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Increasing evidence suggests that transcriptional control and chromatin activities at large involve regulatory RNAs, which likely enlist specific RNA-binding proteins (RBPs). Although multiple RBPs have been implicated in transcription control, it has remained unclear how extensively RBPs directly act on chromatin. We embarked on a large-scale RBP ChIP-seq analysis, revealing widespread RBP presence in active chromatin regions in the human genome. Like transcription factors (TFs), RBPs also show strong preference for hotspots in the genome, particularly gene promoters, where their association is frequently linked to transcriptional output. Unsupervised clustering reveals extensive co-association between TFs and RBPs, as exemplified by YY1, a known RNA-dependent TF, and RBM25, an RBP involved in splicing regulation. Remarkably, RBM25 depletion attenuates all YY1-dependent activities, including chromatin binding, DNA looping, and transcription. We propose that various RBPs may enhance network interaction through harnessing regulatory RNAs to control transcription.
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Cromatina/metabolismo , Proteínas de Unión al ARN/metabolismo , ARN/metabolismo , Transcripción Genética/genética , Factor de Transcripción YY1/metabolismo , Sitios de Unión , Regulación de la Expresión Génica , Genoma Humano/genética , Células Hep G2 , Humanos , Células K562 , Proteínas Nucleares , Regiones Promotoras Genéticas/genética , Unión Proteica , Proteínas de Unión al ARN/genética , RNA-Seq , Transcriptoma , Factor de Transcripción YY1/genéticaRESUMEN
CTCF is crucial for chromatin structure and transcription regulation in early embryonic development. However, the kinetics of CTCF chromatin occupation in preimplantation embryos have remained unclear. In this study, we used CUT&RUN technology to investigate CTCF occupancy in mouse preimplantation development. Our findings revealed that CTCF begins binding to the genome prior to zygotic genome activation (ZGA), with a preference for CTCF-anchored chromatin loops. Although the majority of CTCF occupancy is consistently maintained, we identified a specific set of binding sites enriched in the mouse-specific short interspersed element (SINE) family B2 that are restricted to the cleavage stages. Notably, we discovered that the neuroprotective protein ADNP counteracts the stable association of CTCF at SINE B2-derived CTCF-binding sites. Knockout of Adnp in the zygote led to impaired CTCF binding signal recovery, failed deposition of H3K9me3, and transcriptional derepression of SINE B2 during the morula-to-blastocyst transition, which further led to unfaithful cell differentiation in embryos around implantation. Our analysis highlights an ADNP-dependent restriction of CTCF binding during cell differentiation in preimplantation embryos. Furthermore, our findings shed light on the functional importance of transposable elements (TEs) in promoting genetic innovation and actively shaping the early embryo developmental process specific to mammals.
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Cromatina , Desarrollo Embrionario , Animales , Ratones , Sitios de Unión , Blastocisto/metabolismo , Cromatina/metabolismo , Desarrollo Embrionario/genética , Regulación del Desarrollo de la Expresión Génica , Proteínas de Homeodominio/metabolismo , Mamíferos , Ratones Noqueados , Proteínas del Tejido Nervioso/metabolismo , Cigoto/metabolismoRESUMEN
Many proteins regulate the expression of genes by binding to specific regions encoded in the genome1. Here we introduce a new data set of RNA elements in the human genome that are recognized by RNA-binding proteins (RBPs), generated as part of the Encyclopedia of DNA Elements (ENCODE) project phase III. This class of regulatory elements functions only when transcribed into RNA, as they serve as the binding sites for RBPs that control post-transcriptional processes such as splicing, cleavage and polyadenylation, and the editing, localization, stability and translation of mRNAs. We describe the mapping and characterization of RNA elements recognized by a large collection of human RBPs in K562 and HepG2 cells. Integrative analyses using five assays identify RBP binding sites on RNA and chromatin in vivo, the in vitro binding preferences of RBPs, the function of RBP binding sites and the subcellular localization of RBPs, producing 1,223 replicated data sets for 356 RBPs. We describe the spectrum of RBP binding throughout the transcriptome and the connections between these interactions and various aspects of RNA biology, including RNA stability, splicing regulation and RNA localization. These data expand the catalogue of functional elements encoded in the human genome by the addition of a large set of elements that function at the RNA level by interacting with RBPs.
