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1.
Gene ; 766: 145117, 2021 Jan 15.
Artigo em Inglês | MEDLINE | ID: mdl-32920039

RESUMO

The extracellular vesicles (EVs) of uterine flushing fluids (UFs) mediate intrauterine communication between conceptus and uterus in pigs. The small RNAs of UFs-EVs are widely recognized as important factors that influence embryonic implantation. However, small RNAs expression profiles of porcine UFs-EVs during peri-implantation are still unknown. In this study, cup-shaped EVs of porcine UFs on days 10 (D10), 13 (D13) and 18 (D18) of pregnancy were isolated and characterized. The expression of small RNAs in these EVs was comprehensively profiled through sequencing. A total of 152 known microRNAs (miRNAs), 43 novel miRNAs, 6248 known Piwi-interacting RNAs (piRNAs) and 110 novel piRNAs were identified. Among these small RNAs, RT-qRCR results indicated that ssc-let-7f-5p, ssc-let-7i-5p and ssc-let-7g were differentially expressed during the three stages. Bioinformatics analysis showed that the miRNAs differentially expressed in the three comparisons (D10 vs D13, D13 vs D18 and D10 vs D18) were involved in important processes and pathways related to immunization, endometrial receptivity and embryo development, which play important roles in embryonic implantation. Our results reveal that EVs from porcine UFs contain various small RNAs with potentially vital effects on implantation. This research also provides resources for studies of miRNAs and piRNAs in the cross-talk between embryo and endometrium.


Assuntos
Implantação do Embrião/genética , Vesículas Extracelulares/genética , MicroRNAs/genética , Útero/fisiologia , Animais , Desenvolvimento Embrionário/genética , Endométrio/fisiologia , Feminino , Gravidez , RNA Interferente Pequeno/genética , RNA-Seq/métodos , Suínos
2.
Cell Prolif ; 53(11): e12877, 2020 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-33169503

RESUMO

Cells utilize different means of inter-cellular communication to function properly. Here, we review the crosstalk between cancer cells and their surrounding environment through microRNA (miRNA)-containing extracellular vesicles (EVs). The current findings suggest that the export of miRNAs and uptake of miRNA-containing EVs might be an active process. As post-transcriptional regulators of gene expression, cancer-derived miRNAs that are taken up by normal cells can change the translational profile of the recipient cell towards a transformed proteome. Stromal cells can also deliver miRNAs via EVs to cancer cells to support tumour growth and cancer progression. Therefore, gaining a better understanding of EV-mediated inter-cellular communication in the tumour microenvironment might lead to the development of novel diagnostic and therapeutic strategies.


Assuntos
Vesículas Extracelulares/genética , MicroRNAs/genética , Neoplasias/genética , Animais , Comunicação Celular , Vesículas Extracelulares/patologia , Regulação Neoplásica da Expressão Gênica , Humanos , Neoplasias/patologia , Microambiente Tumoral
3.
PLoS Negl Trop Dis ; 14(9): e0008618, 2020 09.
Artigo em Inglês | MEDLINE | ID: mdl-32986706

RESUMO

Schistosomes are the causative agent of schistosomiasis, which affects more than 200 million people worldwide. Unlike other trematode parasites, schistosomes (along with the Didymozoidae) have evolved separate sexes. Pairing of males and females is a prerequisite for female sexual development and subsequent egg production. However, the mechanisms underlying these processes remain poorly understood. Extracellular vesicles (EVs) have been shown to play important roles in many biological processes. In the present study, we characterized EVs isolated from adult male and female Schistosoma japonicum. Proteomic analyses of the isolated EVs revealed that some proteins are significantly enriched in male or female EVs. RNA-sequencing analysis of a small RNA population associated with EVs identified 18 miRNAs enriched in male and female S. japonicum EVs. Among these, miR-750 was specifically enriched in female EVs. Additionally, the inhibition of miR-750 by a miRNA inhibitor led to decreased egg production in female schistosomes cultured in vitro. Collectively, our results suggest that miR-750 within female EV cargo may be involved in regulating ovary development and egg production in S. japonicum females.


Assuntos
Vesículas Extracelulares/genética , MicroRNAs/genética , Proteínas de Protozoários/genética , Proteínas de Protozoários/isolamento & purificação , Schistosoma japonicum/crescimento & desenvolvimento , Animais , Vesículas Extracelulares/metabolismo , Feminino , Masculino , Camundongos , Camundongos Endogâmicos BALB C , MicroRNAs/antagonistas & inibidores , Óvulo/efeitos dos fármacos , Óvulo/crescimento & desenvolvimento , Proteoma/genética , Coelhos , Schistosoma japonicum/genética , Esquistossomose/parasitologia
4.
Nat Commun ; 11(1): 4489, 2020 09 07.
Artigo em Inglês | MEDLINE | ID: mdl-32895384

RESUMO

We report a covalent chemistry-based hepatocellular carcinoma (HCC)-specific extracellular vesicle (EV) purification system for early detection of HCC by performing digital scoring on the purified EVs. Earlier detection of HCC creates more opportunities for curative therapeutic interventions. EVs are present in circulation at relatively early stages of disease, providing potential opportunities for HCC early detection. We develop an HCC EV purification system (i.e., EV Click Chips) by synergistically integrating covalent chemistry-mediated EV capture/release, multimarker antibody cocktails, nanostructured substrates, and microfluidic chaotic mixers. We then explore the translational potential of EV Click Chips using 158 plasma samples of HCC patients and control cohorts. The purified HCC EVs are subjected to reverse-transcription droplet digital PCR for quantification of 10 HCC-specific mRNA markers and computation of digital scoring. The HCC EV-derived molecular signatures exhibit great potential for noninvasive early detection of HCC from at-risk cirrhotic patients with an area under receiver operator characteristic curve of 0.93 (95% CI, 0.86 to 1.00; sensitivity = 94.4%, specificity = 88.5%).


