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1.
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
2.
Yonsei Med J ; 61(9): 750-761, 2020 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-32882759

RESUMO

PURPOSE: Gastric cancer (GC) is a malignant tumor with a high mortality rate. Drug resistance is a major obstacle to GC therapy. This study aimed to investigate the role and mechanism of exosomal circPRRX1 in doxorubicin resistance in GC. MATERIALS AND METHODS: HGC-27 and AGS cells were exposed to different doses of doxorubicin to construct doxorubicin-resistant cell lines. Levels of circPRRX1, miR-3064-5p, and nonreceptor tyrosine phosphatase 14 (PTPN14) were detected by quantitative real-time PCR or Western blot assay. Then, 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide, transwell, and Western blot assays were used to explore the function of circPRRX1 in GC cells. Interactions among circPRRX1, miR-3064-5p, and PTPN14 were confirmed by dual-luciferase reporter assay. The in vivo function of circPRRX1 was analyzed in a xenograft tumor model. RESULTS: CircPRRX1 was highly expressed in doxorubicin-resistant GC cell lines. Knockdown of circPRRX1 reversed doxorubicin resistance in doxorubicin-resistant GC cells. Additionally, extracellular circPRRX1 was carried by exosomes to spread doxorubicin resistance. CircPRRX1 silencing reduced doxorubicin resistance by targeting miR-3064-5p or regulating PTPN14. In GC patients, high levels of circPRRX1 in serum exosomes were associated with poor responses to doxorubicin treatment. Moreover, depletion of circPRRX1 reduced doxorubicin resistance in vivo. CONCLUSION: CircPRRX1 strengthened doxorubicin resistance by modulating miR-3064-5p/PTPN14 signaling and might be a therapeutic target for GC patients.


Assuntos
Doxorrubicina/farmacologia , Resistencia a Medicamentos Antineoplásicos/genética , MicroRNAs/genética , Neoplasias Gástricas/tratamento farmacológico , Neoplasias Gástricas/metabolismo , Linhagem Celular Tumoral , Exossomos/genética , Exossomos/metabolismo , Exossomos/patologia , Regulação Neoplásica da Expressão Gênica , Proteínas de Homeodomínio , Humanos , MicroRNAs/metabolismo , Proteínas Tirosina Fosfatases não Receptoras/metabolismo , Reação em Cadeia da Polimerase em Tempo Real , Transdução de Sinais/genética , Neoplasias Gástricas/genética , Neoplasias Gástricas/patologia
3.
PLoS One ; 15(8): e0237044, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32745122

RESUMO

Human norovirus (HuNoV) is a leading cause of acute gastroenteritis. Outbreaks normally occur via the fecal-oral route. HuNoV infection is thought to occur by viral particle transmission, but increasing evidence suggests a function for exosomes in HuNoV infection. HuNoV is contained within stool-derived exosomes, and exosome-associated HuNoV has been shown to replicate in human intestinal enteroids. In this study, we examine exosome-associated HuNoV infection of Vero cells and show that exosomes containing HuNoV may attach, infect, and be passaged in Vero cells. These findings support earlier findings and have implications for developing HuNoV disease intervention strategies.


Assuntos
Infecções por Caliciviridae/metabolismo , Infecções por Caliciviridae/transmissão , Exossomos/metabolismo , Animais , Infecções por Caliciviridae/genética , Criança , Pré-Escolar , Chlorocebus aethiops , Enterocolite/virologia , Exossomos/genética , Fezes/virologia , Feminino , Gastroenterite/virologia , Humanos , Masculino , Norovirus/patogenicidade , Células Vero , Vírion
4.
Life Sci ; 258: 118226, 2020 Oct 01.
Artigo em Inglês | MEDLINE | ID: mdl-32771555

RESUMO

AIM: Colorectal carcinoma (CRC) is one of the most prevalent cancers throughout the world. Circulating serum-derived microRNAs (miRNAs, miRs) can be used as non-invasive biomarkers for CRC diagnosis. This study aimed to identify a panel of six serum exosomal miRNAs as novel diagnostic biomarkers for CRC. MAIN METHODS: Exosomes were isolated and characterized from the conditioned media of the human colon adenocarcinoma cells (HCT-116 and Caco2). Sera were isolated from peripheral blood of 45 CRC and also 45 healthy individuals. The expression levels and diagnostic value of candidate circulating miRNAs (miR-19a, miR-20a, miR-150, miR-143, miR-145, and let-7a) were measured through quantitative real-time PCR. Receiver operating characteristic (ROC) curves were applied to evaluate the diagnostic accuracy of selected miRNAs. The association of candidate miRNAs and clinicopathological characteristics e.g. tumor node metastasis (TNM) staging and lymph node metastasis (LNM) were further evaluated. KEY FINDINGS: Circulating serum miR-19a, miR-20a, miR-150, and let-7a were significantly up-regulated in CRC patients, while miR-143 and miR-145 showed a significant down-regulation. The higher levels of miR-143 and miR-145 in patients with TNM stage I-II were detected, whereas miR-19a, miR-20a, miR-150, and let-7a were highly expressed in TNM stage III. The expression levels of miR-19a, miR-20a, and miR-150 were positively correlated with LNM status, while the expression levels of miR-143 and miR-145 were lower in patients with LNM. Area under the ROC curves of miR-19a, miR-20a, miR-150, miR-143, miR-145, and let-7a were 0.87, 0.83, 0.75, 0.76, 0.78 and 0.71, respectively. SIGNIFICANCE: We established a panel of six-circulating miRNA signature (i.e. miR-19a, miR-20a, miR-143, miR-145, miR-150, and let-7a) in serum as a non-invasive biomarker for CRC diagnosis. These findings confirm that serum-derived miRNAs have a strong potential to be a diagnostic biomarker for patients with CRC.


