF and 23 healthy subjects (HSs) (cohort 1), and the ones from plasma derived from 14 patients with IPF and 14 HSs (cohort 2). Exosomal miR expression was performed by quantitative reverse transcription-PCR. The functional role of exosomal miRs was assessed in vitro by transfecting miR mimics in human alveolar epithelial cells and lung fibroblasts. RESULTS: Exosomal miR analysis showed that miR-142-3p was significantly upregulated in sputum and plasma of patients with IPF (8.06-fold, p<0.0001; 1.64 fold, p=0.008, respectively). Correlation analysis revealed a positive association between exosomal miR-142-3p and the percentage of macrophages from sputum of patients with IPF (r=0.576, p=0.012), suggesting macrophage origin of exosomal miR-142-3p upregulation. The overexpression of miR-142-3p in alveolar epithelial cells and lung fibroblasts was able to reduce the expression of transforming growth factor ß receptor 1 (TGFß-R1) and profibrotic genes. Furthermore, exosomes isolated from macrophages present antifibrotic properties due in part to the repression of TGFß-R1 by miR-142-3p transfer in target cells. DISCUSSION: Our results suggest that macrophage-derived exosomes may fight against pulmonary fibrosis progression via the delivery of antifibrotic miR-142-3 p to alveolar epithelial cells and lung fibroblasts.
Assuntos
Células Epiteliais Alveolares/metabolismo , Exossomos/metabolismo , Fibrose Pulmonar Idiopática/metabolismo , Fibrose Pulmonar Idiopática/patologia , Macrófagos/metabolismo , MicroRNAs/metabolismo , Idoso , Idoso de 80 Anos ou mais , Estudos de Casos e Controles , Feminino , Fibroblastos/metabolismo , Humanos , Masculino , Pessoa de Meia-IdadeRESUMO
Epithelial-mesenchymal transitions (EMTs) are high-profile in the field of circulating tumor cells (CTCs). EMT-shifted CTCs are considered to encompass pre-metastatic subpopulations though underlying molecular mechanisms remain elusive. Our previous work identified tissue factor (TF) as an EMT-induced gene providing tumor cells with coagulant properties and supporting metastatic colonization by CTCs. We here report that vimentin, the type III intermediate filament considered a canonical EMT marker, contributes to TF regulation and positively supports coagulant properties and early metastasis. Different evidence further pointed to a new post-transcriptional regulatory mechanism of TF mRNA by vimentin: (1) vimentin silencing accelerated TF mRNA decay after actinomycin D treatment, reflecting TF mRNA stabilization, (2) RNA immunoprecipitation revealed enriched levels of TF mRNA in vimentin immunoprecipitate, (3) TF 3'-UTR-luciferase reporter vector assays implicated the 3'-UTR of TF mRNA in vimentin-dependent TF regulation, and (4) using different TF 3'UTR-luciferase reporter vectors mutated for potential miR binding sites and specific Target Site Blockers identified a key miR binding site in vimentin-dependent TF mRNA regulation. All together, these data support a novel mechanism by which vimentin interferes with a miR-dependent negative regulation of TF mRNA, thereby promoting coagulant activity and early metastasis of vimentin-expressing CTCs.
Assuntos
Neoplasias da Mama/genética , Células Neoplásicas Circulantes/metabolismo , Tromboplastina/genética , Vimentina/genética , Regiões 3' não Traduzidas/genética , Neoplasias da Mama/patologia , Linhagem Celular Tumoral , Dactinomicina/farmacologia , Transição Epitelial-Mesenquimal/genética , Feminino , Regulação Neoplásica da Expressão Gênica/genética , Humanos , MicroRNAs/genética , Metástase Neoplásica , Estabilidade de RNA/efeitos dos fármacos , RNA Mensageiro/genéticaRESUMO
The chemotherapeutic drug epirubicin increases the exosomal export of miR-503 in endothelial cells. To understand the mechanisms behind this process, we transfected endothelial cells with miR-503 carrying a biotin tag. Then, we pulled-down the proteins interacting with miR-503 and studied their role in microRNA exosomal export. A total of four different binding partners were identified by mass spectrometry and validated by western blotting and negative controls, among them ANXA2 and hnRNPA2B1. Using knock-down systems combined with pull-down analysis, we determined that epirubicin mediates the export of miR-503 by disrupting the interaction between hnRNPA2B1 and miR-503. Then, both ANXA2 and miR-503 are sorted into exosomes while hnRNPA2B1 is relocated into the nucleus. The combination of these processes culminates in the increased export of miR-503. These results suggest, for the first time, that RNA-binding proteins can negatively regulate the exosomal sorting of microRNAs.