Assuntos
Biomarcadores Tumorais/isolamento & purificação , Carcinoma Hepatocelular/diagnóstico , Detecção Precoce de Câncer/métodos , Vesículas Extracelulares/genética , Neoplasias Hepáticas/diagnóstico , Idoso , Biomarcadores Tumorais/genética , Carcinoma Hepatocelular/sangue , Carcinoma Hepatocelular/genética , Carcinoma Hepatocelular/patologia , Estudos de Casos e Controles , Química Click/instrumentação , Química Click/métodos , Química Computacional , Simulação por Computador , Diagnóstico Diferencial , Dimetilpolisiloxanos/química , Progressão da Doença , Detecção Precoce de Câncer/instrumentação , Feminino , Células Hep G2 , Humanos , Dispositivos Lab-On-A-Chip , Biópsia Líquida/instrumentação , Biópsia Líquida/métodos , Cirrose Hepática/sangue , Cirrose Hepática/patologia , Neoplasias Hepáticas/sangue , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/patologia , Masculino , Técnicas Analíticas Microfluídicas/instrumentação , Técnicas Analíticas Microfluídicas/métodos , Pessoa de Meia-Idade , Nanoestruturas/química , Nanofios/química , Estadiamento de Neoplasias , RNA Mensageiro/genética , RNA Mensageiro/isolamento & purificação , Curva ROC , Reação em Cadeia da Polimerase Via Transcriptase Reversa/instrumentação , Reação em Cadeia da Polimerase Via Transcriptase Reversa/métodos
5.
Anticancer Res ; 40(10): 5509-5516, 2020 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-32988874

RESUMO

BACKGROUND/AIM: Extracellular vesicles (EVs) can mediate drug resistance within the tumor microenvironment by delivering bioactive molecules, including proteins. Here, we performed a comparative proteomic analysis of EVs secreted by A549 lung cancer cells and their cisplatin-resistant counterparts in order to identify proteins involved in drug resistance. MATERIALS AND METHODS: Cells were co-cultivated using a transwell system to evaluate EV exchange. EVs were isolated by ultracentrifugation and analyzed using microscopy and nanoparticle tracking. EV proteome was analyzed by mass spectrometry. RESULTS: EV-mediated communication was observed between co-cultured A549 and A549/CDDP cells. EVs isolated from both cells were mainly exosome-like structures. Extracellular matrix components, cell adhesion proteins, complement factors, histones, proteasome subunits and membrane transporters were found enriched in the EVs released by cisplatin-resistant cells. CONCLUSION: Proteins identified in this work may have a relevant role in modulating the chemosensitivity of the recipient cells and could represent useful biomarkers to monitor cisplatin response in lung cancer.


Assuntos
Biomarcadores Tumorais/genética , Cisplatino/farmacologia , Neoplasias Pulmonares/tratamento farmacológico , Proteoma/genética , Células A549 , Cisplatino/efeitos adversos , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Resistencia a Medicamentos Antineoplásicos/genética , Exossomos/efeitos dos fármacos , Exossomos/genética , Vesículas Extracelulares/genética , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/patologia , Espectrometria de Massas , Proteômica/métodos , Microambiente Tumoral/efeitos dos fármacos
6.
PLoS One ; 15(8): e0236439, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32813744

RESUMO

Extracellular vesicles (EVs) in human blood are a potential source of biomarkers. To which extent anticoagulation affects their concentration, cellular origin and protein composition is largely unexplored. To study this, blood from 23 healthy subjects was collected in acid citrate dextrose (ACD), citrate or EDTA, or without anticoagulation to obtain serum. EVs were isolated by ultracentrifugation or by size-exclusion chromatography (SEC) for fluorescence-SEC. EVs were analyzed by micro flow cytometry, NTA, TEM, Western blot, and protein mass spectrometry. The plasma EV concentration was unaffected by anticoagulants, but serum contained more platelet EVs. The protein composition of plasma EVs differed between anticoagulants, and between plasma and serum. Comparison to other studies further revealed that the shared EV protein composition resembles the "protein corona" of synthetic nanoparticles incubated in plasma or serum. In conclusion, we have validated a higher concentration of platelet EVs in serum than plasma by contemporary EV methods. Anticoagulation should be carefully described (i) to enable study comparison, (ii) to utilize available sample cohorts, and (iii) when preparing/selecting biobank samples. Further, the similarity of the EV protein corona and that of nanoparticles implicates that EVs carry both intravesicular and extravesicular cargo, which will expand their applicability for biomarker discovery.