Assuntos
Biomarcadores Tumorais/sangue , Biomarcadores Tumorais/genética , Neoplasias Colorretais/sangue , Neoplasias Colorretais/genética , MicroRNAs/sangue , MicroRNAs/genética , Adulto , Idoso , Células CACO-2 , Exossomos/genética , Exossomos/metabolismo , Feminino , Redes Reguladoras de Genes/genética , Células HCT116 , Humanos , Masculino , Pessoa de Meia-Idade
5.
PLoS One ; 15(8): e0237023, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32785244

RESUMO

OBJECTIVE: Melanocytes play a central role in skin homeostasis. In this study, we focus on the function of melanocyte releasing exosomes as well as exosomal microRNAs (miRNAs) and investigate whether ultraviolet B (UVB) irradiation exerts an impact on it. MATERIALS AND METHODS: Exosomes derived from human primary melanocytes were isolated through differential centrifugation and were identified in three ways, including transmission electron microscopy observation, nanoparticle tracking analysis, and Western blot analysis. Melanocytes were irradiated with UVB for the indicated time, and then melanin production and exosome secretion were measured. The exosomal miRNA expression profile of melanocytes were obtained by miRNA sequencing and confirmed by real-time PCR. RESULTS: Exosomes derived from human primary melanocytes were verified. UVB irradiation induced melanin production and increased the exosome release by the melanocytes. In total, 15 miRNAs showed higher levels in UVB-irradiated melanocyte-derived exosomes compared with non-irradiated ones, and the top three upregulated exosomal miRNAs were miR-4488, miR-320d, and miR-7704 (fold change > 4.0). CONCLUSION: It is verified for the first time that UVB irradiation enhanced the secretion of exosomes by melanocytes and changed their exosomal miRNA profile. This findings open a new direction for investigating the communication between melanocytes and other skin cells, and the connection between UVB and skin malignant initiation.


Assuntos
Exossomos/genética , Melanócitos/metabolismo , Melanócitos/efeitos da radiação , Secreções Corporais/metabolismo , Exossomos/metabolismo , Humanos , Melaninas/análise , MicroRNAs/genética , Cultura Primária de Células , Transcriptoma/genética , Raios Ultravioleta/efeitos adversos
6.
Cell Prolif ; 53(10): e12890, 2020 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-32808361

RESUMO

OBJECTIVES: It is of profound significance for clinical bone regeneration to clarify the specific molecular mechanism from which we found that osteogenic differentiation of adipose-derived stem cells (ADSCs) will be probably promoted by exosomes. MATERIALS AND METHODS: By means of lentiviral transfection, miR-130a-3p overexpression and knockdown ADSCs were constructed. Alizarin Red S was used to detect the calcium deposits, and qPCR was used to detect osteogenesis-related genes, to verify the effect of miR-130a-3p on the osteogenic differentiation of ADSCs. CCK-8 was used to detect the effect of miR-130a-3p on the proliferation of ADSCs. The target binding between miR-130a-3p and SIRT7 was verified by dual-luciferase reporter gene assay. Furthermore, the role of Wnt signalling pathway in the regulation of ADSCs osteogenesis and differentiation by miR-130a-3p was further verified by detecting osteogenic-related genes and proteins and alkaline phosphatase activity. RESULTS: (a) Overexpression of miR-130a-3p can enhance the osteogenic differentiation of ADSCs while reducing protein and mRNA levels of SIRT7, a target of miR-130a-3p. (b) Our study further found that overexpression of miR-130a-3p leads to down-regulation of SIRT7 expression with up-regulation of Wnt signalling pathway-associated protein. (c) Overexpression of miR-130a-3p inhibited proliferation of ADSCs, while knockdown promoted it. CONCLUSIONS: The obtained findings indicate that exosomal miR-130a-3p can promote osteogenic differentiation of ADSCs partly by mediating SIRT7/Wnt/ß-catenin axis, which will hence promote the application of exosomal microRNA in the field of bone regeneration.


