RESUMEN
Poor reproducibility within and across studies arising from lack of knowledge regarding the performance of extracellular RNA (exRNA) isolation methods has hindered progress in the exRNA field. A systematic comparison of 10 exRNA isolation methods across 5 biofluids revealed marked differences in the complexity and reproducibility of the resulting small RNA-seq profiles. The relative efficiency with which each method accessed different exRNA carrier subclasses was determined by estimating the proportions of extracellular vesicle (EV)-, ribonucleoprotein (RNP)-, and high-density lipoprotein (HDL)-specific miRNA signatures in each profile. An interactive web-based application (miRDaR) was developed to help investigators select the optimal exRNA isolation method for their studies. miRDar provides comparative statistics for all expressed miRNAs or a selected subset of miRNAs in the desired biofluid for each exRNA isolation method and returns a ranked list of exRNA isolation methods prioritized by complexity, expression level, and reproducibility. These results will improve reproducibility and stimulate further progress in exRNA biomarker development.
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Ácidos Nucleicos Libres de Células/aislamiento & purificación , MicroARN Circulante/aislamiento & purificación , ARN/aislamiento & purificación , Adulto , Líquidos Corporales/química , Línea Celular , Vesículas Extracelulares/metabolismo , Femenino , Voluntarios Sanos , Humanos , Masculino , MicroARNs/aislamiento & purificación , MicroARNs/metabolismo , ARN/metabolismo , Reproducibilidad de los Resultados , Análisis de Secuencia de ARN/métodosRESUMEN
This study demonstrates the potential of using biological nanoparticles to deliver RNA therapeutics targeting programmed death-ligand 1 (PD-L1) as a treatment strategy for cholangiocarcinoma (CCA). RNA therapeutics offer prospects for intracellular immune modulation, but effective clinical translation requires appropriate delivery strategies. Milk-derived nanovesicles were decorated with epithelial cellular adhesion molecule (EpCAM) aptamers and used to deliver PD-L1 small interfering RNA (siRNA) or Cas9 ribonucleoproteins directly to CCA cells. In vitro, nanovesicle treatments reduced PD-L1 expression in CCA cells while increasing degranulation, cytokine release, and tumor cell cytotoxicity when tumor cells were co-cultured with T cells or natural killer cells. Similarly, immunomodulation was observed in multicellular spheroids that mimicked the tumor microenvironment. Combining targeted therapeutic vesicles loaded with siRNA to PD-L1 with gemcitabine effectively reduced tumor burden in an immunocompetent mouse CCA model compared with controls. This proof-of-concept study demonstrates the potential of engineered targeted nanovesicle platforms for delivering therapeutic RNA cargoes to tumors, as well as their use in generating effective targeted immunomodulatory therapies for difficult-to-treat cancers such as CCA.
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Antígeno B7-H1 , Colangiocarcinoma , Inmunoterapia , ARN Interferente Pequeño , Colangiocarcinoma/terapia , Colangiocarcinoma/metabolismo , Colangiocarcinoma/inmunología , Colangiocarcinoma/tratamiento farmacológico , Colangiocarcinoma/patología , Antígeno B7-H1/metabolismo , Antígeno B7-H1/antagonistas & inhibidores , Antígeno B7-H1/genética , Animales , Humanos , Ratones , Línea Celular Tumoral , Inmunoterapia/métodos , ARN Interferente Pequeño/genética , ARN Interferente Pequeño/administración & dosificación , Nanopartículas/química , Neoplasias de los Conductos Biliares/terapia , Neoplasias de los Conductos Biliares/patología , Neoplasias de los Conductos Biliares/metabolismo , Neoplasias de los Conductos Biliares/inmunología , Microambiente Tumoral/inmunología , Modelos Animales de Enfermedad , Desoxicitidina/análogos & derivados , Desoxicitidina/farmacología , GemcitabinaRESUMEN
Extracellular vesicles (EVs) are a heterogeneous group of biological nano-sized vesicles that are released from cells and contribute to intercellular communication. Emerging knowledge about their biogenesis, composition, release, and uptake has resulted in broad interest in elucidating their potential roles in disease pathophysiology. The distinct biological properties of these biological nanoparticles emphasize several appealing advantages for potential therapeutic applications compared with the use of synthetic nanoparticles. When administered systemically, EVs are taken up and sequestered within the liver, further emphasizing opportunities for therapeutic use. Consequently, there is growing interest in their use for liver diseases. EVs can be used directly as therapeutics, and several studies have highlighted the intrinsic therapeutic properties of mesenchymal stem cell-derived EVs for chronic and acute liver diseases. Alternatively, EVs can be modified to facilitate their use for the delivery of therapeutic cargo. In this review, we discuss the cellular sources of EV, provide a concise overview of their potential use in diverse processes, and outline several promising applications for the use of EV-based therapeutics for liver diseases. The use of EV-based therapeutics provides a viable approach to target hepatic pathophysiology.