Assuntos
Biomarcadores/sangue , Proteínas Sanguíneas/isolamento & purificação , Vesículas Extracelulares/genética , Proteoma/genética , Adulto , Plaquetas/química , Proteínas Sanguíneas/genética , Feminino , Citometria de Fluxo/métodos , Voluntários Saudáveis , Humanos , Masculino , Espectrometria de Massas/métodos , Pessoa de Meia-Idade
7.
PLoS One ; 15(7): e0235611, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32634139

RESUMO

Liquid biopsy has become widely applied in clinical medicine along with the progress in innovative technologies, such as next generation sequencing, but the origin of circulating tumor DNA (ctDNA) has not yet been precisely established. We reported bimodal peaks of long fragment circulating free DNA (cfDNA) of 5 kb and short fragment cfDNA of 170 bp in patients with advanced lung cancer, and both contained ctDNA. In this paper, we demonstrate that the total amount of cfDNA is higher when patients with lung cancer have extrathoracic metastases, and the amount of long fragment cfDNA is significantly higher in those patients. To investigate the origin of long fragment cfDNA, conditioned media isolated from lung cancer cell lines was fractionated. Long fragment cfDNA was found concomitant with extracellular vesicles (EVs), but short fragment cfDNA was not observed in any fractions. However, in peripheral blood from a metastatic animal model both fragments were detected even with those same lung cancer cell lines. In human plasma samples, long fragment cfDNA was observed in the same fraction as that from conditioned media, and short fragment cfDNA existed in the supernatant after centrifugation at 100,000g. Concentration of ctDNA in the supernatant was two times higher than that in plasma isolated by the conventional procedure. Long fragment cfDNA associated with tumor progression might therefore be released into peripheral blood, and it is possible that the long fragment cfDNA escapes degradation by co-existing with EVs. Examination of the biological characteristics of long fragment cfDNA is a logical subject of further investigation.


Assuntos
DNA Tumoral Circulante/análise , Neoplasias Pulmonares/diagnóstico , Animais , Estudos de Casos e Controles , Linhagem Celular Tumoral , DNA Tumoral Circulante/química , Meios de Cultivo Condicionados/metabolismo , Modelos Animais de Doenças , Receptores ErbB/genética , Vesículas Extracelulares/genética , Vesículas Extracelulares/metabolismo , Frequência do Gene , Humanos , Biópsia Líquida , Pneumopatias/sangue , Pneumopatias/patologia , Neoplasias Pulmonares/sangue , Neoplasias Pulmonares/patologia , Camundongos , Camundongos Knockout , Mutação , Metástase Neoplásica , Transplante Heterólogo
8.
Am J Physiol Cell Physiol ; 319(2): C419-C431, 2020 08 01.
Artigo em Inglês | MEDLINE | ID: mdl-32639875

RESUMO

Small noncoding microRNAs (miRNAs) are important regulators of skeletal muscle size, and circulating miRNAs within extracellular vesicles (EVs) may contribute to atrophy and its associated systemic effects. The purpose of this study was to understand how muscle atrophy and regrowth alter in vivo serum EV miRNA content. We also associated changes in serum EV miRNA with protein synthesis, protein degradation, and miRNA within muscle, kidney, and liver. We subjected adult (10 mo) F344/BN rats to three conditions: weight bearing (WB), hindlimb suspension (HS) for 7 days to induce muscle atrophy, and HS for 7 days followed by 7 days of reloading (HSR). Microarray analysis of EV miRNA content showed that the overall changes in serum EV miRNA were predicted to target major anabolic, catabolic, and mechanosensitive pathways. MiR-203a-3p was the only miRNA demonstrating substantial differences in HS EVs compared with WB. There was a limited association of EV miRNA content to the corresponding miRNA content within the muscle, kidney, or liver. Stepwise linear regression demonstrated that EV miR-203a-3p was correlated with muscle mass and muscle protein synthesis and degradation across all conditions. Finally, EV miR-203a-3p expression was significantly decreased in human subjects who underwent unilateral lower limb suspension (ULLS) to induce muscle atrophy. Altogether, we show that serum EV miR-203a-3p expression is related to skeletal muscle protein turnover and atrophy. We suggest that serum EV miR-203a-3p content may be a useful biomarker and future work should investigate whether serum EV miR-203a-3p content is mechanistically linked to protein synthesis and degradation.