Assuntos
Exossomos/genética , Células-Tronco Mesenquimais/citologia , MicroRNAs/genética , Osteogênese , Sirtuínas/genética , Via de Sinalização Wnt , Diferenciação Celular , Células Cultivadas , Exossomos/metabolismo , Regulação da Expressão Gênica , Humanos , Células-Tronco Mesenquimais/metabolismo , MicroRNAs/metabolismo , Transdução de Sinais , Sirtuínas/metabolismo , Proteínas Wnt/genética , Proteínas Wnt/metabolismo , beta Catenina/genética , beta Catenina/metabolismo
7.
PLoS Genet ; 16(7): e1008901, 2020 07.
Artigo em Inglês | MEDLINE | ID: mdl-32645003

RESUMO

The RNA exosome is an evolutionarily-conserved ribonuclease complex critically important for precise processing and/or complete degradation of a variety of cellular RNAs. The recent discovery that mutations in genes encoding structural RNA exosome subunits cause tissue-specific diseases makes defining the role of this complex within specific tissues critically important. Mutations in the RNA exosome component 3 (EXOSC3) gene cause Pontocerebellar Hypoplasia Type 1b (PCH1b), an autosomal recessive neurologic disorder. The majority of disease-linked mutations are missense mutations that alter evolutionarily-conserved regions of EXOSC3. The tissue-specific defects caused by these amino acid changes in EXOSC3 are challenging to understand based on current models of RNA exosome function with only limited analysis of the complex in any multicellular model in vivo. The goal of this study is to provide insight into how mutations in EXOSC3 impact the function of the RNA exosome. To assess the tissue-specific roles and requirements for the Drosophila ortholog of EXOSC3 termed Rrp40, we utilized tissue-specific RNAi drivers. Depletion of Rrp40 in different tissues reveals a general requirement for Rrp40 in the development of many tissues including the brain, but also highlight an age-dependent requirement for Rrp40 in neurons. To assess the functional consequences of the specific amino acid substitutions in EXOSC3 that cause PCH1b, we used CRISPR/Cas9 gene editing technology to generate flies that model this RNA exosome-linked disease. These flies show reduced viability; however, the surviving animals exhibit a spectrum of behavioral and morphological phenotypes. RNA-seq analysis of these Drosophila Rrp40 mutants reveals increases in the steady-state levels of specific mRNAs and ncRNAs, some of which are central to neuronal function. In particular, Arc1 mRNA, which encodes a key regulator of synaptic plasticity, is increased in the Drosophila Rrp40 mutants. Taken together, this study defines a requirement for the RNA exosome in specific tissues/cell types and provides insight into how defects in RNA exosome function caused by specific amino acid substitutions that occur in PCH1b can contribute to neuronal dysfunction.


Assuntos
Doenças Cerebelares/genética , Proteínas do Citoesqueleto/genética , Drosophila melanogaster/genética , Complexo Multienzimático de Ribonucleases do Exossomo/genética , Proteínas do Tecido Nervoso/genética , Neurônios/metabolismo , Proteínas de Ligação a RNA/genética , Substituição de Aminoácidos/genética , Animais , Sistemas CRISPR-Cas/genética , Doenças Cerebelares/patologia , Cerebelo/metabolismo , Cerebelo/patologia , Modelos Animais de Doenças , Exossomos/genética , Humanos , Mutação/genética , Neurônios/patologia , RNA/genética
8.
Life Sci ; 257: 118017, 2020 Sep 15.
Artigo em Inglês | MEDLINE | ID: mdl-32603821

RESUMO

AIMS: Mesenchymal stem cell (MSC)-derived exosomes (MSCs-exos) regulate biological functions in different diseases, such as liver fibrosis, diabetes, and ischaemic heart injury. However, the function of MSC-derived exosomes on the intestinal barrier and the underlying mechanisms are poorly characterized. MAIN METHODS: The expression of miR-34a/c-5p, miR-29b-3p and Claudin-3 in human normal intestinal tissues and damaged intestinal tissues was evaluated by RT-qPCR. The effect of MSC-secreted exosomes on Claudins in Caco-2 cells was measured by using confocal microscopy, RT-qPCR and Western blot. Dual luciferase reporter assays and RNA immunoprecipitation (RIP) assays were performed to study the interaction between miR-34a/c-5p, miR-29b-3p and Snail. I/R-induced intestinal damage in rats was used to determine the in vivo effect of MSC-exos on intestinal barrier function. KEY FINDINGS: In this study, we found that miR-34a/c-5p, miR-29b-3p and Claudin-3 were downregulated in damaged human intestinal tissues. MSC-exos increased the expression of Claudin-3, Claudin-2 and ZO-1 in Caco-2 cells. Further studies demonstrated that MSC-exos promoted Claudin-3, Claudin-2 and ZO-1 expression in Caco-2 cells by Snail, which was targeted by miR-34a/c-5p and miR-29b-3p. In vivo experiments showed that MSC-derived exosomes could improve I/R-induced intestinal damage through the Snail/Claudins signaling pathway. SIGNIFICANCE: The findings here suggest a novel molecular basis for the therapy of intestinal barrier dysfunction.


