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The isolation and enrichment of specific extracellular vesicle (EV) subpopulations are essential in the context of precision medicine. However, the current methods predominantly rely on a single-positive marker and are susceptible to interference from soluble proteins or impurities. This limitation represents a significant obstacle to the widespread application of EVs in biological research. Herein, a novel approach that utilizes proximity ligation assay (PLA) and DNA-RNA hybridization are proposed to facilitate the binding of two proteins on the EV membrane in advance enabling the isolation and enrichment of intact EVs with double-positive membrane proteins followed by using functionalized magnetic beads for capture and enzymatic cleavage for isolated EVs release. The isolated subpopulations of EVs can be further utilized for cellular uptake studies, high-throughput small RNA sequencing, and breast cancer diagnosis. Hence, developing and implementing a specialized system for isolating and enriching a specific subpopulation of EVs can enhance basic and clinical research in this field.
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Neoplasias de la Mama , Vesículas Extracelulares , Humanos , Femenino , Proteínas de la Membrana/metabolismo , Neoplasias de la Mama/metabolismo , Vesículas Extracelulares/metabolismo , ARN , Separación InmunomagnéticaRESUMEN
BACKGROUND: There is little data on the role of endoscopic stricturotomy (ES) in treating deep small bowel strictures. We aimed to investigate the efficacy and safety of balloon-assisted enteroscopy-based ES (BAE-based ES) for deep small bowel strictures associated with Crohn's disease (CD). METHODS: This multicentre retrospective cohort study included consecutive patients with CD-associated deep small bowel strictures treated with BAE-based ES between 2017 and 2023. The outcomes included technical success, clinical improvement, surgery-free rate, reintervention-free rate, and adverse events. RESULTS: Twenty-eight patients with CD underwent 58 BAE-based ES procedures for non-passable deep small bowel strictures, with a median follow-up time of 519.5 days (interquartile range, 306-728 days). Fifty-six (96.0%) procedures were technically successful in 26 (92.9%) patients. Twenty patients (71.4%) showed clinical improvement at week 8. The cumulative surgery-free rate at 1 year was 74.8% (95% confidence interval [CI], 60.3-92.9%). A higher body mass index was associated with a decreased need for surgery (hazard ratio = 0.084, 95% CI, 0.016-0.45, P = 0.0036). Postprocedural adverse events (bleeding and perforation) requiring reintervention occurred in 3.4% of the procedures. CONCLUSIONS: The novel BAE-based ES provides high technical success, favorable efficacy, and safety in CD-associated deep small bowel strictures, which may provide an alternative for endoscopic balloon dilation and surgery.
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Enfermedad de Crohn , Obstrucción Intestinal , Humanos , Enfermedad de Crohn/cirugía , Constricción Patológica/etiología , Constricción Patológica/cirugía , Estudios de Cohortes , Estudios Retrospectivos , Resultado del Tratamiento , Obstrucción Intestinal/etiología , Obstrucción Intestinal/cirugía , Dilatación/métodos , Endoscopía Gastrointestinal/métodosRESUMEN
Activated M2-polarized macrophages are drivers of pulmonary fibrosis in several clinical scenarios, including Idiopathic Pulmonary Fibrosis (IPF). In this study, we investigated the effects of targeting the CD206 receptor in M2-like macrophages with a novel synthetic analogue of a naturally occurring Host Defense Peptide (HDP), RP-832c, to decrease profibrotic cytokines. RP-832c selectively binds to CD206 on M2-polarized bone marrow-derived macrophages (BMDM) in vitro, resulting in a time-dependent decrease in CD206 expression and a transient increase in M1-macrophage marker TNF-α. To elucidate the antifibrotic effects of RP-832c, we used a murine model of bleomycin (BLM)-induced early-stage pulmonary fibrosis. RP-832c significantly reduced fibrosis in a dose-dependent manner, and decreased CD206, TGF-ß1, and α-SMA expression in mouse lungs. Similarly, in an established model of lung fibrosis, RP-832c significantly decreased lung fibrosis and significantly decreased inflammatory cytokines TNF-α, IL-6, IL-10, IFN-γ, CXCL1/2, and fibrosis markers TGF-ß1 and MMP-13. In comparison with the FDA-approved drugs Nintedanib and Pirfenidone, RP-832c exhibited a similar reduction in fibrosis compared to Pirfenidone, and to a greater extent than Nintedanib, with no apparent toxicities observed. In summary, our findings showed that inhibiting the profibrotic alternatively activated M2-like macrophages using a novel peptide, RP-832c, could reduce BLM-induced pulmonary fibrosis in mice, warranting the therapeutic potential of this peptide for patients with pulmonary fibrosis.