Assuntos
MicroRNAs/genética , Músculo Esquelético/metabolismo , Atrofia Muscular/genética , Transtornos Musculares Atróficos/genética , Animais , Biomarcadores/metabolismo , Vesículas Extracelulares/genética , Elevação dos Membros Posteriores , Humanos , Rim/metabolismo , Fígado/metabolismo , Análise em Microsséries , Proteínas Musculares/genética , Atrofia Muscular/metabolismo , Atrofia Muscular/patologia , Transtornos Musculares Atróficos/metabolismo , Transtornos Musculares Atróficos/patologia , Ratos
9.
Sci Rep ; 10(1): 9414, 2020 06 10.
Artigo em Inglês | MEDLINE | ID: mdl-32523124

RESUMO

Infiltrative tumor growth into adjacent soft tissues is a major cause of the frequent recurrence and tumor-related death of myxofibrosarcoma (MFS), but no useful biomarkers reflecting tumor burden and infiltrative growth are available. While emerging evidence suggests a diagnostic and functional role of extracellular/circulating microRNA (miRNA) in various malignant diseases, their significance in MFS patients remains unknown. Global miRNA profiling identified four upregulated miRNAs in MFS patient sera and culture media of MFS cells. Among these, serum miR-1260b level was significantly upregulated in patient serum discriminating from healthy individuals and closely correlated with clinical status and tumor dynamics in MFS-bearing mice. In addition, high miR-1260b expression in serum was correlated with radiological tail-like patterns, characteristic of the infiltrative MFS. The extracellular miR-1260b was embedded in tumor-derived extracellular vesicles (EVs) and promoted cellular invasion of MFS through the downregulation of PCDH9 in the adjacent normal fibroblasts. Collectively, circulating miR-1260b expression may represent a novel diagnostic target for tumor monitoring of this highly aggressive sarcoma. Moreover, EV-miR-1260b could act as a transfer messenger to adjacent cells and mediate the infiltrative growth of MFS, providing new insights into the mechanism of infiltrative nature via crosstalk between tumor cells and their microenvironment.


Assuntos
MicroRNA Circulante/genética , Fibrossarcoma/genética , MicroRNAs/genética , Transcriptoma/genética , Idoso , Idoso de 80 Anos ou mais , Animais , Biomarcadores Tumorais/genética , Linhagem Celular Tumoral , Regulação para Baixo/genética , Vesículas Extracelulares/genética , Feminino , Fibroblastos/patologia , Perfilação da Expressão Gênica/métodos , Histiocitoma Fibroso Maligno/genética , Humanos , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Pessoa de Meia-Idade , Recidiva Local de Neoplasia/genética , Neoplasias de Tecidos Moles/genética , Microambiente Tumoral/genética , Regulação para Cima/genética
10.
DNA Cell Biol ; 39(9): 1486-1493, 2020 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-32551866

RESUMO

Extracellular vesicles (EVs) have been known to carry multiple bioactive molecules, including lipids, mRNA/miRNA, and proteins. However, recent studies show that specific DNAs are also packed into EVs secreted by various cells, which are considered as powerful markers for diagnosis and prognosis of disease. DNAs in EVs are derived from parental cells, representing the mutation and even spanning of the whole genomic DNA of parental cells. Interestingly, increasing numbers of studies have found that the genetic materials in different EVs are not only universal but also random and different, which may be related to the size of EVs. In this review, we discuss the different characteristics of DNAs in EVs and the rules of their variation. We hope our review will trigger the continuing exploration of the origins, characteristics, and variations of DNAs in EVs.


Assuntos
DNA/genética , Vesículas Extracelulares/genética , Neoplasias/genética , Animais , Vesículas Extracelulares/metabolismo , Humanos , Mutação
11.
Sci Rep ; 10(1): 7713, 2020 05 07.
Artigo em Inglês | MEDLINE | ID: mdl-32382024

RESUMO

Extracellular vesicles secreted from tumor cells are functional vehicles capable of contributing to intercellular communication and metastasis. A growing number of studies have focused on elucidating the role that tumor-derived extracellular vesicles play in spreading pancreatic cancer to other organs, due to the highly metastatic nature of the disease. We recently showed that small extracellular vesicles secreted from pancreatic cancer cells could initiate malignant transformation of healthy cells. Here, we analyzed the protein cargo contained within these vesicles using mass spectrometry-based proteomics to better understand their makeup and biological characteristics. Three different human pancreatic cancer cell lines were compared to normal pancreatic epithelial cells revealing distinct differences in protein cargo between cancer and normal vesicles. Vesicles from cancer cells contain an enrichment of proteins that function in the endosomal compartment of cells responsible for vesicle formation and secretion in addition to proteins that have been shown to contribute to oncogenic cell transformation. Conversely, vesicles from normal pancreatic cells were shown to be enriched for immune response proteins. Collectively, results contribute to what we know about the cargo contained within or excluded from cancer cell-derived extracellular vesicles, supporting their role in biological processes including metastasis and cancer progression.


Assuntos
Transformação Celular Neoplásica/genética , Neoplasias Pancreáticas/genética , Proteômica , Microambiente Tumoral/genética , Comunicação Celular/genética , Linhagem Celular Tumoral , Exossomos/genética , Vesículas Extracelulares/genética , Vesículas Extracelulares/patologia , Perfilação da Expressão Gênica , Regulação Neoplásica da Expressão Gênica/genética , Humanos , Neoplasias Pancreáticas/patologia
12.
Clin Sci (Lond) ; 134(8): 985-999, 2020 04 30.
Artigo em Inglês | MEDLINE | ID: mdl-32297634