Assuntos
Mucosa Intestinal/metabolismo , MicroRNAs/genética , Animais , Condrócitos/metabolismo , Claudinas/metabolismo , Exossomos/genética , Exossomos/metabolismo , Humanos , Intestinos/fisiologia , Masculino , Células-Tronco Mesenquimais/metabolismo , Células-Tronco Mesenquimais/fisiologia , MicroRNAs/fisiologia , Ratos , Ratos Sprague-Dawley , Traumatismo por Reperfusão/metabolismo , Transdução de Sinais , Fatores de Transcrição da Família Snail/metabolismo
9.
PLoS One ; 15(7): e0236126, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32667939

RESUMO

Pasteurization of donated human milk preserves it for storage and makes it safe for feeding, but at the expense of its composition, nutritional values and functions. Here, we aimed to investigate the impact of Holder Pasteurization (HoP) and High Pressure Processing (HPP) methods on miRNA in human milk and to evaluate impact of these changes on miRNA functions. Milk samples obtained from women in 50th day of lactation (n = 3) were subjected either to HoP, HPP or remained unpasteurized as a control. Subsequently, miRNA was isolated from whole material and exosomal fraction and sequenced with Illumina NextSeq 500. Sequencing data were processed, read counts were mapped to miRNA and analyzed both quantitatively with DESeq2 and functionally with DIANA mirPath v.3. While HPP caused statistically insignificant decrease in number of miRNA reads compared to unprocessed material, HoP led to 82-fold decrease in whole material (p = 0.0288) and 302-fold decrease in exosomes (p = 0.0021) not leaving enough reads for further analysis. Changes in composition of miRNA fraction before and after HPP indicated uneven stability of individual miRNAs under high pressure conditions, with miR-30d-5p identified as relatively stable and miR-29 family as sensitive to HPP. Interestingly, about 2/3 of unprocessed milk miRNA content consists of only 10 distinct miRNAs with miR-148a-3p at the top. Functional analysis of most abundant human milk miRNAs showed their involvement in signaling pathways, cell communication, proliferation and metabolism that are obviously important in rapidly growing infants. Functions of miRNAs which suffered the greatest depletion during HPP were similar to roles of the majority of unprocessed human milk's miRNA, which indicates that those functions may be weakened although not completely lost. Our findings indicate that HPP is less detrimental to human milk miRNAs than HoP and should be considered in further research on recommended processing procedures for human milk banks.


Assuntos
Exossomos/genética , MicroRNAs/análise , MicroRNAs/genética , Pasteurização/métodos , Pressão , Manejo de Espécimes/métodos , Adulto , Feminino , Perfilação da Expressão Gênica , Humanos , Leite Humano , Adulto Jovem
10.
Life Sci ; 257: 118092, 2020 Sep 15.
Artigo em Inglês | MEDLINE | ID: mdl-32681912

RESUMO

AIMS: Competing endogenous RNAs (ceRNAs) play essential roles in cancer pathogenesis and those in exosomes have been the promising biomarkers for cancer diagnose and therapy. We aim to identify potential active ceRNA pairs in cancer blood exosomes by combining TCGA and exoRBase. MAIN METHODS: Two strict screening criteria were implemented, including hypergeometric test on the targets predicted by RNA22 for differential miRNAs and Pearson test on the candidate mRNAs and lncRNAs for each cancer. Then2638292, 4925485 and 70669 ceRNAs in blood exosomes are available for colorectal cancer (CRC), hepatocellular carcinoma (HCC) and pancreatic adenocarcinoma (PAAD), respectively. KEY FINDINGS: A comprehensive functional analysis on differential miRNAs in cancer blood exosomes indicates that they play important roles in development of cancer by degrading or inhibiting the post-transcription translation level of mRNA or by acting as mediators to regulate the expression of mRNA. Topological and biological functional analysis of ceRNA networks demonstrate that hub ceRNAs involve in cancer-related biological pathways and processes, so as to influence the occurrence and development of cancer and would be the potential biomarkers for three cancers. Finally, we designed a web-accessible database, ExoceRNA Atlas (https://www.exocerna-atlas.com/exoceRNA#/) as a repository of ceRNAs in blood exosomes. It can friendly search, browse and visualize ceRNA networks of the query genes along with giving the detailed functional analysis results. The entire ceRNA data can also be freely downloaded. SIGNIFICANCE: ExoceRNA Atlas will serve as a powerful public resource for identifying ceRNAs and greatly deepen our understanding their functions in cancer exosomes.