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Vesículas Extracelulares/trasplante , Hepatopatías/terapia , Sistema de Administración de Fármacos con Nanopartículas/química , Animales , Ensayos Clínicos como Asunto , Modelos Animales de Enfermedad , Vesículas Extracelulares/química , Humanos , Sistema de Administración de Fármacos con Nanopartículas/uso terapéuticoRESUMEN
Extracellular vesicles are endogenous biological nanoparticles that have potential for use as therapeutic nanoparticles or as delivery vehicles for therapeutic agents. Milk nanovesicles (MNV) are extracellular vesicles isolated from bovine milk that have been explored for use as delivery vehicles for RNA therapeutics such as small interfering RNA (siRNA). We performed in vivo toxicological studies of MNV or therapeutic MNV (tMNV) loaded with siRNA as a prelude to their clinical use. Development toxicity was assessed in zebrafish embryos. Acute toxicity was assessed in both mice and zebrafish whereas safety, biochemical, histological and immune effects after multiple dosing were assessed in mice. Zebrafish embryo hatching was accelerated with MNV and tMNV. While acute toxicity or effects on mortality were not observed in zebrafish, developmental effects were observed at high concentrations of MNV. There was a lack of discernable toxicity, mortality and systemic inflammatory or immunological responses in mice following administration of either MNVs or tMNVs. The tolerability and lack of discernable developmental or systemic in vivo toxicity support their use as biological nano-therapeutics. Adoption of a standardized protocol for systematic analysis of in vivo safety and toxicity will facilitate preclinical assessment of EV based formulations for therapeutic use.
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Vesículas Extracelulares/metabolismo , Técnicas de Transferencia de Gen , Leche/toxicidad , ARN Interferente Pequeño/toxicidad , Tratamiento con ARN de Interferencia , Animales , Bovinos , Embrión no Mamífero/efectos de los fármacos , Desarrollo Embrionario , Vesículas Extracelulares/genética , Vesículas Extracelulares/inmunología , Femenino , Masculino , Ratones Endogámicos C57BL , Leche/inmunología , Leche/metabolismo , ARN Interferente Pequeño/genética , ARN Interferente Pequeño/inmunología , ARN Interferente Pequeño/metabolismo , Medición de Riesgo , Bazo/efectos de los fármacos , Bazo/inmunología , Bazo/metabolismo , Pruebas de Toxicidad Aguda , Pez Cebra/embriologíaRESUMEN
Hepatocellular carcinoma is highly resistant to chemotherapy. Here we evaluated the use and efficacy of milk-derived nanovesicles (MNV) as an approach to improve delivery of anticancer agents into HCC cells and intrahepatic tumors. We developed a protocol for isolation of MNVs from skim milk using ultracentrifugation, and characterized using nanoparticle tracking analysis (NTA) and electron microscopy. MNVs were loaded with doxorubicin (dox-MNV) or miR221 antisense oligonucleotides (anti-miR221-MNV), and further evaluated using spectrophotometry, NTA, and zeta potential measurements. HepG2, Hep3B, and PLC/PRF/5 HCC cells in culture were treated with dox-MNV and anti-miR221-MNV and evaluated with drug delivery and anticancer activity. The efficacy of dox-MNV and anti-miR221-MNV to arrest tumor growth in vivo was assessed on intrahepatic tumors induced in nude mice. Cellular uptake studies showed plain and dox-MNV attained saturation within 4 h of treatment. Cytotoxicity studies on HepG2, Hep3B, and PLC/PRF/5 HCC cells with dox-MNV at 1 µM resulted in 20% cell death at 24 h, 50% at 48 h, and 80% at 72 h. HepG2 cells treated with dox-MNV and anti-miR221-MNV exhibited nuclear disintegration, and apoptosis within 24 h. Combination treatment of intrahepatic tumors with dox-MNV and anti-miR221-MNV resulted in marked reduction of tumor size and increased survival rate in nude mice. Our studies demonstrated that MNVs can be effectively used for successful delivery of anticancer agents into HCC cells and intrahepatic tumors. MNV-mediated targeted delivery of anticancer agents could be an efficient modality for the treatment of malignant HCC and might produce a great impact on anticancer therapy.