RESUMO

Small extracellular vesicles (sEVs) as natural membranous vesicles are on the frontiers of nanomedical research, due to their ability to deliver therapeutic molecules such as microRNAs (miRNAs). The miRNA-21 (miR-21) is thought to be involved in the initiation and development of myocardial infarction (MI). Here, we examined whether miR-21 regulation using human peripheral blood-derived sEVs (PB-sEVs) could serve as a potential therapeutic strategy for MI. First, we examined miR-21 levels in hypoxic conditions and validated the ability of PB-sEVs to serve as a potential delivery system for miRNAs. Further, bioinformatics analysis and luciferase assay were performed to identify target genes of miR-21 mechanistically. Among numerous target pathways, we focused on nitrogen metabolism, which remains relatively unexplored compared with other possible miR-21-mediated pathways; hence, we aimed to determine novel target genes of miR-21 related to nitrogen metabolism. In hypoxic conditions, the expression of miR-21 was significantly up-regulated and correlated with nitric oxide synthase 3 (NOS3) levels, which in turn influences cardiac function. The down-regulation of miR-21 expression by PB-sEVs loaded with anti-miR-21 significantly improved survival rates, consistent with the augmentation of cardiac function. However, the up-regulation of miR-21 expression by PB-sEVs loaded with miR-21 reversed these effects. Mechanistically, miR-21 targeted and down-regulated the mRNA and protein expression of striatin (STRN), which could regulate NOS3 expression. In conclusion, we identified a novel therapeutic strategy to improve cardiac function by regulating the expression of miR-21 with PB-sEVs as an miR-21 or anti-miR-21 delivery vehicle and confirmed the miR-21-associated nitrogen metabolic disorders in MI.


Assuntos
Vesículas Extracelulares/química , MicroRNAs/genética , Infarto do Miocárdio/genética , Infarto do Miocárdio/terapia , Animais , Análise Química do Sangue , Vesículas Extracelulares/genética , Vesículas Extracelulares/metabolismo , Feminino , Terapia Genética , Humanos , Masculino , Camundongos Endogâmicos C57BL , MicroRNAs/administração & dosagem , MicroRNAs/metabolismo , Infarto do Miocárdio/metabolismo , Óxido Nítrico Sintase Tipo III/genética , Óxido Nítrico Sintase Tipo III/metabolismo
13.
Nat Commun ; 11(1): 1113, 2020 02 28.
Artigo em Inglês | MEDLINE | ID: mdl-32111843

RESUMO

Extracellular vesicles (EVs) form an endogenous transport system for intercellular transfer of biological cargo, including RNA, that plays a pivotal role in physiological and pathological processes. Unfortunately, whereas biological effects of EV-mediated RNA transfer are abundantly studied, regulatory pathways and mechanisms remain poorly defined due to a lack of suitable readout systems. Here, we describe a highly-sensitive CRISPR-Cas9-based reporter system that allows direct functional study of EV-mediated transfer of small non-coding RNA molecules at single-cell resolution. Using this CRISPR operated stoplight system for functional intercellular RNA exchange (CROSS-FIRE) we uncover various genes involved in EV subtype biogenesis that play a regulatory role in RNA transfer. Moreover we identify multiple genes involved in endocytosis and intracellular membrane trafficking that strongly regulate EV-mediated functional RNA delivery. Altogether, this approach allows the elucidation of regulatory mechanisms in EV-mediated RNA transfer at the level of EV biogenesis, endocytosis, intracellular trafficking, and RNA delivery.


Assuntos
Sistemas CRISPR-Cas , Vesículas Extracelulares/metabolismo , Pequeno RNA não Traduzido/metabolismo , Transporte Biológico , Comunicação Celular , Linhagem Celular , Endocitose/genética , Vesículas Extracelulares/genética , Fluorescência , Genes Reporter/genética , Células HEK293 , Humanos , RNA Guia/genética , RNA Guia/metabolismo , Pequeno RNA não Traduzido/genética
14.
PLoS One ; 15(3): e0229602, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32126572

RESUMO

AIM: This study analyzed microvesicles and exosomes, called as extracellular vesicles (EVs) excreted in serum and cerebrospinal fluid (CSF) from patients with cerebral or gestational toxoplasmosis. METHODS: Clinical samples from 83 individuals were divided into four groups. Group I, 20 sera from healthy individuals and pregnant women (seronegative for toxoplasmosis); group II, 21 sera from seropositive patients for toxoplasmosis (cerebral or gestational forms); group III, 26 CSF samples from patients with cerebral toxoplasmosis/HIV co-infection (CT/HIV) (seropositive for toxoplasmosis); and group IV, 16 CSF samples from seronegative patients for toxoplasmosis, but with HIV infection and other opportunistic infections (OI/HIV). Serum and CSF samples were ultracentrifuged to recover EVs. Next, vesicle size and concentration were characterized by Nanoparticle Tracking Analysis (NTA). RESULTS: Concentrations of serum-derived EVs from toxoplasmosis patients (mean: 2.4 x 1010 EVs/mL) were statically higher than of non-infected individuals (mean: 5.9 x 109 EVs/mL). Concentrations of CSF-derived EVs were almost similar in both groups. CT/HIV (mean: 2.9 x 109 EVs/mL) and OI/HIV (mean: 4.8 x 109 EVs/mL). Analyses by NTA confirmed that CSF-derived EVs and serum-derived EVs had size and shape similar to microvesicles and exosomes. The mean size of EVs was similar in serum and CSF. Thus, the concentration, and not size was able distinguish patients with toxoplasmosis than healthy individuals. Presence of exosomes was also confirmed by transmission electron microscopy and evidence of tetraspanins CD63 and CD9 in immunoblotting. Relative expressions of miR-146a-5p, miR-155-5p, miR-21-5p, miR-29c-3p and miR-125b-5p were estimated in exosomal miRNA extracted of EVs. Serum-derived EVs from group II (cerebral and gestational toxoplasmosis) up-expressed miR-125b-5p and miR-146a-5p. CSF-derived EVs from CT/HIV patients) up-expressed miR-155-5p and miR-21-5p and were unable to express miR-29c-3p. CONCLUSION: These data suggest the participation of EVs and exosomal miRNAs in unbalance of immune response as elevation of TNF-α, IL-6; and downregulation of IFN-γ in cerebral and gestational forms of toxoplasmosis.