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Proteínas de Unión al ARN/química , Proteínas de Unión al ARN/metabolismo , Transcriptoma/genética , Empalme Alternativo/genética , Secuencia de Bases , Sitios de Unión , Línea Celular , Cromatina/genética , Cromatina/metabolismo , Bases de Datos Genéticas , Femenino , Técnicas de Silenciamiento del Gen , Humanos , Espacio Intracelular/genética , Masculino , Unión Proteica , ARN Mensajero/química , ARN Mensajero/genética , ARN Mensajero/metabolismo , Proteínas de Unión al ARN/genética , Especificidad por SustratoRESUMEN
Mutations in several general pre-mRNA splicing factors have been linked to myelodysplastic syndromes (MDSs) and solid tumors. These mutations have generally been assumed to cause disease by the resultant splicing defects, but different mutations appear to induce distinct splicing defects, raising the possibility that an alternative common mechanism is involved. Here we report a chain of events triggered by multiple splicing factor mutations, especially high-risk alleles in SRSF2 and U2AF1, including elevated R-loops, replication stress, and activation of the ataxia telangiectasia and Rad3-related protein (ATR)-Chk1 pathway. We further demonstrate that enhanced R-loops, opposite to the expectation from gained RNA binding with mutant SRSF2, result from impaired transcription pause release because the mutant protein loses its ability to extract the RNA polymerase II (Pol II) C-terminal domain (CTD) kinase-the positive transcription elongation factor complex (P-TEFb)-from the 7SK complex. Enhanced R-loops are linked to compromised proliferation of bone-marrow-derived blood progenitors, which can be partially rescued by RNase H overexpression, suggesting a direct contribution of augmented R-loops to the MDS phenotype.
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Secuencia de Bases/genética , Síndromes Mielodisplásicos/genética , Factores de Empalme de ARN/genética , Puntos de Control del Ciclo Celular/genética , Células HEK293 , Humanos , Mutación , Proteínas Nucleares/genética , Fosfoproteínas/genética , Empalme del ARN/genética , Factores de Empalme de ARN/metabolismo , Ribonucleoproteínas/genética , Factores de Empalme Serina-Arginina/genética , Factor de Empalme U2AF/genéticaRESUMEN
Patients with Hutchinson-Gilford progeria syndrome (HGPS) present with a number of premature aging phenotypes, including DNA damage accumulation, and many of them die of cardiovascular complications. Although vascular pathologies have been reported, whether HGPS patients exhibit cardiac dysfunction and its underlying mechanism is unclear, rendering limited options for treating HGPS-related cardiomyopathy. In this study, we reported a cardiac atrophy phenotype in the LmnaG609G/G609G mice (hereafter, HGPS mice). Using a GFP-based reporter system, we demonstrated that the efficiency of nonhomologous end joining (NHEJ) declined by 50% in HGPS cardiomyocytes in vivo, due to the attenuated interaction between γH2AX and Progerin, the causative factor of HGPS. As a result, genomic instability in cardiomyocytes led to an increase of CHK2 protein level, promoting the LKB1-AMPKα interaction and AMPKα phosphorylation, which further led to the activation of FOXO3A-mediated transcription of atrophy-related genes. Moreover, inhibiting AMPK enlarged cardiomyocyte sizes both in vitro and in vivo. Most importantly, our proof-of-concept study indicated that isoproterenol treatment significantly reduced AMPKα and FOXO3A phosphorylation in the heart, attenuated the atrophy phenotype, and extended the mean lifespan of HGPS mice by ~21%, implying that targeting cardiac atrophy may be an approach to HGPS treatment.