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Antineoplásicos/farmacocinética , Apoptosis/efectos de los fármacos , Carcinoma Hepatocelular/metabolismo , Portadores de Fármacos/farmacocinética , Neoplasias Hepáticas/metabolismo , Nanopartículas/química , Animales , Antineoplásicos/química , Antineoplásicos/farmacología , Línea Celular Tumoral , Doxorrubicina/química , Doxorrubicina/farmacocinética , Doxorrubicina/farmacología , Portadores de Fármacos/química , Portadores de Fármacos/farmacología , Células Hep G2 , Humanos , Ratones , Ratones DesnudosRESUMEN
Circular ribonucleic acids are non-coding ribonucleic acids that can be identified from genome sequencing studies. Although they can be readily detected, their regulation and functional role in human diseases such as cancer are unknown. Using a systematic approach, we analyzed ribonucleic acid-sequencing data from a well-characterized cohort of intrahepatic cholangiocarcinoma to identify genetic pathways related to circular ribonucleic acids. Although the expression of most circular ribonucleic acids was similar in both the cancer and non-cancer tissues, expression of circ2174 was significantly increased in cancer tissues. Network analysis of co-related genes identified several pathways associated with circ2174, and common regulatory mediators between genes in these pathways and circ2174. Among these, alterations in several genes involved in interleukin-16 signaling responses such Lck, interleukin-16, and macrophage inflammatory protein-1-beta were the most prominent. Octamer transcription factor (Oct)-2 was identified as a signal transducer that was common to both circ2174 and interleukin-16. Circ2174 has sequence complementarity to miR149 which can target Oct-2. These data suggest a mechanism whereby circ2174 can act as a sponge to regulate the expression of miR149, and thereby modulate Oct-2 and interleukin-16 signaling pathways in cholangiocarcinoma.
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Colangiocarcinoma/genética , MicroARNs/genética , Factor 2 de Transcripción de Unión a Octámeros/genética , ARN/genética , Colangiocarcinoma/patología , Regulación Neoplásica de la Expresión Génica/genética , Redes Reguladoras de Genes , Humanos , Interleucina-16/genética , ARN Circular , Transducción de SeñalRESUMEN
BACKGROUND: Genetic alterations in chromatin modulators such as BRCA-1 associated protein-1 (BAP1) are the most frequent genetic alteration in intrahepatic cholangiocarcinomas (CCA). We evaluated the contribution of BAP1 expression on tumor cell behavior and therapeutic sensitivity to identify rationale therapeutic strategies. METHODS: The impact of BAP1 expression on sensitivity to therapeutic agents was evaluated in CCA cells with a 7-fold difference in BAP1 expression (KMBC-low, HuCCT1-high) and genetically engineered haplo-insufficient BAP1 knockout cells. We also identified long non-coding RNA genes associated with loss of BAP1 and their role in therapeutic sensitivity. RESULTS: Sensitivity to gemcitabine was greater in low BAP1 expressing or BAP1 knockout cells compared with the high BAP1 expressing cells or control haplo-insufficient cells respectively. Similar results were observed with TSA, olaparib, b-AP15 but not with GSK126. A differential synergistic effect was observed in combinations of gemcitabine with olaparib or GSK126 in KMBC cells and TSA or bAP15 in HuCCT1 cells, indicating BAP1 dependent target-specific synergism and sensitivity to gemcitabine. A BAP1 dependent alteration in expression of lncRNA NEAT-1 was identified by RT-PCR based lncRNA expression profiling, and an inverse relationship between this lncRNA and BAP1 was observed in analysis of the Tumor Cancer Genome Atlas cholangiocarcinoma dataset. Exogenous modulation of NEAT-1 and/or BAP1 expression altered tumor cell phenotype and modulated sensitivity to gemcitabine. CONCLUSIONS: NEAT-1 is a downstream effector of gemcitabine sensitivity in CCA. The expression of BAP1 is a determinant of sensitivity to therapeutic drugs that can be exploited to enhance responses through combination strategies.
