RESUMEN
Communication between interacting organisms via bioactive molecules is widespread in nature and plays key roles in diverse biological processes. Small RNAs (sRNAs) can travel between host plants and filamentous pathogens to trigger transkingdom RNA interference (RNAi) in recipient cells and modulate plant defense and pathogen virulence. However, how fungal pathogens counteract transkingdom antifungal RNAi has rarely been reported. Here we show that a secretory protein VdSSR1 (secretory silencing repressor 1) from Verticillium dahliae, a soil-borne phytopathogenic fungus that causes wilt diseases in a wide range of plant hosts, is required for fungal virulence in plants. VdSSR1 can translocate to plant nucleus and serve as a general suppressor of sRNA nucleocytoplasmic shuttling. We further reveal that VdSSR1 sequesters ALY family proteins, adaptors of the TREX complex, to interfere with nuclear export of the AGO1microRNA (AGO1miRNA) complex, leading to a great attenuation in cytoplasmic AGO1 protein and sRNA levels. With this mechanism, V. dahliae can suppress the accumulation of mobile plant miRNAs in fungal cells and succedent transkingdom silencing of virulence genes, thereby increasing its virulence in plants. Our findings reveal a mechanism by which phytopathogenic fungi antagonize antifungal RNAi-dependent plant immunity and expand the understanding on the complex interaction between host and filamentous pathogens.
Asunto(s)
MicroARNs , Verticillium , Transporte Activo de Núcleo Celular , Antifúngicos , MicroARNs/genética , MicroARNs/metabolismo , Enfermedades de las Plantas/microbiología , Plantas/genética , ARN de Planta , Verticillium/metabolismoRESUMEN
RNA silencing (or RNA interference, RNAi) is a conserved mechanism for regulating gene expression in eukaryotes, which plays vital roles in plant development and response to biotic and abiotic stresses. The discovery of trans-kingdom RNAi and interspecies RNAi provides a theoretical basis for exploiting RNAi-based crop protection strategies. Here, we summarize the canonical RNAi mechanisms in plants and review representative studies associated with plant-pathogen interactions. Meanwhile, we also elaborate upon the principles of host-induced gene silencing, spray-induced gene silencing and microbe-induced gene silencing, and discuss their applications in crop protection, thereby providing help to establish novel RNAi-based crop protection strategies.
Asunto(s)
Protección de Cultivos , Plantas , Interferencia de ARN , Plantas/genética , Eucariontes/genética , ARN Interferente Pequeño/genéticaRESUMEN
Flowering plants and mammals contain imprinted genes that are primarily expressed in the endosperm and placenta in a parent-of-origin manner. In this study, we show that early activation of the geminivirus genes C2 and C3 in Arabidopsis (Arabidopsis thaliana) plants, encoding a viral suppressor of RNA interference and a replication enhancer protein, respectively, is correlated with the transient vegetative expression of VARIANT IN METHYLATION5 (VIM5), an endosperm imprinted gene that is conserved in diverse plant species. VIM5 is a ubiquitin E3 ligase that directly targets the DNA methyltransferases MET1 and CMT3 for degradation by the ubiquitin-26S proteasome proteolytic pathway. Infection with Beet severe curly top virus induced VIM5 expression in rosette leaf tissues, possibly via the expression of the viral replication initiator protein, leading to the early activation of C2 and C3 coupled with reduced symmetric methylation in the C2-3 promoter and the onset of disease symptoms. These findings demonstrate how this small DNA virus recruits a host imprinted gene for the epigenetic activation of viral gene transcription. Our findings reveal a distinct strategy used by plant pathogens to exploit the host machinery in order to inhibit methylation-mediated defense responses when establishing infection.