Assuntos
Bases de Dados Genéticas , Exossomos/genética , Neoplasias/genética , RNA/genética , Humanos , MicroRNAs/genética , Neoplasias/patologia , RNA Longo não Codificante/genética , RNA Mensageiro/genética
11.
Nucleic Acids Res ; 48(15): 8509-8528, 2020 09 04.
Artigo em Inglês | MEDLINE | ID: mdl-32710631

RESUMO

The ribonucleolytic exosome complex is central for nuclear RNA degradation, primarily targeting non-coding RNAs. Still, the nuclear exosome could have protein-coding (pc) gene-specific regulatory activities. By depleting an exosome core component, or components of exosome adaptor complexes, we identify ∼2900 transcription start sites (TSSs) from within pc genes that produce exosome-sensitive transcripts. At least 1000 of these overlap with annotated mRNA TSSs and a considerable portion of their transcripts share the annotated mRNA 3' end. We identify two types of pc-genes, both employing a single, annotated TSS across cells, but the first type primarily produces full-length, exosome-sensitive transcripts, whereas the second primarily produces prematurely terminated transcripts. Genes within the former type often belong to immediate early response transcription factors, while genes within the latter are likely transcribed as a consequence of their proximity to upstream TSSs on the opposite strand. Conversely, when genes have multiple active TSSs, alternative TSSs that produce exosome-sensitive transcripts typically do not contribute substantially to overall gene expression, and most such transcripts are prematurely terminated. Our results display a complex landscape of sense transcription within pc-genes and imply a direct role for nuclear RNA turnover in the regulation of a subset of pc-genes.


Assuntos
Exossomos/genética , Genoma Humano/genética , Fases de Leitura Aberta/genética , RNA/genética , Sítio de Iniciação de Transcrição , Regulação da Expressão Gênica/genética , Células HeLa , Humanos , Anotação de Sequência Molecular , Estabilidade de RNA/genética , RNA Mensageiro/genética , RNA não Traduzido/genética
12.
Life Sci ; 258: 118094, 2020 Oct 01.
Artigo em Inglês | MEDLINE | ID: mdl-32673663

RESUMO

AIMS: Docosahexaenoic acid (DHA) as an omega 3 free fatty acid has been reported to exert anti-angiogenesis effects. However, our current understanding regarding the precise mechanisms of such effects is still limited. Exosomes secreted by cancer cells may act as angiogenesis promoters. The aim of the study was to determine altered expression levels of HIF-1α, TGF-ß, VEGFR, Snail1, Snail2 and SOX2 and their regulating microRNAs in MDA-MB-231 and BT-474 cell lines after treatment with DHA in both normoxic and hypoxic conditions. MAIN METHODS: Human breast cancer cell lines including MDA-MB-231 and BT-474 were treated for 24 h with 100 uM DHA under normoxic and hypoxic conditions. Exosomes were isolated from untreated and treated cells and characterized by transmission electron microscopy (TEM) and western blotting. RNAs from cells and isolated exosomes were extracted and cDNAs were synthesized. Expression levels of miRNAs and their pro-angiogenic target genes were analyzed using quantitative real-time PCR (qRT-PCR). KEY FINDINGS: We showed significant decrease in the expression of pro-angiogenic genes including HIF1-α, TGF-ß, SOX2, Snail1, Snail2 and VEGFR in cells and also their secreted exosomes after treatment with DHA in normoxic and hypoxic conditions. Also the expression levels of tumor suppressor miRs including miR-101, miR-199, miR-342 were increased and the expression levels of oncomiRs including mir-382 and miR-21 were decreased after treatment with DHA in cells and exosomes. SIGNIFICANCE: DHA can alter the expression of pro-angiogenic genes and microRNA contents in breast cancer cells and their derived-exosomes in favor of the inhibition of angiogenesis. Our data demonstrated new insight into DHA's anti-cancer action to target not only breast cancer cells but also their derived exosomes to suppress tumor angiogenesis.


Assuntos
Neoplasias da Mama/irrigação sanguínea , Neoplasias da Mama/genética , Ácidos Docosa-Hexaenoicos/farmacologia , Regulação para Baixo , Exossomos/genética , MicroRNAs/genética , Neovascularização Patológica/genética , Neoplasias da Mama/tratamento farmacológico , Neoplasias da Mama/ultraestrutura , Linhagem Celular Tumoral , Regulação para Baixo/efeitos dos fármacos , Regulação para Baixo/genética , Exossomos/efeitos dos fármacos , Exossomos/ultraestrutura , Feminino , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , MicroRNAs/metabolismo
14.
Cell Prolif ; 53(7): e12833, 2020 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-32525231