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Neoplasias de los Conductos Biliares/genética , Colangiocarcinoma/genética , Resistencia a Antineoplásicos/genética , Regulación Neoplásica de la Expresión Génica , ARN Largo no Codificante/genética , Proteínas Supresoras de Tumor/genética , Ubiquitina Tiolesterasa/genética , Antimetabolitos Antineoplásicos/farmacología , Neoplasias de los Conductos Biliares/metabolismo , Línea Celular Tumoral , Colangiocarcinoma/metabolismo , Desoxicitidina/análogos & derivados , Desoxicitidina/farmacología , Sinergismo Farmacológico , Humanos , Mutación , Proteínas Supresoras de Tumor/metabolismo , Ubiquitina Tiolesterasa/metabolismo , GemcitabinaRESUMEN
Extracellular vesicles (EVs) are membrane-bound vesicles that are released by cells into their extracellular environment, have selective enrichment of specific proteins and RNA, and can mediate intercellular communication. In this review we highlight recent observations of the role of EVs in liver injury, viral hepatitis, alcoholic or nonalcoholic liver disease, biliary tract disease, and liver cancers. Potential applications as markers of diseases or for therapeutic applications are outlined to emphasize the new opportunities that are arising from the study of EVs.
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Comunicación Celular/fisiología , Vesículas Extracelulares/metabolismo , Hepatopatías/metabolismo , Hígado/metabolismo , Animales , Biomarcadores/metabolismo , Exosomas/metabolismo , Humanos , MicroARNs/metabolismoRESUMEN
Hepatic ischemia/reperfusion injury (IRI) and associated inflammation contributes to liver dysfunction and complications after liver surgery and transplantation. Mesenchymal stem cells (MSCs) have been reported to reduce hepatic IRI because of their reparative immunomodulatory effects in injured tissues. Recent studies have highlighted beneficial effects of extracellular vesicles from mesenchymal stem cells (MSC-EV) on tissue injury. The effects of systemically administered mouse bone marrow-derived MSC-EV were evaluated in an experimental murine model of hepatic IRI induced by cross-clamping the hepatic artery and portal vein for 90 minutes followed by reperfusion for periods of up to 6 hours. Compared with controls, intravenous administration of MSC-EV 30 minutes prior to IRI dramatically reduced the extent of tissue necrosis, decreased caspase 3-positive and apoptotic cells, and reduced serum aminotransferase levels. MSC-EV increased hepatic messenger RNA (mRNA) expression of NACHT, LRR, and PYD domains-containing protein 12, and the chemokine (C-X-C motif) ligand 1, and reduced mRNA expression of several inflammatory cytokines such as interleukin 6 during IRI. MSC-EV increased cell viability and suppressed both oxidative injury and nuclear factor kappa B activity in murine hepatocytes in vitro. In conclusion, the administration of extracellular vesicles derived from bone marrow-derived MSCs may ameliorate hepatic IRI by reducing hepatic injury through modulation of the inflammatory response.Liver Transplantation 23 791-803 2017 AASLD.
Asunto(s)
Células de la Médula Ósea/citología , Vesículas Extracelulares , Hígado/patología , Células Madre Mesenquimatosas/citología , Daño por Reperfusión/terapia , Animales , Apoptosis , Caspasa 3/metabolismo , Supervivencia Celular , Quimiocina CXCL1/sangre , Arteria Hepática/patología , Hepatocitos/citología , Hipoxia , Inflamación , Interleucina-6/sangre , Péptidos y Proteínas de Señalización Intracelular/sangre , Trasplante de Células Madre Mesenquimatosas , Ratones , Ratones Endogámicos C57BL , Subunidad p50 de NF-kappa B/sangre , Necrosis , Estrés Oxidativo , Oxígeno/química , Oxígeno/metabolismo , Vena Porta/patología , Especies Reactivas de Oxígeno/metabolismo , Daño por Reperfusión/patología , Transaminasas/sangreRESUMEN
Extracellular vesicles (EVs) are membrane-bound vesicles released from cells into the extracellular environment. There is emerging interest in the use of EVs as potential therapeutic interventions. We sought to evaluate the safety of EVs that may be therapeutically used by performing in vitro toxicological assessments. EVs were obtained from mesenchymal stem cells (MSC-EV) or from bovine milk (BM-EV) by differential ultracentrifugation, and quantitated using nanoparticle tracking analysis. Genotoxic effects, hematological effects, immunological effects and endotoxin production were evaluated at two dose levels. Neither MSC-EVs nor BM-EVs elicited detectable genotoxic effects using either the alkaline comet assay or micronucleus assay. Hemolysis was observed with BM-EVs but not with MSC-EVs. MSC-EVs did not have any significant effect on either spontaneous or collagen-induced platelet aggregation. In contrast, BM-EVs were noted to increase collagen-induced platelet aggregation, even though no spontaneous increase in platelet aggregation was noted. Both types of EVs induced leukocyte proliferation, which was greater with BM-EV. Neither MSC-EVs nor BM-EVs induced HL-60 phagocytosis, although BM-EVs decreased zymosan-induced phagocytosis. Furthermore, neither MSC-EVs nor BM-EVs induced nitric oxide production. Unlike MSC-EVs, BM-EVs tested positive for endotoxin and induced complement activation. There are significant differences in toxicological profiles between MSC-EVs and BM-EVs that may reflect variations in techniques for EV isolation, EV content or cross-species differences. The safety of MSC-EV supports their use for disease therapeutics, whereas detailed safety and toxicological assessment will be necessary before the use of BM-EVs. Copyright © 2016 John Wiley & Sons, Ltd.