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Fibrosis Pulmonar Idiopática , Factor de Crecimiento Transformador beta1 , Animales , Ratones , Bleomicina/efectos adversos , Citocinas , Fibrosis Pulmonar Idiopática/inducido químicamente , Fibrosis Pulmonar Idiopática/metabolismo , Factor de Necrosis Tumoral alfaRESUMEN
African American (AA) women with breast cancer are more likely to have higher inflammation and a stronger overall immune response, which correlate with poorer outcomes. In this report, we applied the nanostring immune panel to identify differences in inflammatory and immune gene expression by race. We observed a higher expression of multiple cytokines in AA patients compared to EA patients, with high expression of CD47, TGFB1, and NFKB1 associated with the transcriptional repressor Kaiso. To investigate the mechanism associated with this expression pattern, we observed that Kaiso depletion results in decreased expression of CD47, and its ligand SIRPA. Furthermore, Kaiso appears to directly bind to the methylated sequences of the THBS1 promotor and repress gene expression. Similarly, Kaiso depletion attenuated tumor formation in athymic nude mice, and these Kaiso-depleted xenograft tissues showed significantly higher phagocytosis and increased infiltration of M1 macrophages. In vitro validation using MCF7 and THP1 macrophages treated with Kaiso-depleted exosomes showed a reduced expression of immune-related markers (CD47 and SIRPA) and macrophage polarization towards the M1 phenotype compared to MCF7 cells treated with exosomes isolated from high-Kaiso cells. Lastly, analysis of TCGA breast cancer patient data demonstrates that this gene signature is most prominent in the basal-like subtype, which is more frequently observed in AA breast cancer patients.
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Aflatoxin B1 (AFB1), the most harmful aflatoxins, is a frequent contamination in feed and food items, raising global concerns in animal production and human public health. Also, AFB1 induces oxidative stress, cytotoxicity, mutations, and DNA lesions through its metabolic transformation into aflatoxin B1-8,9-epoxide (AFBO) by cytochrome P450 (CYP450). Hedyotis diffusa (HD) is a traditional Chinese herbal medicine known for its multiple pharmacological activities, including antioxidant, anti-inflammatory, and immunomodulatory. Yet, the influence of HD on AFB1-induced liver injury in ducks is still unknown. Here, we investigated whether HD positively affects AFB1-induced liver injury in ducks. Results revealed that I) AFB1 caused significant changes in serum biochemical indices and decreased growth performance of ducks (such as ALT, AST, ALP, TP, ALB, final body weight, and body weight gain), whereas HD supplementation at 200 mg/kg mitigated these alterations. II) HD alleviated hepatic histopathological changes and liver index induced by AFB1 in ducks. III) HD significantly attenuated AFB1-induced oxidative stress, as measured by increased antioxidant enzyme activities such as SOD, GPx, and T-AOC and decreased MDA levels. Furthermore, HD reduced the level of AFB1-DNA adduct in duck liver. IV) HD significantly promoted the transcriptional expression of NF-E2-related nuclear factor 2 (Nrf2) and associated genes, including heme oxygenase 1 (HO-1), NAD(P)H dehydrogenase quinone 1 (NQO1), glutamate-cysteine ligase catalytic (GCLC). In conclusion, these results demonstrated that HD could activate the Nrf2 pathway in ducks to reduce the hepatotoxicity driven by AFB1. This finding also provides theoretical and data support for a deeper understanding of the toxic mechanisms of AFB1 and its prevention.