RESUMO

OBJECTIVES: The current study aimed to investigate the mechanism by which exosomes secreted by CHB patients with PNALT and liver inflammation grade (≥A2) affected the development of liver cancer. MATERIALS AND METHODS: Gene expression was assessed by RT-PCR, Western blotting and immunohistochemistry. CCK-8, colony formation, transwell, scratch-wound and flow cytometry assays were used to detect cell viability, proliferation, apoptosis and metastasis. The interaction of TCF21 and HHIP was assessed by co-immunoprecipitation assay. Luciferase reporter was used to detect the combination of TCF21/HHIP and miR-25-3p. Xenograft studies in nude mice manifested tumour growth ability of miR-25-3p. Bioinformatics analyses were conducted using TargetScan, EVmiRNA, TCGA, GEO, DAVID, COEXPEDIA, UALCAN, UCSC and the Human Protein Atlas databases. RESULTS: CHB-PNALT-Exo (≥A2) promoted the proliferation and metastasis of HepG2.2.15 cells. miR-25-3p was upregulated in CHB-PNALT-Exo (≥A2). miR-25-3p overexpression promoted cell proliferation and metastasis and was related to poor survival in patients with CHB-PNALT (≥A2). The cell proliferation- and metastasis-promoting functions of CHB-PNALT-Exo (≥A2) were abolished by miR-25-3p inhibitors. TCF21 directly interacted with HHIP. Inhibition of TCF21 or HHIP promoted cell proliferation and metastasis. Knockdown of TCF21 or HHIP counteracted the effects of CHB-PNALT-Exo (≥A2) containing miR-25-3p inhibitor on cell proliferation, metastasis and the expression of Ki67, E-cadherin and caspase-3/-9. CONCLUSIONS: Transfer of miR-25-3p by CHB-PNALT-Exo promoted the development of liver cancer by inhibiting the co-expression of TCF21 and HHIP.


Assuntos
Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Proteínas de Transporte/genética , Exossomos/genética , Hepatite B Crônica/genética , Neoplasias Hepáticas/genética , Fígado/patologia , Glicoproteínas de Membrana/genética , MicroRNAs/genética , Adulto , Animais , Apoptose/genética , Proliferação de Células/genética , Sobrevivência Celular , Progressão da Doença , Regulação para Baixo/genética , Exossomos/patologia , Feminino , Regulação Neoplásica da Expressão Gênica/genética , Hepatite B Crônica/patologia , Humanos , Inflamação/genética , Inflamação/patologia , Neoplasias Hepáticas/patologia , Masculino , Camundongos , Camundongos Nus , Pessoa de Meia-Idade , Regulação para Cima/genética , Adulto Jovem
15.
Life Sci ; 257: 118024, 2020 Sep 15.
Artigo em Inglês | MEDLINE | ID: mdl-32598931

RESUMO

AIMS: Cancer-derived exosomes carrying tumor-derived molecules such as miRNAs and proteins related to various phenotypes have been detected in both the bloodstream and other biofluids of patients with different cancers. Thus, our main purpose here was to determine the role of the exosomal microRNA-454 (miR-454) derived by MDA-MB-231 in self-renewal of cancer stem cells (CSCs) in ovarian cancer (OC). MATERIALS AND METHODS: Extraction of MDA-MB-231 cells-derived exosomes (231-derived exosomes) was conducted to treat CD44+/CD133+ SKOV3 and CoC1 cells to observe cell growth and stemness. Next, the differentially expressed miRNAs in SKOV3 cells after exosome treatment were filtered using microarray analysis. Subsequently, the cell viability was detected after reducing the exosomal miR-454 and the addition of a Wnt pathway inhibitor C59. Finally, the pro-tumorigenic function of exosomes on OC cells in vivo was investigated. KEY FINDINGS: After co-culture with 231-derived exosomes, the stemness of CSCs were promoted. Subsequently, the reduction of exosomal miR-454 weakened the roles of exosomes on cell stemness. Proline-rich transmembrane protein 2 (PRRT2) was substantiated as a target gene of miR-454 in SKOV3 and CoC1 cells. C59 reversed the repressive role of exosomes in stemness of CSCs. When being evaluated in a mouse model, exosomal miR-454 led to an efficacious effect in suppressing the tumor weight and volume in vivo. SIGNIFICANCE: Altogether, 231-derived exosomes carrying miR-454 disrupted the Wnt pathway by targeting PRRT2, thereby promoting CSC stemness in vitro and OC cell growth in vivo.