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Efectos Colaterales y Reacciones Adversas Relacionados con Medicamentos , Vesículas Extracelulares , Pruebas de Toxicidad/métodos , Animales , Técnicas de Cultivo de Célula , Línea Celular , Cricetulus , Vesículas Extracelulares/química , Humanos , Células Madre Mesenquimatosas/metabolismo , Ratones , Leche/químicaRESUMEN
Resistance to adverse environmental conditions, such as hypoxia, contributes to the reduced efficacy of anticancer therapies and tumor progression. Although deregulated expression of many long noncoding RNA (lncRNA) occurs in human cancers, the contribution of such RNA to tumor responses to hypoxia are unknown. RNA expression profiling identified several hypoxia-responsive lncRNAs, including the long intergenic noncoding RNA, regulator of reprogramming (linc-RoR), which is also increased in expression in malignant liver cancer cells. Linc-RoR expression was increased in hypoxic regions within tumor cell xenografts in vivo. Tumor cell viability during hypoxia was reduced by small interfering RNA (siRNA) to linc-RoR. Compared with controls, siRNA to linc-RoR decreased phosphorylation of p70S6K1 (RPS6KB1), PDK1 and HIF-1α protein expression and increased expression of the linc-RoR target microRNA-145 (miR-145). Linc-RoR was highly expressed in extracellular RNA released by hepatocellular cancer (HCC) cells during hypoxia. Incubation with extracellular vesicle preparations containing extracellular RNA increased linc-RoR, HIF-1α expression and cell survival in recipient cells. These studies show that linc-RoR is a hypoxia-responsive lncRNA that is functionally linked to hypoxia signaling in HCC through a miR-145-HIF-1α signaling module. Furthermore, this work identifies a mechanistic role for the extracellular transfer of linc-RoR in intercellular signaling to promote cell survival during hypoxic stress.
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Carcinoma Hepatocelular/metabolismo , Hipoxia de la Célula/fisiología , Subunidad alfa del Factor 1 Inducible por Hipoxia/metabolismo , Neoplasias Hepáticas/metabolismo , ARN Largo no Codificante/metabolismo , Animales , Carcinoma Hepatocelular/genética , Carcinoma Hepatocelular/patología , Línea Celular Tumoral , Supervivencia Celular/fisiología , Exosomas , Expresión Génica , Células Hep G2 , Xenoinjertos , Humanos , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/patología , Masculino , Ratones , Ratones Desnudos , MicroARNs/genética , MicroARNs/metabolismo , ARN Largo no Codificante/genética , Transducción de SeñalRESUMEN
BACKGROUND: Impaired natural killer (NK) cell-mediated antitumor responses contribute to the growth of liver tumors. Expression of a disintegrin and metalloprotease 9 (ADAM9) increases shedding of membrane-bound major histocompatibility complex class I chain-related protein A and results in evasion from NK cell-mediated cytolysis. ADAM9 is also involved in angiogenesis and tumor progression and is a target of miR-126-3p, a tumor suppressor that is downregulated and alters tumor cell behavior in the liver and other cancers. We evaluated the restoration of miR-126-3p and modulation of the miR-126-3p/ADAM9 axis as a therapeutic approach to simultaneously enhance NK cell-mediated cytolysis while targeting both tumor cells and their microenvironment. METHODS: Precursor miRNAs were loaded into milk-derived nanovesicles to generate therapeutic vesicles (therapeutic milk-derived nanovesicles) for the restoration of functional miR-126-3p in recipient cancer cells. RESULTS: Administration of therapeutic milk-derived nanovesicles increased miR-126-3p expression and reduced ADAM9 expression in target cells and was associated with an increase in membrane-bound major histocompatibility complex class I chain-related protein A. This enhanced NK cell cytolysis in adherent tumor cells and in multicellular tumor spheroids while also impairing angiogenesis and modulating macrophage chemotaxis. Moreover, IV administration of therapeutic milk-derived nanovesicles with adoptive transfer of NK cells reduced tumor burden in orthotopic hepatocellular cancer xenografts in mice. CONCLUSION: A directed RNA therapeutic approach can mitigate NK cell immune evasion, reduce angiogenesis, and alter the tumor cell phenotype through the restoration of miR-126-3p in liver tumor cells. The pleiotropic effects elicited by this multi-targeted approach to modulate the local tumor microenvironment support its use for the treatment of liver cancer.