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Aflatoxina B1 , Enfermedad Hepática Inducida por Sustancias y Drogas , Medicamentos Herbarios Chinos , Hedyotis , Hígado , Factor 2 Relacionado con NF-E2 , Animales , Humanos , Aflatoxina B1/toxicidad , Antioxidantes/farmacología , Antioxidantes/uso terapéutico , Peso Corporal , Patos , Hedyotis/química , Hígado/efectos de los fármacos , Hígado/metabolismo , Factor 2 Relacionado con NF-E2/genética , Factor 2 Relacionado con NF-E2/metabolismo , Transducción de Señal , Enfermedad Hepática Inducida por Sustancias y Drogas/terapia , Medicamentos Herbarios Chinos/farmacología , Medicamentos Herbarios Chinos/uso terapéuticoRESUMEN
Breast cancer has become the leading cause of global cancer incidence and a serious threat to women's health. Accurate diagnosis and early treatment are of great importance to prognosis. Although clinically used diagnostic approaches can be used for cancer screening, accurate diagnosis of breast cancer is still a critical unmet need. Here, we report a 4-plex droplet digital PCR technology for simultaneous detection of four small extracellular vesicle (sEV)-derived mRNAs (PGR, ESR1, ERBB2 and GAPDH) in combination with machine learning (ML) algorithms to improve breast cancer diagnosis. We evaluate the diagnsotic results with and without the assistance of the ML models. The results indicate that ML-assisted analysis exhibits higher diagnostic performance even using a single marker for breast cancer diagnosis, and demonstrate improved diagnostic performance under the best combination of biomarkers and suitable ML diagnostic model. Therefore, multiple sEV-derived mRNAs analysis coupled with ML not only provides the best combination of markers for breast cancer diagnosis, but also significantly improves the diagnostic efficiency of breast cancer.
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Técnicas Biosensibles , Neoplasias de la Mama , Vesículas Extracelulares , Neoplasias de la Mama/diagnóstico , Neoplasias de la Mama/genética , Femenino , Humanos , Aprendizaje Automático , Reacción en Cadena de la Polimerasa , ARN Mensajero/genéticaRESUMEN
Activated M2 polarized macrophages are drivers of pulmonary fibrosis in several clinical scenarios such as Acute Respiratory Disease Syndrome (ARDS) and Idiopathic Pulmonary Fibrosis (IPF), through the production of inflammatory and fibrosis-inducing cytokines. In this study, we investigated the effect of targeting the CD206 receptor with a novel fragment of a Host Defense Peptide (HDP), RP-832c to decrease cytokines that cause fibrosis. RP-832c selectively binds to CD206 on M2 polarized bone marrow derived macrophages (BMDM) in vitro , resulting in a time-dependent decrease in CD206 expression, and a transient increase in M1 marker TNFα, which resolves over a 24hr period. To elucidate the antifibrotic effect of RP-832c, we used a murine model of bleomycin (BLM) -induced early-stage pulmonary fibrosis. RP-832c significantly reduced bleomycin-induced fibrosis in a dosage dependent manner, as well as decreased CD206, TGF-ß1 and α-SMA expression in mouse lungs. Interestingly we did not observe any changes in the resident alveolar macrophage marker CD170 expression. Similarly, in an established model of lung fibrosis, RP-832c significantly decreased fibrosis in the lung, as well as significantly decreased inflammatory cytokines TNFα, IL-6, IL-10, INF-γ, CXCL1/2, and fibrosis markers TGF-ß1 and MMP-13. In comparison with FDA approved drugs, Nintedanib and Pirfenidone, RP-832c exhibited a similar reduction in fibrosis compared to Pirfenidone, and to a greater extent than Nintedanib, with no apparent toxicities observed on body weight or blood chemistry. In summary, RP-832c is a potential agent to mitigate the overactivity of M2 macrophages in pathogenesis several pulmonary fibrotic diseases, including SARS-CoV-2 induced lung fibrosis.