Assuntos
Neoplasias da Mama/genética , MicroRNAs/genética , Células-Tronco Neoplásicas/metabolismo , Neoplasias da Mama/patologia , Carcinoma Epitelial do Ovário/genética , Ciclo Celular , Linhagem Celular Tumoral , Movimento Celular/genética , Proliferação de Células/genética , Técnicas de Cocultura , Resistencia a Medicamentos Antineoplásicos/genética , Exossomos/genética , Exossomos/metabolismo , Feminino , Regulação Neoplásica da Expressão Gênica/genética , Humanos , Proteínas de Membrana/metabolismo , MicroRNAs/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Neoplasias Ovarianas/patologia , Via de Sinalização Wnt/genética , Proteína Wnt1/metabolismo , Proteína Wnt2/genética , Proteína Wnt2/metabolismo
16.
Anticancer Res ; 40(6): 3091-3096, 2020 06.
Artigo em Inglês | MEDLINE | ID: mdl-32487603

RESUMO

BACKGROUND/AIM: Exosomes are produced by normal and cancer cells. Exosomes are found in the serum of cancer patients and have been used for diagnosis and prognosis. Recently tears from non-cancer patients have been found to contain exosomes. In the present report we describe tears from advanced breast-cancer patients. MATERIALS AND METHODS: We found oncogenic miRNAs in the exosomes isolated from tear fluids obtained from five patients with metastatic breast cancer and compared them with tear exosomes form eight healthy volunteers. RESULTS: Tear exosomes had a significantly higher quantity of exosome markers than serum exosomes (CD9, CD63). Tear exosomes were subjected to quantitative reverse-transcription polymerase reaction (qRT-PCR), and western blot analysis to elucidate the status of miRNAs, previously reported in serum from patients with metastatic breast cancer. qRT-PCR and western-blot analysis revealed that breast-cancer-specific miR-21 and miR-200c were highly expressed in tear exosomes from metastatic breast cancer patients in contrast to tear exosomes from healthy volunteers. CONCLUSION: Tear exosomes can be a potential source of diagnostic and prognostic biomarkers for metastatic breast cancer, and possibly other cancers or diseases.


Assuntos
Neoplasias da Mama/genética , Carcinogênese/genética , Exossomos/genética , MicroRNAs/metabolismo , Adulto , Idoso , Neoplasias da Mama/patologia , Feminino , Humanos , Pessoa de Meia-Idade , Metástase Neoplásica
17.
Yakugaku Zasshi ; 140(5): 625-631, 2020.
Artigo em Japonês | MEDLINE | ID: mdl-32378662

RESUMO

Recently, mRNAs and microRNAs (miRNAs) have been identified in exosomes, which can be taken up by neighboring or distant cells. These exosomal-miRNAs may regulate gene expression in recipient cells. miRNAs are a type of non-coding RNA that induce post-transcriptional gene silencing of their target genes and regulate a wide range of biological processes, including apoptosis, differentiation, metabolism, and cell proliferation. According to recent reports, aberrant expression of miRNAs is associated with most pathological disease processes, including carcinogenesis. Therefore circulating onco-miRs are considered as significant therapeutic targets for cancer therapy. However, there is no report to regulate the function of miRNAs in exosomes. In this study, we developed novel drug delivery system using anti-exosome antibody-oligonucleotide conjugates (ExomiR-Tracker) for functional inhibition of circulating miRNAs. The "ExomiR-Tracker" is the world's first innovative molecule that has targeting property for exosome-recipient cells and specifically delivers nucleic acid medicines to the target cells. We found that ExomiR-Tracker can bind to the surface of exosomes and that the complexes are introduced into exosome-recipient cells then inhibit the activity of miRNA. We showed that ExomiR-Tracker can accumulate in cancerous tumors after intravenous administration. Existing technologies have difficulties for introducing anti-miR into exosomes and extremely low possibility to deliver anti-miR to exosome-recipient cells after intravenous administration. However, we successfully developed useful inhibition technology against exosomal-miRNA.


Assuntos
Sistemas de Liberação de Medicamentos , Exossomos , Imunoconjugados/administração & dosagem , MicroRNAs , Neoplasias/tratamento farmacológico , Ácidos Nucleicos/administração & dosagem , Sistemas de Liberação de Medicamentos/métodos , Desenho de Fármacos , Exossomos/genética , Expressão Gênica , Humanos , MicroRNAs/genética , Neoplasias/genética
18.
Nucleic Acids Res ; 48(12): 6839-6854, 2020 07 09.
Artigo em Inglês | MEDLINE | ID: mdl-32449937

RESUMO

SERRATE/ARS2 is a conserved RNA effector protein involved in transcription, processing and export of different types of RNAs. In Arabidopsis, the best-studied function of SERRATE (SE) is to promote miRNA processing. Here, we report that SE interacts with the nuclear exosome targeting (NEXT) complex, comprising the RNA helicase HEN2, the RNA binding protein RBM7 and one of the two zinc-knuckle proteins ZCCHC8A/ZCCHC8B. The identification of common targets of SE and HEN2 by RNA-seq supports the idea that SE cooperates with NEXT for RNA surveillance by the nuclear exosome. Among the RNA targets accumulating in absence of SE or NEXT are miRNA precursors. Loss of NEXT components results in the accumulation of pri-miRNAs without affecting levels of miRNAs, indicating that NEXT is, unlike SE, not required for miRNA processing. As compared to se-2, se-2 hen2-2 double mutants showed increased accumulation of pri-miRNAs, but partially restored levels of mature miRNAs and attenuated developmental defects. We propose that the slow degradation of pri-miRNAs caused by loss of HEN2 compensates for the poor miRNA processing efficiency in se-2 mutants, and that SE regulates miRNA biogenesis through its double contribution in promoting miRNA processing but also pri-miRNA degradation through the recruitment of the NEXT complex.