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Envejecimiento Prematuro , Progeria , Humanos , Ratones , Animales , Progeria/metabolismo , Corazón , Daño del ADN , Inestabilidad Genómica , Proteínas Quinasas Activadas por AMP/genética , Lamina Tipo A/genética , Lamina Tipo A/metabolismoRESUMEN
The pathogenesis of COVID-19 is still elusive, which impedes disease progression prediction, differential diagnosis, and targeted therapy. Plasma cell-free RNAs (cfRNAs) carry unique information from human tissue and thus could point to resourceful solutions for pathogenesis and host-pathogen interactions. Here, we performed a comparative analysis of cfRNA profiles between COVID-19 patients and healthy donors using serial plasma. Analyses of the cfRNA landscape, potential gene regulatory mechanisms, dynamic changes in tRNA pools upon infection, and microbial communities were performed. A total of 380 cfRNA molecules were up-regulated in all COVID-19 patients, of which seven could serve as potential biomarkers (AUC > 0.85) with great sensitivity and specificity. Antiviral (NFKB1A, IFITM3, and IFI27) and neutrophil activation (S100A8, CD68, and CD63)-related genes exhibited decreased expression levels during treatment in COVID-19 patients, which is in accordance with the dynamically enhanced inflammatory response in COVID-19 patients. Noncoding RNAs, including some microRNAs (let 7 family) and long noncoding RNAs (GJA9-MYCBP) targeting interleukin (IL6/IL6R), were differentially expressed between COVID-19 patients and healthy donors, which accounts for the potential core mechanism of cytokine storm syndromes; the tRNA pools change significantly between the COVID-19 and healthy group, leading to the accumulation of SARS-CoV-2 biased codons, which facilitate SARS-CoV-2 replication. Finally, several pneumonia-related microorganisms were detected in the plasma of COVID-19 patients, raising the possibility of simultaneously monitoring immune response regulation and microbial communities using cfRNA analysis. This study fills the knowledge gap in the plasma cfRNA landscape of COVID-19 patients and offers insight into the potential mechanisms of cfRNAs to explain COVID-19 pathogenesis.
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COVID-19 , Ácidos Nucleicos Libres de Células , ARN/sangre , COVID-19/sangre , COVID-19/genética , Ácidos Nucleicos Libres de Células/sangre , Síndrome de Liberación de Citoquinas , Humanos , SARS-CoV-2RESUMEN
Retinal detachment (RD) is a sight-threatening condition that occurs in several retinal diseases. Microglia that reside in retina are activated after RD and are involved in the death of photoreceptor cells. The involvement of microglial pyroptosis in the early pathological process of RD is still unclear. It has been shown that VX-765, an inhibitor of caspase-1, may exert neuroprotective effects by targeting microglial pyroptosis in nervous system disease; however, whether it plays a role in RD is uncertain. This study detected and localized pyroptosis to specific cells by immunofluorescence co-staining and flow cytometry in rat RD models. The majority of gasdermin D N-terminal (GSDMD-N)-positive cells exhibited IBA1-positive or P2RY12-positive microglia in the early stage of RD, indicating the pyroptosis of microglia. Administration of VX-765 shifted the microglia phenotype from M1 to M2, inhibited microglial migration toward the outer nuclear layer (ONL) post-RD, and most importantly, inhibited microglial pyroptosis. The thickness of ONL increased with VX-765 administration, and the photoreceptors were more structured and orderly under hematoxylin and eosin staining and transmission electron microscopy, revealing the protective effects of VX-765 on photoreceptors. Overall, this study demonstrates that inflammation induced by pyroptosis of microglia is the early pathological process of RD. VX-765 may serve as a candidate therapeutic approach for the treatment of RD by targeting microglia.