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Células Endoteliais/metabolismo , Exossomos/metabolismo , Ribonucleoproteínas Nucleares Heterogêneas Grupo A-B/metabolismo , MicroRNAs/metabolismo , Anexina A2/metabolismo , Células Endoteliais da Veia Umbilical Humana , Humanos , Transporte de RNARESUMO
Increasing evidence suggests the potential role of extracellular vesicles (EVs) in many lung diseases. According to their subcellular origin, secretion mechanism, and size, EVs are currently classified into three subpopulations: exosomes, microvesicles, and apoptotic bodies. Exosomes are released in most biofluids, including airway fluids, and play a key role in intercellular communication via the delivery of their cargo (e.g., microRNAs (miRNAs)) to target cell. In a physiological context, lung exosomes present protective effects against stress signals which allow them to participate in the maintenance of lung homeostasis. The presence of air pollution alters the composition of lung exosomes (dysregulation of exosomal miRNAs) and their homeostatic property. Indeed, besides their potential as diagnostic biomarkers for lung diseases, lung exosomes are functional units capable of dysregulating numerous pathophysiological processes (including inflammation or fibrosis), resulting in the promotion of lung disease progression. Here, we review recent studies on the known and potential role of lung exosomes/exosomal miRNAs, in the maintaining of lung homeostasis on one hand, and in promoting lung disease progression on the other. We will also discuss using exosomes as prognostic/diagnostic biomarkers as well as therapeutic tools for lung diseases.
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SSc is a rare disease of unknown origin associated with multiple organ involvement. One of the major complications that drives the mortality of SSc patients is interstitial lung disease. The course of SSc-interstitial lung disease progression has a wide spectrum. Since the treatment is based on aggressive immunosuppression it should not be given to stable or non-progressing disease. The correct identification of disease with high risk of progression remains a challenge for early therapeutic intervention, and biomarkers remain urgently needed. In fact, eight categories of biomarkers have been identified and classified according to the different biological pathways involved. The purpose of this article is to describe the main biomarkers thought to be of interest with clinical value in the diagnosis and prognosis of SSc-interstitial lung disease.
Assuntos
Biomarcadores/sangue , Doenças Pulmonares Intersticiais/diagnóstico , Doenças Pulmonares Intersticiais/etiologia , Escleroderma Sistêmico/complicações , Escleroderma Sistêmico/diagnóstico , Proteínas de Fase Aguda/metabolismo , Fator de Crescimento do Tecido Conjuntivo/sangue , Citocinas/sangue , Progressão da Doença , Humanos , Metaloproteinases da Matriz/sangue , Mucina-1/sangue , Prognóstico , Proteína A Associada a Surfactante Pulmonar/sangue , Proteína D Associada a Surfactante Pulmonar/sangueRESUMO
Idiopathic pulmonary fibrosis (IPF) is a progressive fibrosing interstitial lung disease of unknown aetiology which leads rapidly to death. As diagnosis of IPF is complex, we aimed to characterise microRNA (miRNA) content of exosomes from sputum of patients with IPF. Using miRNA quantitative PCR array, we found a substantial dysregulation of sputum exosomal miRNA levels between patients with IPF and healthy subjects and identified a unique signature of three miRNAs. Interestingly, we found a negative correlation between miR-142-3p and diffusing capacity of the lungs for carbon monoxide/alveolar volume. This is the first characterisation of miRNA content of sputum-derived exosomes in IPF that identified promising biomarkers for diagnosis and disease severity.
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Exossomos/metabolismo , Fibrose Pulmonar Idiopática/diagnóstico , Fibrose Pulmonar Idiopática/metabolismo , MicroRNAs/metabolismo , Escarro/metabolismo , Biomarcadores/metabolismo , Estudos de Casos e Controles , Feminino , Humanos , Fibrose Pulmonar Idiopática/etiologia , Masculino , Sensibilidade e EspecificidadeRESUMO
The development of new lymphatic vessels occurs in many cancerous and inflammatory diseases through the binding of VEGF-C to its receptors, VEGFR-2 and VEGFR-3. The regulation of VEGFR-2/VEGFR-3 heterodimerisation and its downstream signaling in lymphatic endothelial cells (LECs) remain poorly understood. Here, we identify the endocytic receptor, uPARAP, as a partner of VEGFR-2 and VEGFR-3 that regulates their heterodimerisation. Genetic ablation of uPARAP leads to hyperbranched lymphatic vasculatures in pathological conditions without affecting concomitant angiogenesis. In vitro, uPARAP controls LEC migration in response to VEGF-C but not VEGF-A or VEGF-CCys156Ser. uPARAP restricts VEGFR-2/VEGFR-3 heterodimerisation and subsequent VEGFR-2-mediated phosphorylation and inactivation of Crk-II adaptor. uPARAP promotes VEGFR-3 signaling through the Crk-II/JNK/paxillin/Rac1 pathway. Pharmacological Rac1 inhibition in uPARAP knockout mice restores the wild-type phenotype. In summary, our study identifies a molecular regulator of lymphangiogenesis, and uncovers novel molecular features of VEGFR-2/VEGFR-3 crosstalk and downstream signaling during VEGF-C-driven LEC sprouting in pathological conditions.