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Neoplasias Hepáticas , MicroARNs , Humanos , Animales , Ratones , Microambiente Tumoral/genética , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/terapia , MicroARNs/genética , Traslado Adoptivo , Proteínas de la Membrana/genética , Proteínas ADAMRESUMEN
Individuals carrying (GGGGCC) expanded repeats in the C9orf72 gene represent a significant portion of patients suffering from amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). Elucidating how these expanded repeats cause "c9FTD/ALS" has since become an important goal of the field. Toward this end, we sought to investigate whether epigenetic changes are responsible for the decrease in C9orf72 expression levels observed in c9FTD/ALS patients. We obtained brain tissue from ten c9FTD/ALS individuals, nine FTD/ALS cases without a C9orf72 repeat expansion, and nine disease control participants, and generated fibroblastoid cell lines from seven C9orf72 expanded repeat carriers and seven participants carrying normal alleles. Chromatin immunoprecipitation using antibodies for histone H3 and H4 trimethylated at lysines 9 (H3K9), 27 (H3K27), 79 (H3K79), and 20 (H4K20) revealed that these trimethylated residues bind strongly to C9orf72 expanded repeats in brain tissue, but not to non-pathogenic repeats. Our finding that C9orf72 mRNA levels are reduced in the frontal cortices and cerebella of c9FTD/ALS patients is consistent with trimethylation of these histone residues, an event known to repress gene expression. Moreover, treating repeat carrier-derived fibroblasts with 5-aza-2-deoxycytidine, a DNA and histone demethylating agent, not only decreased C9orf72 binding to trimethylated histone residues, but also increased C9orf72 mRNA expression. Our results provide compelling evidence that trimethylation of lysine residues within histones H3 and H4 is a novel mechanism involved in reducing C9orf72 mRNA expression in expanded repeat carriers. Of importance, we show that mutant C9orf72 binding to trimethylated H3K9 and H3K27 is detectable in blood of c9FTD/ALS patients. Confirming these exciting results using blood from a larger cohort of patients may establish this novel epigenetic event as a biomarker for c9FTD/ALS.
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Esclerosis Amiotrófica Lateral/genética , Degeneración Lobar Frontotemporal/genética , Histonas/genética , Proteínas/genética , Adulto , Alelos , Esclerosis Amiotrófica Lateral/metabolismo , Proteína C9orf72 , Metilación de ADN , Epigénesis Genética , Degeneración Lobar Frontotemporal/metabolismo , Expresión Génica , Histonas/metabolismo , Humanos , Proteínas/metabolismoRESUMEN
Extracellular vesicles (EVs) and their contents are gaining recognition as important mediators of intercellular communication through the transfer of bioactive molecules, such as noncoding RNA. The present study comprehensively assessed the microRNA (miRNA/miR) content within EVs released from HepG2 liver cancer (LC) cells and LX2 hepatic stellate cells (HSCs) and determined the contribution of EV miRNA to intercellular communication. Using both transwell and spheroid cocultures of LC cells and HSCs, miR1263p within EV was established as a mediator of HSC to LC cell communication that influenced tumor cell migration and invasion, as well as the growth of multicellular LC/HSC spheroids. Manipulation of miR1263p either by enforced expression using premiR1263p or by inhibition using antimiR1263p did not alter tumor cell viability, proliferation or sensitivity to either sorafenib or regorafenib. By contrast, enforced expression of miR1263p decreased tumorcell migration. Knockdown of miR1263p in tumor cells increased disintegrin and metalloproteinase domaincontaining protein 9 (ADAM9) expression and in HSCs increased collagen1A1 accumulation with an increase in compactness of multicellular spheroids. Within LC/HSC spheroids, ADAM9 and vascular endothelial growth factor expression was increased by silencing of miR1263p but diminished with the restoration of miR1263p. These studies implicate miR1263p in functional effects on migration, invasion and spheroid growth of tumor cells in the presence of HSCs, and thereby demonstrate functional EVRNAbased intercellular signaling between HSCs and LC cells that is directly relevant to tumorcell behavior.