Asunto(s)
Arabidopsis/genética , Arabidopsis/virología , Geminiviridae/patogenicidad , Enfermedades de las Plantas/virología , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , ADN (Citosina-5-)-Metiltransferasas/genética , ADN (Citosina-5-)-Metiltransferasas/metabolismo , Metilación de ADN , ADN-Citosina Metilasas/genética , ADN-Citosina Metilasas/metabolismo , Epigénesis Genética , Regulación de la Expresión Génica de las Plantas , Impresión Genómica , Interacciones Huésped-Patógeno/genética , Enfermedades de las Plantas/genética , Hojas de la Planta/genética , Hojas de la Planta/virología , Plantas Modificadas Genéticamente , Regiones Promotoras Genéticas , Ubiquitina-Proteína Ligasas/genética , Ubiquitina-Proteína Ligasas/metabolismo , Proteínas Virales/genéticaRESUMEN
Nicotinamide adenine dinucleotide (NAD) is a critical cosubstrate for enzymes involved in supplying energy to the brain. Nicotinamide riboside (NR), an NAD+ precursor, emerges as a neuroprotective factor after chronic brain insults. However, researchers have not determined whether it improves cognition after acute ischemia. In the present study, mice with middle cerebral artery occlusion were treated with NR chloride (NRC, 300 mg/kg, IP., 20 min after reperfusion). The results of the Morris water maze test revealed better recovery of learning and memory function in the NRC-treated group. Acute NRC treatment decreased hippocampal infarct volume, reduced neuronal loss and apoptosis in the hippocampus. Western blot and high-performance liquid chromatography assays of hippocampal tissues revealed that the activation of Sirtin-1 and adenosine 5' monophosphate-activated protein kinase was increased, the NAD content was elevated, and the production of adenosine triphosphate was strengthened by NRC. Collectively, acute NRC treatment increased the energy supply, reduced the neuronal loss and apoptosis, protected the hippocampus and ultimately promoted the recovery of cognitive function after brain ischemia.
Asunto(s)
Cloruros , NAD , Animales , Cognición , Hipocampo/metabolismo , Infarto de la Arteria Cerebral Media/metabolismo , Ratones , NAD/metabolismo , Niacinamida/análogos & derivados , Compuestos de PiridinioRESUMEN
Host-induced gene silencing (HIGS) based on trans-kingdom RNA interference (RNAi) has been successfully exploited to engineer host resistance to pests and pathogens, including fungi and oomycetes. However, revealing the mechanisms underlying trans-kingdom RNAi between hosts and pathogens lags behind applications. The effectiveness and durability of trans-kingdom silencing of pathogenic genes are uncharacterized. In this study, using our transgenic 35S-VdH1i cotton plants in which dsVdH1-derived small RNAs (siVdH1) accumulated, small RNA sequencing analysis revealed that siVdH1s exclusively occur within the double-stranded (ds)VdH1 region, and no transitive siRNAs were produced beyond this region in recovered hyphae of Verticillium dahliae (V. dahliae). Accordingly, we found that VdH1 silencing was reduced over time in recovered hyphae cultured in vitro, inferring that once the fungus got rid of the 35S-VdH1i cotton plants would gradually regain their pathogenicity. To explore whether continually exporting dsRNAs/siRNAs from transgenic plants into recipient fungal cells guaranteed the effectiveness and stability of HIGS, we created GFP/RFP double-labeled V. dahliae and transgenic Arabidopsis expressing dsGFP (35S-GFPi plants). Confocal images visually demonstrate the efficient silencing of GFP in V. dahliae that colonized host vascular tissues. Taken together, our results demonstrate that HIGS effectively triggers long-lasting trans-kingdom RNAi during plant vasculature V. dahliae interactions, despite no amplification or transitivity of RNAi being noted in this soil-borne fungal pathogen.
Asunto(s)
Arabidopsis , Verticillium , Arabidopsis/genética , Arabidopsis/microbiología , Resistencia a la Enfermedad/genética , Genes Fúngicos , Gossypium/genética , Enfermedades de las Plantas/genética , Enfermedades de las Plantas/microbiología , Plantas Modificadas Genéticamente/genética , ARN Interferente Pequeño/genética , Verticillium/genéticaRESUMEN
RNA silencing (or RNA interference, RNAi) is a conserved mechanism for regulating gene expression in eukaryotes. The discovery of natural trans-kingdom RNAi indicated that small RNAs act as signaling molecules and enable communication between organisms in different kingdoms. The phenomenon and potential mechanisms of trans-kingdom RNAi are among the most exciting research topics. To better understand trans-kingdom RNAi, we review the history of the discovery and elucidation of RNAi mechanisms. Based on canonical RNAi mechanisms, we summarize the major points of divergence around RNAi pathways in the main eukaryotes' kingdoms, including plants, animals, and fungi. We review the representative incidents associated with the mechanisms and applications of trans-kingdom RNAi in crop protection, and discuss the critical factors that should be considered to develop successful trans-kingdom RNAi-based crop protection strategies.