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Extracellular vesicles (EVs) loaded with proteins, nucleic acids, membrane lipids, and other virulence factors could participate in pathogenic processes in some fungi such as Cryptococcus neoformans and Candida albicans. However, the specific characteristics of EVs derived from Talaromyces marneffei (TM) still have not been figured out yet. In the present study, it has been observed that TM-derived EVs were a heterogeneous group of nanosized membrane vesicles (30-300 nm) under nanoparticle tracking analysis and transmission electron microscopy. The DiI-labeled EVs could be taken up by RAW 264.7 macrophage cells. Incubation of EVs with macrophages would result in increased expression levels of reactive oxygen species, nitric oxide, and some inflammatory factors including interleukin-1ß, interleukin-6, interleukin-10, and tumor necrosis factor. Furthermore, the expression of co-stimulatory molecules (CD80, CD86, and MHC-II) was also increased in macrophages stimulated with EVs. The level of inflammatory factors secreted by macrophages showed a significant decrease when EVs were hydrolyzed by protease, while that of DNA and RNA hydrolase treatment remained unchanged. Subsequently, some virulence factors in EVs including heat shock protein, mannoprotein 1, and peroxidase were determined by liquid chromatography-tandem mass spectrometry. Taken together, our results indicated that the TM-derived EVs could mediate inflammatory response and its protein would play a key role in regulating the function of RAW 264.7 macrophage cells.
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Atrial fibrillation (AF) is a highly prevalent arrhythmia with pronounced morbidity and mortality. Inward-rectifier K+ current (IK1) is believed to be an important regulator of reentrant-spiral dynamics and a major component of AF-related electrical remodeling. MicroRNA-26 (miR-26) is predicted to target the gene encoding KIR2.1, KCNJ2. We found that miR-26 was downregulated in atrial samples from AF animals and patients and this downregulation was accompanied by upregulation of IK1/KIR2.1 protein. miR-26 overexpression suppressed expression of KCNJ2/KIR2.1. In contrast, miR-26 knockdown, inhibition, or binding-site mutation enhanced KCNJ2/KIR2.1 expression, establishing KCNJ2 as a miR-26 target. Knockdown of endogenous miR-26 promoted AF in mice, whereas adenovirus-mediated expression of miR-26 reduced AF vulnerability. Kcnj2-specific miR-masks eliminated miR-26-mediated reductions in Kcnj2, abolishing miR-26's protective effects, while coinjection of a Kcnj2-specific miR-mimic prevented miR-26 knockdown-associated AF in mice. Nuclear factor of activated T cells (NFAT), a known actor in AF-associated remodeling, was found to negatively regulate miR-26 transcription. Our results demonstrate that miR-26 controls the expression of KCNJ2 and suggest that this downregulation may promote AF.
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Fibrilación Atrial/metabolismo , Regulación de la Expresión Génica , MicroARNs/metabolismo , Canales de Potasio de Rectificación Interna/metabolismo , Animales , Sitios de Unión , Células Cultivadas , Perros , Regulación hacia Abajo , Femenino , Humanos , Masculino , Ratones , Ratones Endogámicos C57BL , Mutación , Miocitos Cardíacos/citología , Factores de Transcripción NFATC/metabolismo , Potasio/química , Ratas , Transcripción GenéticaRESUMEN
The human ether-à-go-go-1 (h-eag1) K(+) channel is expressed in a variety of cell lines derived from human malignant tumors and in clinical samples of several different cancers, but is otherwise absent in normal tissues. It was found to be necessary for cell cycle progression and tumorigenesis. Specific inhibition of h-eag1 expression leads to inhibition of tumor cell proliferation. We report here that h-eag1 expression is controlled by the p53-miR-34-E2F1 pathway through a negative feed-forward mechanism. We first established E2F1 as a transactivator of h-eag1 gene through characterizing its promoter region. We then revealed that miR-34, a known transcriptional target of p53, is an important negative regulator of h-eag1 through dual mechanisms by directly repressing h-eag1 at the post-transcriptional level and indirectly silencing h-eag1 at the transcriptional level via repressing E2F1. There is a strong inverse relationship between the expression levels of miR-34 and h-eag1 protein. H-eag1antisense antagonized the growth-stimulating effects and the upregulation of h-eag1 expression in SHSY5Y cells, induced by knockdown of miR-34, E2F1 overexpression, or inhibition of p53 activity. Therefore, p53 negatively regulates h-eag1 expression by a negative feed-forward mechanism through the p53-miR-34-E2F1 pathway. Inactivation of p53 activity, as is the case in many cancers, can thus cause oncogenic overexpression of h-eag1 by relieving the negative feed-forward regulation. These findings not only help us understand the molecular mechanisms for oncogenic overexpression of h-eag1 in tumorigenesis but also uncover the cell-cycle regulation through the p53-miR-34-E2F1-h-eag1 pathway. Moreover, these findings place h-eag1 in the p53-miR-34-E2F1-h-eag1 pathway with h-eag as a terminal effecter component and with miR-34 (and E2F1) as a linker between p53 and h-eag1. Our study therefore fills the gap between p53 pathway and its cellular function mediated by h-eag1.