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Chronic pancreatitis (CP) is marked by progressive fibrosis and the activation of pancreatic stellate cells (PSCs), accompanied by the destruction of pancreatic parenchyma, leading to the loss of acinar cells (ACs). Few research studies have explored the mechanism by which damaged ACs (DACs) contribute to PSCs activation and pancreatic fibrosis. Currently, there are no effective drugs for curing CP or limiting the progression of pancreatic fibrosis. In this research, co-culture with intact acinar cells (IACs) suppressed PSC activation, while co-culture with DACs did the opposite. Krüppel-like factor 4 (KLF4) was significantly upregulated in DACs and was established as the key molecule that switches ACs from PSCs-suppressor to PSCs-activator. We revealed the exosomes of IACs contributed to the anti-activated function of IACs-CS on PSCs. MiRNome profiling showed that let-7 family is significantly enriched in IAC-derived exosomes (>30% miRNome), which partially mediates IACs' suppressive impacts on PSCs. Furthermore, it has been observed that the enrichment of let-7 in exosomes was influenced by the expression level of KLF4. Mechanistic studies demonstrated that KLF4 in ACs upregulated Lin28A, thereby decreasing let-7 levels in AC-derived exosomes, and thus promoting PSCs activation. We utilized an adeno-associated virus specifically targeting KLF4 in ACs (shKLF4-pAAV) to suppress PSCs activation in CP, resulting in reduced pancreatic fibrosis. IAC-derived exosomes hold potential as potent weapons against PSCs activation via let-7s, while activated KLF4/Lin28A signaling in DACs diminished such functions. ShKLF4-pAAV holds promise as a novel therapeutic approach for CP.
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Células Acinares , Exosomas , Fibrosis , Factor 4 Similar a Kruppel , MicroARNs , Células Estrelladas Pancreáticas , Pancreatitis Crónica , Factor 4 Similar a Kruppel/metabolismo , Animales , Células Estrelladas Pancreáticas/metabolismo , Células Estrelladas Pancreáticas/patología , Exosomas/metabolismo , Pancreatitis Crónica/metabolismo , Pancreatitis Crónica/genética , Pancreatitis Crónica/patología , MicroARNs/genética , Células Acinares/metabolismo , Células Acinares/patología , Dependovirus/genética , Ratones , Humanos , Factores de Transcripción de Tipo Kruppel/metabolismo , Factores de Transcripción de Tipo Kruppel/genética , Modelos Animales de Enfermedad , Vectores Genéticos/genética , Vectores Genéticos/administración & dosificación , Masculino , Técnicas de Cocultivo , Páncreas/metabolismo , Páncreas/patología , Terapia Genética/métodosRESUMEN
R-loop, a three-stranded RNA/DNA structure, has been linked to induced genome instability and regulated gene expression. To enable precision analysis of R-loops in vivo, we develop an RNase-H-based approach; this reveals predominant R-loop formation near gene promoters with strong G/C skew and propensity to form G-quadruplex in non-template DNA, corroborating with all biochemically established properties of R-loops. Transcription perturbation experiments further indicate that R-loop induction correlates to transcriptional pausing. Interestingly, we note that most mapped R-loops are each linked to a nearby free RNA end; by using a ribozyme to co-transcriptionally cleave nascent RNA, we demonstrate that such a free RNA end coupled with a G/C-skewed sequence is necessary and sufficient to induce R-loop. These findings provide a topological solution for RNA invasion into duplex DNA and suggest an order for R-loop initiation and elongation in an opposite direction to that previously proposed.
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ADN/química , Ácidos Nucleicos Heterodúplex/química , Regiones Promotoras Genéticas/fisiología , ARN/química , Ribonucleasa H/química , Transcripción Genética , ADN/biosíntesis , Células HEK293 , Humanos , Células K562 , Ácidos Nucleicos Heterodúplex/metabolismo , ARN/biosíntesisRESUMEN
DNA-RNA hybrids play various roles in many physiological progresses, but how this chromatin structure is dynamically regulated during spermatogenesis remains largely unknown. Here, we show that germ cell-specific knockout of Rnaseh1, a specialized enzyme that degrades the RNA within DNA-RNA hybrids, impairs spermatogenesis and causes male infertility. Notably, Rnaseh1 knockout results in incomplete DNA repair and meiotic prophase I arrest. These defects arise from the altered RAD51 and DMC1 recruitment in zygotene spermatocytes. Furthermore, single-molecule experiments show that RNase H1 promotes recombinase recruitment to DNA by degrading RNA within DNA-RNA hybrids and allows nucleoprotein filaments formation. Overall, we uncover a function of RNase H1 in meiotic recombination, during which it processes DNA-RNA hybrids and facilitates recombinase recruitment.