Asunto(s)
Hongos , Plantas , Animales , Hongos/genética , Plantas/genética , Interferencia de ARN , ARN Interferente Pequeño/genéticaRESUMEN
BACKGROUND AND PURPOSE: We aimed to demonstrate the tolerability and feasibility and the effect of remote ischemic post-conditioning on cognitive functioning in patients with post-stroke cognitive impairment. METHODS: This was a single-center, randomized, outcome-blinded, placebo-controlled trial, randomized 1:1 to receive 4 cycles of remote ischemic post-conditioning or a sham procedure for 7 days. The primary outcome measure was tolerability and feasibility of remote ischemic post-conditioning. Secondary outcomes to measure the neurological function with national institute of health stroke scale and the cognitive impairment with Montreal Cognitive Assessment scale and Alzheimer's disease assessment scale-cognitive (at baseline, 90 days, 180 days). RESULTS: 48 patients (24 RIPC and 24 Control) were recruited. remote ischemic post-conditioning was well tolerated with 90 out of 96 cycles completed in full. 4 patients experienced vascular events in the control group: 3 cerebrovascular and 1 cardiovascular event versus only 2 cerebrovascular events in the RIPC group. We showed the similar result in the neurological function with national institute of health stroke scale score with no statistically significant differences between RIPC and control group at baseline (P = 0.796) and 90 days (Pâ¯=â¯0.401) and 180 days (Pâ¯=â¯0.695). But compare with baseline, it was significantly difference in the control and RIPC group at 90 days (P < 0.05) and 180 days (P < 0.05). The comparison of Montreal Cognitive Assessment scale between two groups both showed that P > 0.05 at baseline which was no statistical difference, but P < 0.05 at 90 days and 180 days which were significant statistical difference. The comparison of Alzheimer's disease assessment scale-cognitive between two groups showed that P > 0.05 at baseline (Pâ¯=â¯0.955) and 90 days (Pâ¯=â¯0.138) was no statistical difference, but Pâ¯=â¯0.005<0.05 at 180 days was significant statistical difference. CONCLUSIONS: The remote ischemic post-conditioning for post-stroke cognitive impairment was well tolerated, safe and feasible. The remote ischemic post-conditioning may improve neurological and cognitive outcomes in patients with post-stroke cognitive impairment. A larger trial is warranted. (Clinical Trial Registration-URL: http://www.clinicaltrials.gov. Unique identifier: ChiCTR1800015231.).
Asunto(s)
Cognición , Disfunción Cognitiva/terapia , Poscondicionamiento Isquémico , Accidente Cerebrovascular/complicaciones , Anciano , Anciano de 80 o más Años , China , Disfunción Cognitiva/diagnóstico , Disfunción Cognitiva/etiología , Disfunción Cognitiva/psicología , Estudios de Factibilidad , Femenino , Humanos , Poscondicionamiento Isquémico/efectos adversos , Masculino , Persona de Mediana Edad , Proyectos Piloto , Recuperación de la Función , Accidente Cerebrovascular/diagnóstico , Accidente Cerebrovascular/psicología , Factores de Tiempo , Resultado del TratamientoRESUMEN
BACKGROUND: Salmonella bongori infect mainly cold-blooded hosts, but infections by S. bongori in warm-blooded hosts have been reported. We hypothesized that S. bongori might have diverged into distinct phylogenetic lineages, with some being able to infect warm-blooded hosts. RESULTS: To inspect the divergence status of S. bongori, we first completely sequenced the parakeet isolate RKS3044 and compared it with other sequenced S. bongori strains. We found that RKS3044 contained a novel T6SS encoded in a pathogenicity island-like structure, in addition to a T6SS encoded in SPI-22, which is common to all S. bongori strains so far reported. This novel T6SS resembled the SPI-19 T6SS of the warm-blooded host infecting Salmonella Subgroup I lineages. Genomic sequence comparisons revealed different genomic sequence amelioration events among the S. bongori strains, including a unique CTAG tetranucleotide degeneration pattern in RKS3044, suggesting non-overlapping gene pools between RKS3044 and other S. bongori lineages/strains leading to their independent accumulation of genomic variations. We further proved the existence of a clear-cut genetic boundary between RKS3044 and the other S. bongori lineages/strains analyzed in this study. CONCLUSIONS: The warm-blooded host-infecting S. bongori strain RKS3044 has diverged with distinct genomic features from other S. bongori strains, including a novel T6SS encoded in a previously not reported pathogenicity island-like structure and a unique genomic sequence degeneration pattern. These findings alert cautions about the emergence of new pathogens originating from non-pathogenic ancestors by acquiring specific pathogenic traits.