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Proliferación Celular/efectos de los fármacos , Canales de Potasio Éter-A-Go-Go/metabolismo , Benzotiazoles/farmacología , Western Blotting , Línea Celular Tumoral , Inmunoprecipitación de Cromatina , Factor de Transcripción E2F1/genética , Factor de Transcripción E2F1/metabolismo , Ensayo de Cambio de Movilidad Electroforética , Canales de Potasio Éter-A-Go-Go/genética , Humanos , Imidazoles/farmacología , MicroARNs/genética , MicroARNs/metabolismo , Oligodesoxirribonucleótidos Antisentido/farmacología , Piperazinas/farmacología , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Tolueno/análogos & derivados , Tolueno/farmacología , Proteína p53 Supresora de Tumor/genética , Proteína p53 Supresora de Tumor/metabolismoRESUMEN
In cancers with wild-type (WT) p53 status, the function of p53 is inhibited through direct interaction with Mdm2 oncoprotein, a negative feedback loop to limit the function of p53. In response to cellular stress, p53 escapes the p53:Mdm2 negative feedback to accumulate rapidly to induce cell cycle arrest and apoptosis. We demonstrate herein that an microRNA miR-605 is a new component in the p53 gene network, being transcriptionally activated by p53 and post-transcriptionally repressing Mdm2. Activation of p53 upregulated miR-605 via interacting with the promoter region of the gene. Overexpression of miR-605 directly decreased Mdm2 expression at the post-transcriptional level but indirectly increased the transcriptional activity of p53 on miR-34a via downregulating Mdm2; knockdown of miR-605 did the opposite. Mdm2 inhibitor upregulated expression of both miR-34a and miR-605, which was mitigated by p53 inhibitor. miR-605 preferentially induced apoptosis in WT p53-expressing cells, an effect abolished by p53 inhibition. These results indicate that miR-605 acts to interrupt p53:Mdm2 interaction to create a positive feedback loop aiding rapid accumulation of p53 to facilitate its function in response to stress.
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Apoptosis/fisiología , Retroalimentación Fisiológica/fisiología , Regulación de la Expresión Génica/fisiología , MicroARNs/metabolismo , Proteínas Proto-Oncogénicas c-mdm2/metabolismo , Estrés Fisiológico/fisiología , Proteína p53 Supresora de Tumor/metabolismo , Análisis de Varianza , Apoptosis/genética , Western Blotting , Caspasa 3/metabolismo , Línea Celular Tumoral , Inmunoprecipitación de Cromatina , Proteína Quinasa Dependiente de GMP Cíclico Tipo I , Proteínas Quinasas Dependientes de GMP Cíclico/genética , Ensayo de Cambio de Movilidad Electroforética , Ensayo de Inmunoadsorción Enzimática , Femenino , Regulación de la Expresión Génica/genética , Humanos , Etiquetado Corte-Fin in Situ , Luciferasas , MicroARNs/genética , Plásmidos/genética , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Estrés Fisiológico/genética , Sales de Tetrazolio , TiazolesRESUMEN
Cardiac hypertrophy is characterized by electrical remolding with increased risk of arrhythmogenesis. Enhanced abnormal automaticity of ventricular cells may contribute to hypertrophic arrhythmias. The pacemaker current I(f), carried by the hyperpolarization-activated channels encoded mainly by the HCN2 and HCN4 genes in the heart, plays an important role in rhythmogenesis. Their expressions reportedly increase in hypertrophic and failing hearts, contributing to arrhythmogenesis under these conditions. However, how their expressions are controlled remained unclear. We performed a study to characterize the regulatory elements and transcriptional control of HCN2 and HCN4 genes. We identified the transcription start sites by 5'RACE and core promoter regions of these genes using luciferase reporter assay, and revealed the ubiquitous Sp1 protein as a common transactivator of HCN2 and HCN4 genes. We further unraveled robust increases in HCN2/HCN4 transcripts and protein levels, using real-time RT-PCR and Western blot analyses, in a rat model of left ventricular hypertrophy and in angiotensin II-induced neonatal ventricular hypertrophy. The upregulation of HCN2 and HCN4 transcription was accompanied by pronounced elevations of Sp1 and silencing of Sp1 by siRNA prevented overexpression of HCN2/HCN4 in hypertrophic cardiomyocytes. Our data indicate that Sp1 drives HCN2/HCN4 transcription and determines the functional level of HCN2/HCN4 mRNAs, and upregulation of Sp1 underlie the abnormal re-expression of HCN2/HCN4 genes in hypertrophied myocytes. This study also provides the first evidence for the role of Sp1 in the reactivation of 'fetal' cardiac genes, HCN2 and HCN4, in ventricular myocytes, and thereby in the pathological electrical remodeling in hypertrophied myocytes.