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Meiosis , Ribonucleasa H , Humanos , Masculino , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , ADN/genética , ADN/metabolismo , Recombinasa Rad51/genética , Recombinasa Rad51/metabolismo , Recombinasas/genética , Espermatocitos/metabolismo , Ribonucleasa H/metabolismoRESUMEN
Doxorubicin (DOX) is an anthracycline antibiotic used as an antitumor treatment. However, its clinical application is limited due to severe side effects such as cardiotoxicity. In recent years, numerous studies have demonstrated that cellular aging has become a therapeutic target for DOX-induced cardiomyopathy. However, the underlying mechanism and specific molecular targets of DOX-induced cardiomyocyte aging remain unclear. Poly (ADP-ribose) polymerase (PARP) is a family of protein post-translational modification enzymes in eukaryotic cells, including 18 members. PARP-1, the most well-studied member of this family, has become a potential molecular target for the prevention and treatment of various cardiovascular diseases, such as DOX cardiomyopathy and heart failure. PARP-1 and PARP-2 share 69% homology in the catalytic regions. However, they do not entirely overlap in function. The role of PARP-2 in cardiovascular diseases, especially in DOX-induced cardiomyocyte aging, is less studied. In this study, we found for the first time that down-regulation of PARP-2 can inhibit DOX-induced cellular aging in cardiomyocytes. On the contrary, overexpression of PARP-2 can aggravate DOX-induced cardiomyocyte aging and injury. Further research showed that PARP-2 inhibited the expression and activity of SIRT1, which in turn was involved in the development of DOX-induced cardiomyocyte aging and injury. Our findings provide a preliminary experimental basis for establishing PARP-2 as a new target for preventing and treating DOX cardiomyopathy and related drug development.
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Senescencia Celular , Doxorrubicina , Miocitos Cardíacos , Poli(ADP-Ribosa) Polimerasas , Sirtuina 1 , Doxorrubicina/efectos adversos , Doxorrubicina/farmacología , Miocitos Cardíacos/efectos de los fármacos , Miocitos Cardíacos/patología , Miocitos Cardíacos/metabolismo , Sirtuina 1/metabolismo , Sirtuina 1/genética , Animales , Senescencia Celular/efectos de los fármacos , Poli(ADP-Ribosa) Polimerasas/metabolismo , Poli(ADP-Ribosa) Polimerasas/genética , Ratas , Cardiotoxicidad/patología , Cardiotoxicidad/metabolismo , Cardiotoxicidad/prevención & control , Cardiotoxicidad/etiología , Apoptosis/efectos de los fármacos , Ratas Sprague-Dawley , Antibióticos Antineoplásicos/toxicidad , Antibióticos Antineoplásicos/efectos adversos , Antibióticos Antineoplásicos/farmacología , Cardiomiopatías/inducido químicamente , Cardiomiopatías/patología , Cardiomiopatías/metabolismo , Cardiomiopatías/genética , HumanosRESUMEN
BACKGROUND: Lactylation, a novel contributor to post-translational protein modifications, exhibits dysregulation across various tumors. Nevertheless, its intricate involvement in colorectal carcinoma, particularly for non-histone lactylation and its intersection with metabolism and immune evasion, remains enigmatic. METHODS: Employing immunohistochemistry on tissue microarray with clinical information and immunofluorescence on colorectal cell lines, we investigated the presence of global lactylation and its association with development and progression in colorectal cancer as well as its functional location. Leveraging the AUCell algorithm alongside correlation analysis in single-cell RNA sequencing data, as well as cox-regression and lasso-regression analysis in TCGA dataset and confirmed in GEO dataset, we identified a 23-gene signature predicting colorectal cancer prognosis. Subsequently, we analyzed the associations between the lactylation related gene risk and clinical characteristics, mutation landscapes, biological functions, immune cell infiltration, immunotherapy responses, and drug sensitivity. Core genes were further explored for deep biological insights through bioinformatics and in vitro experiments. RESULTS: Our study innovatively reveals a significant elevation of global lactylation in colorectal cancer, particularly in malignant tumors, confirming it as an independent prognostic factor for CRC. Through a comprehensive analysis integrating tumor tissue arrays, TCGA dataset, GEO dataset, combining in silico investigations and in vitro experiments, we identified a 23-gene Lactylation-Related Gene risk model capable of predicting the prognosis of colorectal cancer patients. Noteworthy variations were observed in clinical characteristics, biological functions, immune cell infiltration, immune checkpoint expression, immunotherapy responses and drug sensitivity among distinct risk groups. CONCLUSIONS: The Lactylation-Related Gene risk model exhibits significant potential for improving the management of colorectal cancer patients and enhancing therapeutic outcomes, particularly at the intersection of metabolism and immune evasion. This finding underscores the clinical relevance of global lactylation in CRC and lays the groundwork for mechanism investigation and targeted therapeutic strategies given the high lactate concentration in CRC.
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Neoplasias Colorrectales , Inmunoterapia , Humanos , Pronóstico , Algoritmos , Línea Celular , Neoplasias Colorrectales/genética , Microambiente TumoralRESUMEN
BACKGROUND: Spinal cord injury (SCI) is characterized by extensive demyelination and inflammatory responses. Facilitating the clearance of lipid droplets (LDs) within microglia contributes to creating a microenvironment that favors neural recovery and provides essential materials for subsequent remyelination. Therefore, investigating MicroRNAs (miRNAs) that regulate lipid homeostasis after SCI and elucidating their potential mechanisms in promoting LDs clearance in microglia have become focal points of SCI research. METHODS: We established a subacute C5 hemicontusion SCI model in mice and performed transcriptomic sequencing on the injury epicenter to identify differentially expressed genes and associated pathways. Confocal imaging was employed to observe LDs accumulation. Multi-omics analyses were conducted to identify differentially expressed mRNA and miRNA post-SCI. Pathway enrichment analysis and protein-protein interaction network construction were performed using bioinformatics methods, revealing miR-223-Abca1 as a crucial miRNA-mRNA pair in lipid metabolism regulation. BV2 microglia cell lines overexpressing miR-223 were engineered, and immunofluorescence staining, western blot, and other techniques were employed to assess LDs accumulation, relevant targets, and inflammatory factor expression, confirming its role in regulating lipid homeostasis in microglia. RESULTS: Histopathological results of our hemicontusion SCI model confirmed LDs aggregation at the injury epicenter, predominantly within microglia. Our transcriptomic analysis during the subacute phase of SCI in mice implicated ATP-binding cassette transporter A1 (Abca1) as a pivotal gene in lipid homeostasis, cholesterol efflux and microglial activation. Integrative mRNA-miRNA multi-omics analysis highlighted the crucial role of miR-223 in the neuroinflammation process following SCI, potentially through the regulation of lipid metabolism via Abca1. In vitro experiments using BV2 cells overexpressing miR-223 demonstrated that elevated levels of miR-223 enhance ABCA1 expression in myelin debris and LPS-induced BV2 cells. This promotes myelin debris degradation and LDs clearance, and induces a shift toward an anti-inflammatory M2 phenotype. CONCLUSIONS: In summary, our study unveils the critical regulatory role of miR-223 in lipid homeostasis following SCI. The mechanism by which this occurs involves the upregulation of ABCA1 expression, which facilitates LDs clearance and myelin debris degradation, consequently alleviating the lipid burden, and inhibiting inflammatory polarization of microglia. These findings suggest that strategies to enhance miR-223 expression and target ABCA1, thereby augmenting LDs clearance, may emerge as appealing new clinical targets for SCI treatment.