Asunto(s)
Islas Genómicas , Periquitos/microbiología , Salmonella/clasificación , Secuenciación Completa del Genoma/métodos , Animales , Evolución Molecular , Especiación Genética , Tamaño del Genoma , Genoma Bacteriano , Humanos , Filogenia , Salmonella/genética , Salmonella/patogenicidad , Factores de Virulencia/genéticaRESUMEN
OBJECTIVE: To evaluate whether the macrophage migration inhibitory factor (MIF) level in serum of ischemic stroke patients was associated with their clinical severity and early outcome. METHODS: During February 2017-March 2018, consecutive patients admitted to our hospital because of first-ever ischemic stroke were identified. The prognostic value of MIF was set for predicting the outcome of these patients at discharge. The results were compared with existing methods, including National Institutes of Health Stroke Scale (NIHSS) score and validated indicators. RESULTS: 289 patients were enrolled. The serum level of all patients was determined (median: 20.6â¯ng/ml). At admission, 131 patients (45.3%) were evaluated as minor stroke (NIHSSâ¯<â¯5). When serum level of MIF was increased by each 1â¯ng/ml, the unadjusted and adjusted risk of moderate-to-high clinical severity was elevated by 5% (ORâ¯=â¯1.05 [95% CI: 1.01-1.09], Pâ¯=â¯0.006) and 3% (1.03 [1.00-1.08], Pâ¯=â¯0.02), respectively. At discharge, 82 patients (28.4%) had poor functional outcomes. The median serum level of MIF was lower in group with good outcomes than that observed in poor outcomes (19.4[15.8-24.2] vs. 24.0[19.9-29.4]â¯ng/ml; Pâ¯<â¯0.001). When serum level of MIF was increased by each 1â¯ng/ml, the unadjusted and adjusted risk of poor outcomes was elevated by 9% (1.09 [1.05-1.13], Pâ¯<â¯0.001) and 6% (1.06 [1.02-1.10], Pâ¯<â¯0.01), respectively. CONCLUSIONS: High MIF levels are independently related to the moderate to high clinical severity in ischemic stroke patients, as well as the poor outcome at discharge.
Asunto(s)
Isquemia Encefálica/sangre , Isquemia Encefálica/patología , Oxidorreductasas Intramoleculares/sangre , Factores Inhibidores de la Migración de Macrófagos/sangre , Accidente Cerebrovascular/sangre , Accidente Cerebrovascular/patología , Anciano , Femenino , Hospitalización , Humanos , Masculino , Persona de Mediana Edad , Pronóstico , Índice de Severidad de la EnfermedadRESUMEN
Spinal cord injury (SCI) is a debilitating, costly, and common pathological condition that affects the function of central nervous system (CNS). To date, there are few promising therapeutic strategies available for SCI. To look for a suitable therapeutic strategy, we have developed a sublethal hypoxic preconditioning procedure using Fluorescence-activated cell sorting (FACS) analysis, LDH releasing, and cell viability assays in vitro. Meanwhile, we have examined the benefits of neural stem cells (NSCs) transplantation prior to hypoxic preconditioning on functional recovery and potential mechanism via MRI screening, H&E, and Nissl staining, immunofluorescence staining and Elisa assays. Our data showed that transplantation of hypoxic prconditioned NSCs could enhance neuronal survival, especially 5-TH+ and ChAT+ neurons, in the injured spinal cord to reinforce functional benefits. The hypoxia exposure upregulated HIF-1α, neurotrophic and growth factors including neurotrophin-3 (NT-3), glial cell-derived neurotrophic factor (GDNF), and brain-derived neurotrophic factor (BDNF) in vitro and in vivo. Furthermore, functional recovery, including locomotor and hypersensitivities to mechanical and thermal stimulation assessed via behavioral and sensory tests, improved significantly in rats with engraftment of NSCs after hypoxia exposure from day 14 post-SCI, compared with the control and N-NSCs groups. In short, the approach employed in this study could result in functional recovery via upregulating neurotrophic and growth factors, which implies that hypoxic preconditioning strategy could serve as an effective and feasible strategy for cell-based therapy in the treatment of SCI in rats.