Assuntos
Complicações Parasitárias na Gravidez/sangue , Complicações Parasitárias na Gravidez/líquido cefalorraquidiano , Toxoplasmose Cerebral/sangue , Toxoplasmose Cerebral/líquido cefalorraquidiano , Toxoplasmose/complicações , Micropartículas Derivadas de Células/genética , Micropartículas Derivadas de Células/patologia , Exossomos/genética , Exossomos/patologia , Vesículas Extracelulares/genética , Vesículas Extracelulares/patologia , Feminino , Expressão Gênica , Infecções por HIV/sangue , Infecções por HIV/líquido cefalorraquidiano , Infecções por HIV/complicações , Voluntários Saudáveis , Humanos , MicroRNAs/sangue , MicroRNAs/líquido cefalorraquidiano , MicroRNAs/genética , Microscopia Eletrônica de Transmissão , Gravidez , Complicações Parasitárias na Gravidez/genética , Toxoplasmose/sangue , Toxoplasmose/líquido cefalorraquidiano , Toxoplasmose Cerebral/genética
15.
Biochim Biophys Acta Gene Regul Mech ; 1863(7): 194519, 2020 07.
Artigo em Inglês | MEDLINE | ID: mdl-32142907

RESUMO

Extracellular vesicles (EVs) are released by cells from all kingdoms and represent one form of cell-cell interaction. This universal system of communication blurs cells type boundaries, offering an new avenue for pathogens to infect their hosts. EVs carry with them an arsenal of virulence factors that have been the focus of numerous studies. During the last years, the RNA content of EVs has also gained increasing attention, particularly in the context of infection. Secreted RNA in EVs (evRNA) from several bacterial pathogens have been characterised but the exact mechanisms promoting pathogenicity remain elusive. In this review, we evaluate the potential of such transcripts to directly interact with targets in infected cells and, by this, represent a novel angle of host cell manipulation during bacterial infection. This article is part of a Special Issue entitled: RNA and gene control in bacteria edited by Dr. M. Guillier and F. Repoila.


Assuntos
Bactérias/patogenicidade , Infecções Bacterianas/microbiologia , Vesículas Extracelulares/metabolismo , Interações Hospedeiro-Patógeno , RNA Bacteriano/genética , Animais , Bactérias/metabolismo , Infecções Bacterianas/metabolismo , Vesículas Extracelulares/genética , Humanos , RNA Bacteriano/metabolismo
16.
Biochim Biophys Acta Mol Basis Dis ; 1866(6): 165753, 2020 06 01.
Artigo em Inglês | MEDLINE | ID: mdl-32126269

RESUMO

BACKGROUND: Obstructive sleep apnea syndrome (OSAS) is associated to intermittent hypoxia (IH) and is an aggravating factor of non-alcoholic fatty liver disease (NAFLD). We investigated the effects of hypoxia in both in vitro and in vivo models of NAFLD. METHODS: Primary rat hepatocytes treated with free fatty acids (FFA) were subjected to chemically induced hypoxia (CH) using the hypoxia-inducible factor-1 alpha (HIF-1α) stabilizer cobalt chloride (CoCl2). Triglyceride (TG) content, mitochondrial superoxide production, cell death rates, cytokine and inflammasome components gene expression and protein levels of cleaved caspase-1 were assessed. Also, Kupffer cells (KC) were treated with conditioned medium (CM) and extracellular vehicles (EVs) from hypoxic fat-laden hepatic cells. The choline deficient L-amino acid defined (CDAA)-feeding model used to assess the effects of IH on experimental NAFLD in vivo. RESULTS: Hypoxia induced HIF-1α in cells and animals. Hepatocytes exposed to FFA and CoCl2 exhibited increased TG content and higher cell death rates as well as increased mitochondrial superoxide production and mRNA levels of pro-inflammatory cytokines and of inflammasome-components interleukin-1ß, NLRP3 and ASC. Protein levels of cleaved caspase-1 increased in CH-exposed hepatocytes. CM and EVs from hypoxic fat-laden hepatic cells evoked a pro-inflammatory phenotype in KC. Livers from CDAA-fed mice exposed to IH exhibited increased mRNA levels of pro-inflammatory and inflammasome genes and increased levels of cleaved caspase-1. CONCLUSION: Hypoxia promotes inflammatory signals including inflammasome/caspase-1 activation in fat-laden hepatocytes and contributes to cellular crosstalk with KC by release of EVs. These mechanisms may underlie the aggravating effect of OSAS on NAFLD. [Abstract word count: 257].