Assuntos
Proteínas de Arabidopsis/genética , MicroRNAs/genética , RNA Helicases/genética , Processamento Pós-Transcricional do RNA/genética , Proteínas de Ligação a RNA/genética , Arabidopsis/genética , Proteínas de Ligação ao Cálcio/genética , Núcleo Celular/genética , Exossomos/genética , Regulação da Expressão Gênica de Plantas/genética , Mutação/genética , Precursores de RNA/genética , Estabilidade de RNA/genética , Ribonuclease III/genética
19.
Cell Prolif ; 53(6): e12828, 2020 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-32391938

RESUMO

Exosomes, small extracellular vesicles ranging from 30 to 150 nm, are secreted by various cell types, including tumour cells. Recently, microRNAs (miRNAs) were identified to be encapsulated and hence protected from degradation within exosomes. These exosomal miRNAs can be horizontally transferred to target cells, in which they subsequently modulate biological processes. Increasing evidence indicates that exosomal miRNAs play a critical role in modifying the microenvironment of lung cancers, possibly facilitating progression, invasion, angiogenesis, metastasis and drug resistance. In this review, we summarize the novel findings on exosomal miRNA functions during lung cancer initiation and progression. In addition, we highlight their potential role and challenges as biomarkers in lung cancer diagnosis, prognosis and drug resistance and as therapeutic agents.


Assuntos
Exossomos/genética , Neoplasias Pulmonares/genética , MicroRNAs/fisiologia , Biomarcadores Tumorais , Proliferação de Células , Resistencia a Medicamentos Antineoplásicos , Humanos , Neoplasias Pulmonares/irrigação sanguínea , Neoplasias Pulmonares/diagnóstico , Neoplasias Pulmonares/patologia , Metástase Neoplásica , Neovascularização Patológica
20.
Life Sci ; 255: 117857, 2020 Aug 15.
Artigo em Inglês | MEDLINE | ID: mdl-32470446

RESUMO

AIMS: To explore the pro-metastatic role of exosomes derived from highly invasive pancreatic cancer cell and the associated aberrant expression of exosomal microRNAs (miRNAs). MAIN METHODS: Weakly invasive PC-1 cells were treated with exosomes of highly invasive PC-1.0 cells to determine the pro-metastatic effect of PC-1.0 derived exosomes. The exosomal miRNA profile was further investigated using high-throughput sequencing. The level of miR-125b-5p in highly and weakly invasive pancreatic cancer cells was further determined. Pancreatic cancer cells transfected with miR-125b-5p mimic and inhibitor were used to explore the effect of miR-125b-5p on migration, invasion and epithelial-to-mesenchymal transition (EMT). Treatment with PC-1.0 derived exosome and Western blot assay were performed to validate STARD13 as a target of exosomal miR-125b-5p in pancreatic cancer. KEY FINDINGS: PC-1.0 derived exosomes promoted the migration and invasion of weakly invasive PC-1 cells. miRNA sequencing revealed 62 miRNAs upregulated in PC-1.0 derived exosomes. miR-125b-5p most significantly promoted migration and invasion and was associated with metastasis in pancreatic cancer. Further, miR-125b-5p was upregulated in highly invasive pancreatic cancer cells and increased migration, invasion, and EMT. Moreover, its upregulation was associated with activation of MEK2/ERK2 signaling. The tumor suppressor STARD13 was directly targeted by miR-125b-5p in pancreatic cancer, which was associated with good prognosis and was suppressed by exosomes derived from highly invasive cancer cells. SIGNIFICANCE: This study explored the pro-metastatic role of exosomes derived from highly invasive pancreatic cancer cells and the associated aberrant expression of exosomal miRNAs, which may help to elucidate the metastatic mechanism of pancreatic cancer.


Assuntos
Exossomos/genética , Proteínas Ativadoras de GTPase/genética , MicroRNAs/genética , Neoplasias Pancreáticas/patologia , Proteínas Supressoras de Tumor/genética , Linhagem Celular Tumoral , Movimento Celular/genética , Transição Epitelial-Mesenquimal/genética , Humanos , Invasividade Neoplásica/genética , Metástase Neoplásica/genética , Neoplasias Pancreáticas/genética , Fenótipo , Regulação para Cima
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