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Vesículas Extracelulares , Neoplasias Hepáticas , MicroARNs , Humanos , Microambiente Tumoral/genética , Factor A de Crecimiento Endotelial Vascular/metabolismo , Neoplasias Hepáticas/patología , Comunicación Celular/genética , MicroARNs/metabolismo , Células Estrelladas Hepáticas/metabolismo , Células Estrelladas Hepáticas/patología , Vesículas Extracelulares/metabolismo , Proteínas de la Membrana/metabolismo , Proteínas ADAM/metabolismo , Proteínas ADAM/farmacologíaRESUMEN
Extracellular vesicles (EVs) show promise for targeted drug delivery but face production challenges with low yields. Cell-derived nanovesicles (CDNVs) made by reconstituting cell membranes could serve as EV substitutes. In this study, CDNVs were generated from mesenchymal stem cells by extrusion. Their proteomic composition, in vitro and in vivo toxicity, and capacity for loading RNA or proteins were assessed. Compared with EVs, CDNVs were produced at higher yields, were comprised of a broader range of proteins, and showed no detrimental effects on cell proliferation, DNA damage, or nitric oxide production in vitro or on developmental toxicity in vivo. CDNVs could be efficiently loaded with RNA and engineered to modify surface proteins. The feasibility of generating immunomodulatory CDNVs was demonstrated by preparing CDNVs with enhanced surface expression of PD1, which could bind to PD-L1 expressing tumor cells, enhance NK and T cell degranulation, and increase immune-mediated tumor cell death. These findings demonstrate the adaptability and therapeutic promise of CDNVs as promising substitutes for natural EVs that can be engineered to enhance immunomodulation.
RESUMEN
Cancer stem cells (CSC) within non-small cell lung carcinoma (NSCLC) tumors drive NSCLC progression, metastasis, relapse, and intrinsic chemoresistance. Understanding the mechanisms that support the malignant phenotypes of NSCLC CSCs may provide insights for improved NSCLC therapeutic interventions. Here, we report that expression of RAB27B, a small GTPase, is significantly upregulated in NSCLC CSCs when compared with bulk cancer cells (BCC). Short hairpin RNA-mediated knockdown of RAB27B leads to a loss of stem cell marker gene expression and reduced NSCLC spheroid growth, clonal expansion, transformed growth, invasion, and tumorigenicity. We find that NSCLC CSCs secrete significantly more extracellular vesicles (EV) than BCCs, and that this is RAB27B-dependent. Furthermore, CSC-derived EVs, but not BCC-derived EVs, induce spheroid growth, clonal expansion, and invasion in BCCs. Finally, RAB27B is required for CSC-derived EV-induced stemness in BCCs. Taken together, our results indicate that RAB27B is required for maintenance of a highly tumorigenic, cancer-initiating, invasive stem-like cell population in NSCLC and RAB27B is involved in propagating EV-mediated communication from NSCLC CSCs to BCCs. Our findings further suggest that inhibition of RAB27B-dependent EV secretion may be a potential therapeutic strategy for NSCLC. Significance: Expression of RAB27B in CSCs leads to elevated levels of EVs that mediate communication between CSCs and BCCs that maintains a stem-like phenotype in NSCLC cells.