Asunto(s)
Precondicionamiento Isquémico , Locomoción , Factores de Crecimiento Nervioso/metabolismo , Células-Madre Neurales/metabolismo , Recuperación de la Función , Traumatismos de la Médula Espinal , Trasplante de Células Madre , Animales , Femenino , Células-Madre Neurales/patología , Ratas , Ratas Sprague-Dawley , Traumatismos de la Médula Espinal/metabolismo , Traumatismos de la Médula Espinal/patología , Traumatismos de la Médula Espinal/fisiopatología , Traumatismos de la Médula Espinal/terapiaRESUMEN
Satellite RNAs (satRNAs) are small noncoding subviral RNA pathogens in plants that depend on helper viruses for replication and spread. Despite many decades of research, the origin of satRNAs remains unknown. In this study we show that a ß-glucuronidase (GUS) transgene fused with a Cucumber mosaic virus (CMV) Y satellite RNA (Y-Sat) sequence (35S-GUS:Sat) was transcriptionally repressed in N. tabacum in comparison to a 35S-GUS transgene that did not contain the Y-Sat sequence. This repression was not due to DNA methylation at the 35S promoter, but was associated with specific DNA methylation at the Y-Sat sequence. Both northern blot hybridization and small RNA deep sequencing detected 24-nt siRNAs in wild-type Nicotiana plants with sequence homology to Y-Sat, suggesting that the N. tabacum genome contains Y-Sat-like sequences that give rise to 24-nt sRNAs capable of guiding RNA-directed DNA methylation (RdDM) to the Y-Sat sequence in the 35S-GUS:Sat transgene. Consistent with this, Southern blot hybridization detected multiple DNA bands in Nicotiana plants that had sequence homology to Y-Sat, suggesting that Y-Sat-like sequences exist in the Nicotiana genome as repetitive DNA, a DNA feature associated with 24-nt sRNAs. Our results point to a host genome origin for CMV satRNAs, and suggest novel approach of using small RNA sequences for finding the origin of other satRNAs.
Asunto(s)
Satélite del Virus del Mosaico del Pepino/genética , Cucumovirus/genética , Glucuronidasa/genética , Nicotiana/genética , Cucumovirus/patogenicidad , Metilación de ADN/genética , Silenciador del Gen , Genoma de Planta , Virus Helper/genética , Plantas Modificadas Genéticamente , ARN Interferente Pequeño , Análisis de Secuencia de ARN , Nicotiana/virología , TransgenesRESUMEN
The microRNA family, miR-30, plays diverse roles in regulating key aspects of neoplastic transformation, metastasis, and clinical outcomes in different types of tumors. Accumulating evidence proves that miR-30 family is pivotal in the breast cancer development by controlling critical signaling pathways and relevant oncogenes. Here, we review the roles of miR-30 family members in the tumorigenesis, metastasis, and drug resistance of breast cancer, and their application to predict the prognosis of breast cancer patients. We think miR-30 family members would be promising biomarkers for breast cancer and may bring a novel insight in molecular targeted therapy of breast cancer.
Asunto(s)
Neoplasias de la Mama/genética , MicroARNs/genética , Biomarcadores de Tumor/genética , Biomarcadores de Tumor/metabolismo , Neoplasias de la Mama/metabolismo , Neoplasias de la Mama/patología , Carcinogénesis/genética , Carcinogénesis/metabolismo , Resistencia a Antineoplásicos , Femenino , Humanos , MicroARNs/metabolismo , Metástasis de la Neoplasia , Transducción de Señal/genéticaRESUMEN
Cisplatin is a widely used chemotherapeutic agent in breast cancer treatments with inevitable rapidly acquired resistance or intrinsically resistance. Enormous evidence points to the bioprocesses of resistant formation consisting of diverse miRNAs direct and indirect actions on relevant encoding genes. In this report, we overview detailed information on the miRNAs effect on cisplatin-induced resistance, including alterations in cell survival, modification of DNA damage response, changes in cellular uptake or efflux of the drug, altered DNA methylation, and perturbations in the miRNA biogenesis pathway. This will provide potential miRNA-targeted strategies for the treatment of breast cancer therapy and requires further clinical application.