Assuntos
Subunidade alfa do Fator 1 Induzível por Hipóxia/genética , Hipóxia/genética , Proteína 3 que Contém Domínio de Pirina da Família NLR/genética , Hepatopatia Gordurosa não Alcoólica/genética , Apneia Obstrutiva do Sono/genética , Animais , Caspase 1/genética , Deficiência de Colina/genética , Deficiência de Colina/metabolismo , Deficiência de Colina/patologia , Cobalto/toxicidade , Modelos Animais de Doenças , Vesículas Extracelulares/genética , Vesículas Extracelulares/metabolismo , Vesículas Extracelulares/patologia , Ácidos Graxos não Esterificados/farmacologia , Regulação da Expressão Gênica/efeitos dos fármacos , Hepatócitos/metabolismo , Hepatócitos/patologia , Humanos , Hipóxia/induzido quimicamente , Hipóxia/metabolismo , Hipóxia/patologia , Inflamassomos/genética , Inflamação/induzido quimicamente , Inflamação/genética , Inflamação/metabolismo , Inflamação/patologia , Interleucina-1beta/genética , Macrófagos do Fígado/metabolismo , Macrófagos do Fígado/patologia , Camundongos , Hepatopatia Gordurosa não Alcoólica/complicações , Hepatopatia Gordurosa não Alcoólica/metabolismo , Hepatopatia Gordurosa não Alcoólica/patologia , Ratos , Apneia Obstrutiva do Sono/etiologia , Apneia Obstrutiva do Sono/metabolismo , Apneia Obstrutiva do Sono/patologia , Triglicerídeos/genética
17.
Nat Commun ; 11(1): 719, 2020 02 05.
Artigo em Inglês | MEDLINE | ID: mdl-32024826

RESUMO

Lipid overload results in lipid redistribution among metabolic organs such as liver, adipose, and muscle; therefore, the interplay between liver and other organs is important to maintain lipid homeostasis. Here, we show that liver responds to lipid overload first and sends hepatocyte-derived extracellular vesicles (EVs) targeting adipocytes to regulate adipogenesis and lipogenesis. Geranylgeranyl diphosphate synthase (Ggpps) expression in liver is enhanced by lipid overload and regulates EV secretion through Rab27A geranylgeranylation. Consistently, liver-specific Ggpps deficient mice have reduced fat adipose deposition. The levels of several EV-derived miRNAs in the plasma of non-alcoholic fatty liver disease (NAFLD) patients are positively correlated with body mass index (BMI), and these miRNAs enhance adipocyte lipid accumulation. Thus, we highlight an inter-organ mechanism whereby the liver senses different metabolic states and sends corresponding signals to remodel adipose tissue to adapt to metabolic changes in response to lipid overload.


Assuntos
Tecido Adiposo/metabolismo , Vesículas Extracelulares/metabolismo , Hepatócitos/metabolismo , Metabolismo dos Lipídeos/fisiologia , Fígado/metabolismo , Hepatopatia Gordurosa não Alcoólica/sangue , Adipócitos/metabolismo , Adipócitos/patologia , Tecido Adiposo/patologia , Animais , Índice de Massa Corporal , Dieta Hiperlipídica/efeitos adversos , Vesículas Extracelulares/genética , Farnesiltranstransferase/genética , Humanos , Lipogênese , Fígado/citologia , Fígado/efeitos dos fármacos , Masculino , Camundongos Endogâmicos C57BL , Camundongos Knockout , MicroRNAs/sangue , Complexos Multienzimáticos/genética , Hepatopatia Gordurosa não Alcoólica/genética , Proteínas rab27 de Ligação ao GTP/genética , Proteínas rab27 de Ligação ao GTP/metabolismo
18.
Int J Mol Sci ; 21(4)2020 Feb 11.
Artigo em Inglês | MEDLINE | ID: mdl-32054041

RESUMO

Extracellular vesicles (EVs) are secreted from most cell types and utilized in a complex network of near and distant cell-to-cell communication. Insight into this complex nanoscopic interaction in the development, progression and treatment of oral squamous cell carcinoma (OSCC) and precancerous oral mucosal disorders, termed oral potentially malignant disorders (OPMDs), remains of interest. In this review, we comprehensively present the current state of knowledge of EVs in OSCC and OPMDs. A systematic literature search strategy was developed and updated to December 17, 2019. Fifty-five articles were identified addressing EVs in OSCC and OPMDs with all but two articles published from 2015, highlighting the novelty of this research area. Themes included the impact of OSCC-derived EVs on phenotypic changes, lymph-angiogenesis, stromal immune response, mechanisms of therapeutic resistance as well as utility of EVs for drug delivery in OSCC and OPMD. Interest and progress of knowledge of EVs in OSCC and OPMD has been expanding on several fronts. The oral cavity presents a unique and accessible microenvironment for nanoparticle study that could present important models for other solid tumours.