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Carcinoma de Pulmón de Células no Pequeñas , Vesículas Extracelulares , Neoplasias Pulmonares , Humanos , Carcinoma de Pulmón de Células no Pequeñas/genética , Línea Celular Tumoral , Recurrencia Local de Neoplasia/metabolismo , Vesículas Extracelulares/genética , Neoplasias Pulmonares/genética , Células Madre Neoplásicas/metabolismo , FenotipoRESUMEN
UNLABELLED: Hepatocellular carcinoma (HCC) is characterized by a propensity for multifocality, growth by local spread, and dysregulation of multiple signaling pathways. These features may be determined by the tumoral microenvironment. The potential of tumor cells to modulate HCC growth and behavior by secreted proteins has been extensively studied. In contrast, the potential for genetic modulation is poorly understood. We investigated the role and involvement of tumor-derived nanovesicles capable of altering gene expression and characterized their ability to modulate cell signaling and biological effects in other cells. We show that HCC cells can produce nanovesicles and exosomes that differ in both RNA and protein content from their cells of origin. These can be taken up and internalized by other cells and can transmit a functional transgene. The microRNA (miRNA) content of these exosomes was examined, and a subset highly enriched within exosomes was identified. A combinatorial approach to identify potential targets identified transforming growth factor ß activated kinase-1 (TAK1) as the most likely candidate pathway that could be modulated by these miRNAs. Loss of TAK1 has been implicated in hepatocarcinogenesis and is a biologically plausible target for intercellular modulation. We show that HCC cell-derived exosomes can modulate TAK1 expression and associated signaling and enhance transformed cell growth in recipient cells. CONCLUSION: Exosome-mediated miRNA transfer is an important mechanism of intercellular communication in HCC cells. These observations identify a unique intercellular mechanism that could potentially contribute to local spread, intrahepatic metastases, or multifocal growth in HCC.
Asunto(s)
Carcinoma Hepatocelular/patología , Proliferación Celular , Exosomas/metabolismo , Neoplasias Hepáticas/patología , MicroARNs/genética , Carcinoma Hepatocelular/genética , Comunicación Celular/genética , Comunicación Celular/fisiología , Exosomas/genética , Femenino , Humanos , Neoplasias Hepáticas/genética , Masculino , Invasividad Neoplásica/genética , Invasividad Neoplásica/patología , Reacción en Cadena en Tiempo Real de la Polimerasa , Muestreo , Transducción de Señal/fisiología , Células Tumorales Cultivadas , Microambiente TumoralRESUMEN
The therapeutic activities elicited by mesenchymal stem cells (MSC) are in part mediated through paracrine action by the release of extracellular vesicles (EV) and secreted proteins. Collectively, these MSC-derived factors, referred to as the secretome product (SP), are intrinsically therapeutic and represent an attractive alternative to cell-based therapies. Herein, we developed a lyopreservation protocol to extend the shelf-life of the MSC-SP without compromising the structural or functional integrity of the vesicular components. The SP isolated from normoxia- and anoxia-exposed MSC elicited protective effects in an in vitro model of oxidative injury and the bioactivity was retained in the lyophilized samples. Three separate formulations of MSC-SP were isolated by tangential flow filtration using sucrose, trehalose, and mannitol as lyoprotectant agents. The MSC-SPs were lyophilized using a manifold protocol and the structural and functional integrity were assessed. The trehalose formulation of SP exhibited the highest EV and protein recovery after manifold-based lyophilization. To facilitate development as a therapeutic, a shelf lyophilization protocol was developed which markedly enhanced the recovery of EV and proteins. In conclusion, lyophilization represents an efficient method to preserve the structural and functional integrity of the MSC-SP and can be used to develop an off-the-shelf therapeutic.
RESUMEN
Long noncoding RNAs (lncRNAs) are implicated in many physiological or disease processes and alterations in their expression may contribute to the development of various diseases. Accurate quantitation of lncRNA can be useful in measuring changes in expression in different settings such as in the circulation where the measurement of lncRNA may be useful as a biomarker of disease. However, the low levels of lncRNA expression require the use of highly sensitive detection technologies for accurate quantitation. Digital polymerase chain reaction (dPCR) is a sensitive method for absolute quantification of lncRNA and can be useful for measurement of gene expression when transcript levels are low. By providing a direct measurement without normalization, the use of dPCR may provide advantages for quantitation of low-abundance targets.
Asunto(s)
Expresión Génica , ARN Largo no Codificante/genética , Reacción en Cadena en Tiempo Real de la Polimerasa , Biomarcadores , Línea Celular Tumoral , Análisis de Datos , Regulación de la Expresión Génica , Humanos , Reacción en Cadena en Tiempo Real de la Polimerasa/métodosRESUMEN
RNA sequencing using nanopore sequencing is a powerful method for transcriptome analysis. The approach is appropriate for comprehensive profiling of the wide range of long noncoding RNAs. Use of nanopore-based sequencing can provide information on novel transcripts, sequence polymorphisms, and splicing variants, and thus has advantages over other gene expression profiling methods such as microarrays. Circulating extracellular long noncoding RNAs are of particular interest because of their potential use as biomarkers. Here, we describe a protocol for cDNA-PCR sequencing of circulating RNA for biomarker discovery in whole blood samples using commercially available kits and nanopore sequencing.