Asunto(s)
Antineoplásicos/farmacología , Neoplasias de la Mama/genética , Cisplatino/farmacología , Resistencia a Antineoplásicos/genética , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , MicroARNs/genética , Animales , Neoplasias de la Mama/tratamiento farmacológico , Femenino , HumanosRESUMEN
The high resistant rate of Adriamycin (Adr) is associated with a poor prognosis of breast cancer in women worldwide. Since miR-222 might contribute to chemoresistance in many cancer types, in this study, we aimed to investigate its efficacy in breast cancer through PTEN/Akt/p27 kip1 pathway. Firstly, in vivo, we verified that miR-222 was upregulated in chemoresistant tissues after surgery compared with the paired preneoadjuvant samples of 21 breast cancer patients. Then, human breast cancer Adr-resistant cell line (MCF-7/Adr) was constructed to validate the pathway from the parental sensitive cell line (MCF-7/S). MCF-7/Adr and MCF-7/S were transfected with miR-222 mimics, miR-222 inhibitors, or their negative controls, respectively. The results showed that inhibition of miR-222 in MCF-7/Adr significantly increased the expressions of PTEN and p27 kip1 and decreased phospho-Akt (p-Akt) both in mRNA and protein levels (p < 0.05) by using quantitative real-time PCR (qRT-PCR) and western blot. MTT and flow cytometry suggested that lower expressed miR-222 enhanced apoptosis and decreased the IC50 of MCF-7/Adr cells. Additionally, immunofluorescence demonstrated that the subcellular location of p27 kip1 was dislocated resulting from the alteration of miR-222. Conversely, in MCF-7/S transfected with miR-222 mimics, upregulation of miR-222 is associated with decreasing PTEN and p27 kip1 and increasing Akt accompanied by less apoptosis and higher IC50. Importantly, Adr resistance induced by miR-222 overexpression through PTEN/Akt/p27 was completely blocked by LY294002, an Akt inhibitor. Taken together, these data firstly elucidated that miR-222 could reduce the sensitivity of breast cancer cells to Adr through PTEN/Akt/p27 kip1 signaling pathway, which provided a potential target to increase the sensitivity to Adr in breast cancer treatment and further improved the prognosis of breast cancer patients.
Asunto(s)
Neoplasias de la Mama/tratamiento farmacológico , Neoplasias de la Mama/genética , Inhibidor p27 de las Quinasas Dependientes de la Ciclina/metabolismo , Doxorrubicina/farmacología , Resistencia a Antineoplásicos/genética , MicroARNs/genética , Fosfohidrolasa PTEN/metabolismo , Proteínas Proto-Oncogénicas c-akt/metabolismo , Antibióticos Antineoplásicos/farmacología , Apoptosis , Western Blotting , Neoplasias de la Mama/patología , Proliferación Celular , Inhibidor p27 de las Quinasas Dependientes de la Ciclina/genética , Femenino , Citometría de Flujo , Técnica del Anticuerpo Fluorescente , Regulación Neoplásica de la Expresión Génica , Humanos , Células MCF-7 , Fosfohidrolasa PTEN/genética , Pronóstico , Proteínas Proto-Oncogénicas c-akt/genética , ARN Mensajero/genética , Reacción en Cadena en Tiempo Real de la Polimerasa , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Transducción de SeñalRESUMEN
While adriamycin (adr) offers improvement in survival for breast cancer (BCa) patients, unfortunately, drug resistance is almost inevitable. Mounting evidence suggests that exosomes act as a vehicle for genetic cargo and constantly shuttle biologically active molecules including microRNAs (miRNAs) between heterogeneous populations of tumor cells, engendering a resistance-promoting niche for cancer progression. Our recent study showed that exosomes from docetaxel-resistance BCa cells could modulate chemosensitivity by delivering miRNAs. Herein, we expand on our previous finding and explore the relevance of exosome-mediated miRNA delivery in resistance transmission of adr-resistant BCa sublines. We now demonstrated the selective packing of miRNAs within the exosomes (A/exo) derived from adr-resistant BCa cells. The highly expressed miRNAs in A/exo were significantly increased in recipient fluorescent sensitive cells (GFP-S) after A/exo incorporation. Gene ontology analysis of predicted targets showed that the top 30 most abundant miRNAs in A/exo were involved in crucial biological processes. Moreover, A/exo not only loaded miRNAs for its production and release but also carried miRNAs associated with Wnt signaling pathway. Furthermore, A/exo co-culture assays indicated that miRNA-containing A/exo was able to increase the overall resistance of GFP-S to adr exposure and regulate gene levels in GFP-S. Our results reinforce our earlier reports that adr-resistant BCa cells could manipulate a more deleterious microenvironment and transmit resistance capacity through altering gene expressions in sensitive cells by transferring specific miRNAs contained within exosomes.