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Canales Catiónicos Regulados por Nucleótidos Cíclicos/genética , Hipertrofia Ventricular Izquierda/genética , Inmunoglobulinas/metabolismo , Canales Iónicos/genética , Proteínas Musculares/genética , Miocitos Cardíacos/metabolismo , Canales de Potasio/genética , Angiotensina II/metabolismo , Angiotensina II/farmacología , Animales , Secuencia de Bases , Línea Celular , Canales Catiónicos Regulados por Nucleótidos Cíclicos/metabolismo , Humanos , Canales Regulados por Nucleótidos Cíclicos Activados por Hiperpolarización , Hipertrofia Ventricular Izquierda/metabolismo , Hipertrofia Ventricular Izquierda/patología , Inmunoglobulinas/genética , Canales Iónicos/metabolismo , Datos de Secuencia Molecular , Proteínas Musculares/metabolismo , Canales de Potasio/metabolismo , Regiones Promotoras Genéticas , ARN Mensajero/metabolismo , ARN Interferente Pequeño/metabolismo , Ratas , Transcripción Genética , Regulación hacia ArribaRESUMEN
Anti-miRNA antisense inhibitors (AMOs) have demonstrated their utility in miRNA research and potential in miRNA therapy. Here we report a modified AMO approach in which multiple antisense units are engineered into a single unit that is able to simultaneously silence multiple-target miRNAs, the multiple-target AMO or MTg-AMO. We validated the technique with two separate MTg-AMOs: anti-miR-21/anti-miR-155/anti-miR-17-5p and anti-miR-1/anti-miR-133. We first verified the ability of the MTg-AMOs to antagonize the repressive actions of their target miRNAs using luciferase reporter activity assays and to specifically knock down the levels of their target miRNAs using real-time RT-PCR methods. We then used the MTg-AMO approach to identify several tumor suppressors-TGFBI, APC and BCL2L11 as the target genes for oncogenic miR-21, miR-155 and miR-17-5p, respectively, and two cardiac ion channel genes HCN2 (encoding a subunit of cardiac pacemaker channel) and CACNA1C (encoding the alpha-subunit of cardiac L-type Ca(2+) channel) for the muscle-specific miR-1 and miR-133. We further demonstrated that the MTg-AMO targeting miR-21, miR-155 and miR-17-5p produced a greater inhibitory effect on cancer cell growth, compared with the regular single-target AMOs. Moreover, while using the regular single-target AMOs excluded HCN2 as a target gene for either miR-1 or miR-133, the MTg-AMO approach is able to reveal HCN2 as the target for both miR-1 and miR-133. Our findings suggest the MTg-AMO as an improved approach for miRNA target finding and for studying function of miRNAs. This approach may find its broad application for exploring biological processes involving multiple miRNAs and multiple genes.