Assuntos
Linfangiogênese , Glicoproteínas de Membrana/metabolismo , Receptores de Superfície Celular/metabolismo , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/metabolismo , Receptor 3 de Fatores de Crescimento do Endotélio Vascular/metabolismo , Animais , Linhagem Celular Tumoral , Dimerização , Células Endoteliais/metabolismo , Células Endoteliais/patologia , Feminino , Humanos , Vasos Linfáticos/metabolismo , Vasos Linfáticos/patologia , Masculino , Glicoproteínas de Membrana/genética , Camundongos , Receptores de Superfície Celular/genética , Transdução de Sinais , Fator C de Crescimento do Endotélio Vascular/metabolismo , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/química , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/genética , Receptor 3 de Fatores de Crescimento do Endotélio Vascular/química , Receptor 3 de Fatores de Crescimento do Endotélio Vascular/genéticaRESUMO
Exosomes are small extracellular vesicles of around 100 nm of diameter produced by most cell types. These vesicles carry nucleic acids, proteins, lipids, and other biomolecules and function as carriers of biological information in processes of extracellular communication. The content of exosomes is regulated by the external and internal microenvironment of the parent cell, but the intrinsic mechanisms of loading of molecules into exosomes are still not completely elucidated. In this study, by the use of next-generation sequencing we have characterized in depth the RNA composition of healthy endothelial cells and exosomes and provided an accurate profile of the different coding and noncoding RNA species found per compartment. We have also discovered a set of unique genes preferentially included (or excluded) into vesicles. Moreover, after studying the enrichment of RNA motifs in the genes unequally distributed between cells and exosomes, we have detected a set of enriched sequences for several classes of RNA. In conclusion, our results provide the basis for studying the involvement of RNA-binding proteins capable of recognizing RNA sequences and their role in the export of RNAs into exosomes.
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Exossomos/metabolismo , RNA/classificação , Sequência de Bases , Exossomos/genética , Sequenciamento de Nucleotídeos em Larga Escala , Células Endoteliais da Veia Umbilical Humana/metabolismo , Humanos , Motivos de Nucleotídeos/genética , RNA/genética , RNA/metabolismo , Análise de Sequência de RNARESUMO
Exosomes are communication mediators participating in the intercellular exchange of proteins, metabolites and nucleic acids. Recent studies have demonstrated that exosomes are characterized by a unique proteomic composition that is distinct from the cellular one. The mechanisms responsible for determining the proteome content of the exosomes remain however obscure. In the current study we employ ultrastructural approach to validate a novel exosomal protein myoferlin. This is a multiple C2-domain containing protein, known for its conserved physiological function in endocytosis and vesicle fusion biology. Emerging studies demonstrate that myoferlin is frequently overexpressed in cancer, where it promotes cancer cell migration and invasion. Our data expand these findings by showing that myoferlin is a general component of cancer cell derived exosomes from different breast and pancreatic cancer cell lines. Using proteomic analysis, we demonstrate for the first time that myoferlin depletion in cancer cells leads to a significantly modulated exosomal protein load. Such myoferlin-depleted exosomes were also functionally deficient as shown by their reduced capacity to transfer nucleic acids to human endothelial cells (HUVEC). Beyond this, myoferlin-depleted cancer exosomes also had a significantly reduced ability to induce migration and proliferation of HUVEC. The present study highlights myoferlin as a new functional player in exosome biology, calling for novel strategies to target this emerging oncogene in human cancer.
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Neoplasias da Mama/metabolismo , Proteínas de Ligação ao Cálcio/metabolismo , Exossomos/metabolismo , Proteínas de Membrana/metabolismo , Proteínas Musculares/metabolismo , Neoplasias Pancreáticas/metabolismo , Western Blotting , Neoplasias da Mama/genética , Neoplasias da Mama/ultraestrutura , Proteínas de Ligação ao Cálcio/genética , Movimento Celular , Proliferação de Células , Exossomos/genética , Exossomos/ultraestrutura , Feminino , Células Endoteliais da Veia Umbilical Humana/metabolismo , Humanos , Células MCF-7 , Masculino , Proteínas de Membrana/genética , Microscopia Eletrônica , Proteínas Musculares/genética , Neovascularização Fisiológica , Neoplasias Pancreáticas/genética , Neoplasias Pancreáticas/ultraestrutura , Mapas de Interação de Proteínas , Proteômica/métodos , Interferência de RNA , Transdução de Sinais , TransfecçãoRESUMO
Control of mRNA levels, a fundamental aspect in the regulation of gene expression, is achieved through a balance between mRNA synthesis and decay. E26-related gene (Erg) proteins are canonical transcription factors whose previously described functions are confined to the control of mRNA synthesis. Here, we report that ERG also regulates gene expression by affecting mRNA stability and identify the molecular mechanisms underlying this function in human cells. ERG is recruited to mRNAs via interaction with the RNA-binding protein RBPMS, and it promotes mRNA decay by binding CNOT2, a component of the CCR4-NOT deadenylation complex. Transcriptome-wide mRNA stability analysis revealed that ERG controls the degradation of a subset of mRNAs highly connected to Aurora signaling, whose decay during S phase is necessary for mitotic progression. Our data indicate that control of gene expression by mammalian transcription factors may follow a more complex scheme than previously anticipated, integrating mRNA synthesis and degradation.