Asunto(s)
Neoplasias de la Mama/tratamiento farmacológico , Resistencia a Antineoplásicos/genética , Exosomas/genética , MicroARNs/genética , Apoptosis/efectos de los fármacos , Neoplasias de la Mama/genética , Neoplasias de la Mama/patología , Técnicas de Cocultivo , Docetaxel , Doxorrubicina/administración & dosificación , Exosomas/efectos de los fármacos , Femenino , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Humanos , Células MCF-7 , MicroARNs/biosíntesis , Taxoides/administración & dosificación , Microambiente Tumoral/genética , Vía de Señalización Wnt/efectos de los fármacosRESUMEN
Breast cancer (BCa) is one of the major deadly cancers in women. However, treatment of BCa is still hindered by the acquired-drug resistance. It is increasingly reported that exosomes take part in the development, metastasis, and drug resistance of BCa. However, the specific role of exosomes in drug resistance of BCa is poorly understood. In this study, we investigate whether exosomes transmit drug resistance through delivering miR-222. We established an adriamycin-resistant variant of Michigan Cancer Foundation-7 (MCF-7) breast cancer cell line (MCF-7/Adr) from a drug-sensitive variant (MCF-7/S). Exosomes were isolated from cell supernatant by ultracentrifugation. Cell viability was assessed by MTT assay and apoptosis assay. Individual miR-222 molecules in BCa cells were detected by fluorescence in situ hybridization (FISH). Then, FISH was combined with locked nucleic acid probes and enzyme-labeled fluorescence (LNA-ELF-FISH). Individual miR-222 could be detected as bright photostable fluorescent spots and then the quantity of miR-222 per cell could be counted. Stained exosomes were taken in by the receipt cells. MCF-7/S acquired drug resistance after co-culture with exosomes from MCF-7/Adr (A/exo) but did not after co-culture with exosomes from MCF-7/S (S/exo). The quantity of miR-222 in A/exo-treated MCF-7/S was significantly greater than in S/exo-treated MCF-7/S. MCF-7/S transfected with miR-222 mimics acquired adriamycin resistance while MCF-7/S transfected with miR-222 inhibitors lost resistance. In conclusion, exosomes are effective in transmitting drug resistance and the delivery of miR-222 via exosomes may be a mechanism.
Asunto(s)
Doxorrubicina/farmacología , Resistencia a Antineoplásicos/efectos de los fármacos , Exosomas/genética , MicroARNs/genética , Antibióticos Antineoplásicos/farmacología , Apoptosis/efectos de los fármacos , Apoptosis/genética , Neoplasias de la Mama/genética , Neoplasias de la Mama/patología , Supervivencia Celular/efectos de los fármacos , Supervivencia Celular/genética , Resistencia a Antineoplásicos/genética , Exosomas/ultraestructura , Femenino , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Humanos , Hibridación Fluorescente in Situ , Células MCF-7 , Microscopía Electrónica de Transmisión , Microscopía FluorescenteRESUMEN
We aimed to explore the different protective effects of tirofiban on myocardial ischemia-reperfusion (IR) injury in New Zealand white rabbits by comparing the results from different administration methods. Fifty New Zealand white rabbits were randomly divided into a sham group (group A, n = 10) and four IR groups (group B, IR group with injection of physiological saline; group C, tirofiban administered through marginal ear vein after reperfusion; group D, tirofiban injected through coronary ostia before reperfusion; group E, tirofiban injected through coronary artery after blood flow restoration; all n = 10). Myocardial IR injury models were prepared in IR groups. An automatic biochemical analyzer (HITACHI 7020, Japan) was applied for testing serum creatine kinase-MB levels. The myeloperoxidase activity, malondialdehyde levels, nitric oxide synthase activity, and nitric oxide (NO) volume were detected 180 minutes after reperfusion. The myocardial apoptosis was identified using the terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling technique, and the protein expressions of B-cell lymphoma-2, Bcl-2 associated X, and aquaporin-1 were measured through Western blot. The highest and lowest ST-segment resolution among the IR groups was observed in groups E and B, respectively. The creatine kinase-MB levels at 60, 120, and 180 minutes in group E was greatly decreased than in groups B, C, and D. Compared with the sham group, the IR groups demonstrated evidently elevated myeloperoxidase activity, malondialdehyde levels, inducible NOS activity, NO volume, myocardial apoptotic index, and aquaporin-1 expressions; among the IR groups, these indicators were increased and decreased most in groups B and E, respectively. The B-cell lymphoma-2/Bcl-2 associated X ratio in the IR groups were evidently higher than the sham group, with the highest and lowest rate in groups E and B, respectively. Tirofiban injection through coronary artery after blood flow restoration has a better protective effect against myocardial IR injury than tirofiban administration through coronary ostia before reperfusion and tirofiban injection through the auricular vein after reperfusion.