Assuntos
Carcinoma de Células Escamosas/patologia , Vesículas Extracelulares/patologia , Neoplasias Bucais/patologia , Animais , Carcinogênese/genética , Carcinogênese/patologia , Carcinoma de Células Escamosas/genética , Carcinoma de Células Escamosas/terapia , Sistemas de Liberação de Medicamentos/métodos , Vesículas Extracelulares/genética , Humanos , MicroRNAs/genética , Neoplasias Bucais/genética , Neoplasias Bucais/terapia , Neovascularização Patológica/genética , Neovascularização Patológica/patologia , Neovascularização Patológica/terapia , Lesões Pré-Cancerosas/genética , Lesões Pré-Cancerosas/patologia , Lesões Pré-Cancerosas/terapia , Transcriptoma
19.
Sci Rep ; 10(1): 2860, 2020 02 18.
Artigo em Inglês | MEDLINE | ID: mdl-32071370

RESUMO

Colorectal cancer (CRC) is the most common type cancers in the world. CRC occurs sporadically in the majority of cases, indicating the predominant cause of the disease are environmental factors. Diet-induced changes in gut-microbiome are recently supposed to contribute on epidemics of CRC. This study was aimed to investigate the association of metagenomics and metabolomics in gut extracellular vesicles (EVs) of CRC and healthy subjects. A total of 40 healthy volunteers and 32 patients with CRC were enrolled in this study. Metagenomic profiling by sequencing 16 S rDNA was performed for assessing microbial codiversity. We explored the small molecule metabolites using gas chromatography-time-of-flight mass spectrometry. In total, stool EVs were prepared from 40 healthy volunteers and 32 patients with CRC. Metagenomic profiling demonstrated that bacterial phyla, particularly of Firmicutes and Proteobacteria, were significantly altered in patients with colorectal cancer. Through metabolomics profiling, we determined seven amino acids, four carboxylic acids, and four fatty acids; including short-chain to long chain fatty acids that altered in the disease group. Binary logistic regression was further tested to evaluate the diagnostic performance. In summary, the present findings suggest that gut flora dysbiosis may result in alternation of amino acid metabolism, which may be correlated with the pathogenesis of CRC.


Assuntos
Neoplasias Colorretais/diagnóstico , Vesículas Extracelulares/genética , Fezes/microbiologia , Metabolômica , Bactérias/classificação , Bactérias/genética , Neoplasias Colorretais/genética , Neoplasias Colorretais/microbiologia , Neoplasias Colorretais/patologia , Disbiose/genética , Disbiose/microbiologia , Disbiose/patologia , Microbioma Gastrointestinal/genética , Regulação Neoplásica da Expressão Gênica/genética , Humanos , Metagenoma/genética , Metagenômica
20.
PLoS One ; 15(2): e0229610, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32101583

RESUMO

Extracellular vesicles (EVs) have attracted increasing attention because of their potential roles in various biological processes and medical applications. However, isolation of EVs is technically challenging mainly due to their small and heterogeneous size and contaminants that are often co-isolated. We have thus designed a two-step magnetic bead-based (2MBB) method for isolation a subset of EVs as well as their microRNAs from samples of a limited amount. The process involves utilizing magnetic beads coated with capture molecules that recognize EV surface markers, such as CD63. Captured EVs could be eluted from beads or lyzed directly for subsequent analysis. In this study, we used a second set of magnetic beads coated with complementary oligonucleotides to isolate EV-associated microRNAs (EV-miRNAs). The efficiencies of 2MBB processes were assessed by reverse transcription-polymerase chain reaction (RT-PCR) with spiked-in exogenous cel-miR-238 molecules. Experimental results demonstrated the high efficiency in EV enrichment (74 ± 7%, n = 4) and miRNA extraction (91 ± 4%, n = 4). Transmission electron micrographs (TEM) and nanoparticle tracking analysis (NTA) show that captured EVs enriched by 2MBB method could be released and achieved a higher purity than the differential ultracentrifugation (DUC) method (p < 0.001, n = 3). As a pilot study, EV-miR126-3p and total circulating cell-free miR126-3p (cf-miR126-3p) in eight clinical plasma samples were measured and compared with the level of protein markers. Compared to cf-miR126-3p, a significant increase in correlations between EV-miR126-3p and cardiac troponin I (cTnI) and N-terminal propeptide of B-type natriuretic peptide (NT-proBNP) was detected. Furthermore, EV-miR126-3p levels in plasma samples from healthy volunteers (n = 18) and high-risk cardiovascular disease (CVD) patients (n = 10) were significantly different (p = 0.006), suggesting EV-miR126 may be a potential biomarker for cardiovascular diseases. 2MBB technique is easy, versatile, and provides an efficient means for enriching EVs and EV-associated nucleic acid molecules.


Assuntos
Doenças Cardiovasculares/genética , Vesículas Extracelulares/genética , Separação Imunomagnética/métodos , Biomarcadores/metabolismo , Doenças Cardiovasculares/metabolismo , Vesículas Extracelulares/metabolismo , Humanos , Fenômenos Magnéticos , MicroRNAs/análise , MicroRNAs/genética , MicroRNAs/isolamento & purificação , MicroRNAs/metabolismo , Projetos Piloto , Curva ROC
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