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MicroARNs/antagonistas & inhibidores , Oligodesoxirribonucleótidos Antisentido/química , Interferencia de ARN , Animales , Línea Celular Tumoral , Proliferación Celular , Células Cultivadas , Genes Supresores de Tumor , Ingeniería Genética , Humanos , Canales Iónicos/genética , MicroARNs/química , Neoplasias/patología , Neoplasias/terapia , RatasRESUMEN
BACKGROUND: Inhibition of individual K(+) currents causes functionally based compensatory increases in other K(+) currents that minimize changes in action potential duration, a phenomenon known as repolarization reserve. The possibility that sustained K(+) channel inhibition may induce remodeling of ion current expression has not been tested. Accordingly, we assessed the effects of sustained inhibition of one K(+) current on various other cardiac ionic currents. METHODS AND RESULTS: Adult canine left ventricular cardiomyocytes were incubated in primary culture and paced at a physiological rate (1 Hz) for 24 hours in the presence or absence of the highly selective rapid delayed-rectifier K(+) current (I(Kr)) blocker dofetilide (5 nmol/L). Sustained dofetilide exposure led to shortened action potential duration and increased repolarization reserve (manifested as a reduced action potential duration-prolonging response to I(Kr) blockade). These repolarization changes were accompanied by increased slow delayed-rectifier (I(Ks)) density, whereas I(Kr), transient-outward (I(to)), inward-rectifier (I(K1)), L-type Ca(2+) (I(CaL)), and late Na(+) current remained unchanged. The mRNA expression corresponding to KvLQT1 and minK (real-time polymerase chain reaction) was unchanged, but their protein expression (Western blot) was increased, suggesting posttranscriptional regulation. To analyze possible mechanisms, we quantified the muscle-specific microRNA subtypes miR-133a and miR-133b, which can posttranscriptionally regulate and repress KvLQT1 protein expression without affecting mRNA expression. The expression levels of miR-133a and miR-133b were significantly decreased in cells cultured in dofetilide compared with control, possibly accounting for KvLQT1 protein upregulation. CONCLUSIONS: Sustained reductions in I(Kr) may lead to compensatory upregulation of I(Ks) through posttranscriptional upregulation of underlying subunits, likely mediated (at least partly) by microRNA changes. These results suggest that feedback control of ion channel expression may influence repolarization reserve.
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Canal de Potasio KCNQ1/biosíntesis , Miocardio/metabolismo , Miocitos Cardíacos/metabolismo , Fenetilaminas/farmacología , Bloqueadores de los Canales de Potasio/farmacología , Sulfonamidas/farmacología , Regulación hacia Arriba/efectos de los fármacos , Animales , Estimulación Cardíaca Artificial , Células Cultivadas , Perros , Ventrículos Cardíacos/citología , Ventrículos Cardíacos/metabolismo , Humanos , MicroARNs/metabolismo , Miocardio/citología , Miocitos Cardíacos/citología , Potasio/metabolismo , ARN Mensajero/biosíntesis , Sodio/metabolismoRESUMEN
Cardiac hypertrophy is characterized by electrical remolding with increased risk of arrhythmogenesis. Enhanced abnormal automaticity of ventricular cells contributes critically to hypertrophic arrhythmias. The pacemaker current I(f), carried by the hyperpolarization-activated channels encoded mainly by the HCN2 and HCN4 genes in the heart, plays an important role in determining cardiac automaticity. Their expressions reportedly increase in hypertrophic and failing hearts, contributing to arrhythmogenesis under these conditions. We performed a study on post-transcriptional regulation of expression of HCN2 and HCN4 genes by microRNAs. We experimentally established HCN2 as a target for repression by the muscle-specific microRNAs miR-1 and miR-133 and established HCN4 as a target for miR-1 only. We unraveled robust increases in HCN2 and HCN4 protein levels in a rat model of left ventricular hypertrophy and in angiotensin II-induced neonatal ventricular hypertrophy. The up-regulation of HCN2/HCN4 was accompanied by pronounced reduction of miR-1/miR-133 levels. Forced expression of miR-1/miR-133 by transfection prevented overexpression of HCN2/HCN4 in hypertrophic cardiomyocytes. The serum-responsive factor protein level was found significantly decreased in hypertrophic hearts, and silencing of this protein by RNA interference resulted in increased levels of miR-1/miR-133 and concomitant increases in HCN2 and HCN4 protein levels. We conclude that down-regulation of miR-1 and miR-133 expression contributes to re-expression of HCN2/HCN4 and thereby the electrical remodeling process in hypertrophic hearts. Our study also sheds new light on the cellular function and pathological role of miR-1/miR-133 in the heart.