Asunto(s)
Fibrinolíticos/administración & dosificación , Daño por Reperfusión Miocárdica/tratamiento farmacológico , Tirosina/análogos & derivados , Animales , Apoptosis/efectos de los fármacos , Circulación Coronaria/efectos de los fármacos , Vasos Coronarios/metabolismo , Modelos Animales de Enfermedad , Fibrinolíticos/farmacología , Etiquetado Corte-Fin in Situ , Inyecciones , Daño por Reperfusión Miocárdica/complicaciones , Óxido Nítrico/metabolismo , Conejos , Distribución Aleatoria , Tirofibán , Tirosina/administración & dosificación , Tirosina/farmacologíaRESUMEN
Transport through the cell membrane can be divided into active, passive and vesicular types (exosomes). Exosomes are nano-sized vesicles released by a variety of cells. Emerging evidence shows that exosomes play a critical role in cancers. Exosomes mediate communication between stroma and cancer cells through the transfer of nucleic acid and proteins. It is demonstrated that the contents and the quantity of exosomes will change after occurrence of cancers. Over the last decade, growing attention has been paid to the role of exosomes in the development of breast cancer, the most life-threatening cancer in women. Breast cancer could induce salivary glands to secret specific exosomes, which could be used as biomarkers in the diagnosis of early breast cancer. Exosome-delivered nucleic acid and proteins partly facilitate the tumorigenesis, metastasis and resistance of breast cancer. Exosomes could also transmit anti-cancer drugs outside breast cancer cells, therefore leading to drug resistance. However, exosomes are effective tools for transportation of anti-cancer drugs with lower immunogenicity and toxicity. This is a promising way to establish a drug delivery system.
Asunto(s)
Neoplasias de la Mama/patología , Resistencia a Antineoplásicos/fisiología , Exosomas/metabolismo , Invasividad Neoplásica/patología , Animales , Antineoplásicos/administración & dosificación , Neoplasias de la Mama/tratamiento farmacológico , Sistemas de Liberación de Medicamentos , Femenino , Humanos , Metástasis de la NeoplasiaRESUMEN
BACKGROUND: Currently, exosomes that act as mediators of intercellular communication are being researched extensively. Our previous studies confirmed that these exosomes contain microRNAs (miRNAs) that could alter chemo-susceptibility, which is partly attributed to the successful intercellular transfer of multidrug resistance (MDR)-specific miRNAs. We also confirmed that ß-elemene could influence MDR-related miRNA expression and regulate the expression of the target genes PTEN and Pgp, which may lead to the reversal of the chemoresistant breast cancer (BCA) cells. We are the first to report these findings, and we propose the following logical hypothesis: ß-elemene can mediate MDR-related miRNA expression in cells, thereby affecting the exosome contents, reducing chemoresistance transmission via exosomes, and reversing the drug resistance of breast cancer cells. METHODS: MTT-cytotoxic, miRNA microarray, real-time quantitative PCR, Dual Luciferase Activity Assay, and Western blot analysis were performed to investigate the impact of ß-elemene on the expression of chemoresistance specific miRNA and PTEN as well as Pgp in chemoresistant BCA exosomes. RESULTS: Drug resistance can be reversed by ß-elemene related to exosomes. There were 104 differentially expressed miRNAs in the exosomes of two chemoresistant BCA cells: adriacin (Adr) - resistant MCF-7 cells (MCF-7/Adr) and docetaxel (Doc) - resistant MCF-7 cells (MCF-7/Doc) that underwent treatment. Of these, 31 miRNAs were correlated with the constant changes in the MDR. The expression of miR-34a and miR-452 can lead to changes in the characteristics of two chemoresistant BCA exosomes: MCF-7/Adr exosomes (A/exo) and MCF-7/Doc exosomes (D/exo). The PTEN expression affected by ß-elemene was significantly increased, and the Pgp expression affected by ß-elemene was significantly decreased in both cells and exosomes. ß-elemene induced a significant increase in the apoptosis rate in both MCF-7/Doc and MCF-7/Adr cells. CONCLUSIONS: Drug resistance can be reversed by ß-elemene, which can alter the expression of some MDR-related miRNAs, including PTEN and Pgp in MCF-7/Adr and MCF-7/Doc in cells. It can therefore affect the exosome contents and induce the reduction of resistance transmission via exosomes.