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Cardiomegalia/genética , Canales Catiónicos Regulados por Nucleótidos Cíclicos/genética , Regulación hacia Abajo , Canales Iónicos/genética , MicroARNs/genética , Proteínas Musculares/genética , Canales de Potasio/genética , Animales , Animales Recién Nacidos , Secuencia de Bases , Células Cultivadas , Ventrículos Cardíacos/metabolismo , Canales Regulados por Nucleótidos Cíclicos Activados por Hiperpolarización , Masculino , Ratas , Ratas Sprague-DawleyRESUMEN
Cardiac tissues contain cells susceptible to and cells resistant to apoptosis, and this difference is important for normal morphogenesis during development and for abnormal loss of cells during pathogenesis such as myocardial infarction and heart failure. While efforts have been made to understand the cellular and intercellular events of apoptotic cells, the signaling mechanisms in cells surviving from apoptotic injuries have been overlooked. Understanding signal transduction processes in cells with apoptosis resistance is of crucial importance to develop better strategies of preserving post-mitotic cells. To this end, we performed studies in neonatal rat ventricular myocytes using oxidative stress (H(2)O(2)) as an apoptotic inducer. We identified a population of cells bearing higher resistance to apoptosis and found that the cells that survived from apoptotic insults had markedly higher levels of AKT and STAT3. Inhibition of AKT activity by a dominant negative AKT construct or by a PI3K inhibitor reduced active NF-kappaB and STAT3 expression without significantly altering the activity of the latter. Activation of AKT by a constitutively activated AKT construct caused the opposite effects. Direct activation of NF-kappaB also enhanced STAT3 expression, an effect abrogated by NF-kappaB inhibitor. On the other hand, knockdown of STAT3 by siRNA or inhibition of STAT3 activity by decoy oligodeoxynucleotides or by JAK2 inhibitor diminished AKT expression. In conclusion, cardiomyocytes possess an apoptosis-resistant property as a cytoprotection mechanism which is likely conferred by mutual transactivation between AKT/NF-kappaB and JAK2/STAT3, a novel crosstalk between the two signaling pathways within the networking governing the cell fate.
Asunto(s)
Apoptosis , Miocitos Cardíacos/metabolismo , Estrés Oxidativo , Proteínas Quinasas/metabolismo , Factores de Transcripción STAT/metabolismo , Transducción de Señal , Activación Transcripcional , Animales , Apoptosis/efectos de los fármacos , Células Cultivadas , Peróxido de Hidrógeno/farmacología , Quinasas Janus/metabolismo , Miocitos Cardíacos/citología , Miocitos Cardíacos/efectos de los fármacos , FN-kappa B/metabolismo , Estrés Oxidativo/efectos de los fármacos , Fosfatidilinositol 3-Quinasas/metabolismo , Proteínas Proto-Oncogénicas c-akt/metabolismo , Ratas , Transducción de Señal/efectos de los fármacosRESUMEN
The long QT syndrome genes human ether-a-go-go-related gene (HERG1) and voltage-gated K+ channel, KQT-like subfamily, member 1, gene (KCNQ1), encoding K+ channels critical to the repolarization rate and repolarization reserve in cardiac cells, and thereby the likelihood of arrhythmias, are both composed of two isoforms: HERG1a and HERG1b and KCNQ1a and KCNQ1b, respectively. Expression of these genes is dynamic, depending on the differentiation status and disease states. We identified their core promoter regions and transcription start sites. Our data suggest that HERG1a and HERG1b, and KCNQ1a and KCNQ1b, represent independent transcripts instead of being alternatively spliced variants of the same gene, for they each have their own transcription start sites and their own promoter regions. We obtained data pointing to the potential role of stimulating protein 1 (Sp1) in the transactivation of these genes. We compared expression profiling of these genes across a variety of human tissues. Consistent with the general lack of cis elements for cardiac-specific transcription factors and the presence of multiple sites for ubiquitous Sp1 sites in the core promoter regions of HERG1a/HERG1b and KCNQ1a/KCNQ1b genes, the transcripts demonstrated widespread distribution across a variety of human tissues. We further revealed that the mRNA levels of all HERG1 and KCNQ1 isoforms were asymmetrically distributed within the heart, being more abundant in the right atria and ventricles relative to the left atria and ventricles. These findings open up an opportunity for studying interventricular gradients of slow and rapid delayed rectifier K+ current and of cardiac repolarization as well. Our study might help us understand the molecular mechanisms for arrhythmias since heterogeneity of ion channel activities is an important substrate for arrhythmogenesis.