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Transportador 1 de Casete de Unión a ATP , Gotas Lipídicas , Ratones Endogámicos C57BL , MicroARNs , Microglía , Traumatismos de la Médula Espinal , Regulación hacia Arriba , Traumatismos de la Médula Espinal/metabolismo , Traumatismos de la Médula Espinal/patología , MicroARNs/metabolismo , MicroARNs/genética , Microglía/metabolismo , Microglía/patología , Animales , Transportador 1 de Casete de Unión a ATP/metabolismo , Transportador 1 de Casete de Unión a ATP/genética , Gotas Lipídicas/metabolismo , Ratones , Línea Celular , Masculino , Metabolismo de los Lípidos/genéticaRESUMEN
BACKGROUND: Limonin shows promise in alleviating non-alcoholic fatty liver disease. We investigated the mechanisms of limonin against non-alcoholic steatohepatitis (NASH) using network pharmacology and molecular docking. METHODS: Public databases provided NASH- and limonin-associated targets. VennDiagram identified potential limonin targets for NASH. Enrichment analysis explored the limonin-NASH relationship. PPI network analysis, CytoHubba models, and bioinformatics identified hub genes for NASH treatment. Molecular docking assessed limonin's binding ability to hub targets. RESULTS: We found 37 potential limonin targets in NASH, involved in oxidative stress, inflammation, and signaling pathways. PPI network analysis revealed seven hub genes (STAT3, NFKBIA, MTOR, TLR4, CASP8, PTGS2, NFKB1) as NASH treatment targets. Molecular docking confirmed limonin's binding to STAT3, CASP8, and PTGS2. Animal experiments on high-fat diet mice showed limonin reduced hepatic steatosis, lipid accumulation, and expression of p-STAT3/STAT3, CASP8, and PTGS2. CONCLUSION: Limonin's therapeutic effects in NASH may stem from its antioxidant and anti-inflammatory properties. STAT3, CASP8, and PTGS2 are potential key targets for NASH treatment, warranting further investigation.
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Limoninas , Enfermedad del Hígado Graso no Alcohólico , Humanos , Ratones , Animales , Enfermedad del Hígado Graso no Alcohólico/tratamiento farmacológico , Enfermedad del Hígado Graso no Alcohólico/genética , Enfermedad del Hígado Graso no Alcohólico/metabolismo , Farmacología en Red , Ciclooxigenasa 2/uso terapéutico , Limoninas/farmacología , Limoninas/uso terapéutico , Simulación del Acoplamiento MolecularRESUMEN
Panax notoginseng has the effect of stimulating circulation to end stasis. Our study was designed to evaluate the anti-thrombotic effect of protoparaxotriol saponins (PTS) from Panax notoginseng and the involved mechanisms. A thrombosis model was constructed, and the anti-thrombotic activity of PTS was determined by erythrocyte staining, heart rate, and blood flow velocity. In addition, quantitative real-time polymerase chain reaction (qPCR) was used to identify changes in the expression of genes related to coagulation, inflammation, and apoptosis. PTS alleviated arachidonic acid (AA)-induced caudal vein thrombosis, restored blood flow, and increased the area of cardiac erythrocyte staining, heart rate and blood flow velocity. It reduced the ponatinib-induced cerebral thrombus area and decreased the intensity of erythrocyte staining. The qPCR data showed that the anti-thrombotic effect of PTS was mediated by suppression of genes related to coagulation, inflammation and apoptosis, and also involved inhibition of NF-κB and PI3K/Akt pathways.
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Cancer, as a public health issue, is the leading cause of death worldwide. Tetrahydroisoquinoline derivatives have effective biological activities and can be used as potential therapeutic agents for antitumor drugs. In this work, we designed and synthesized a series of novel tetrahydroisoquinoline compounds and evaluated their antitumor activity in vitro on several representative human cancer cell lines. The results showed that the vast majority of compounds showed good inhibitory activities against the cancer cell lines of HCT116, MDA-MB-231, HepG2, and A375.
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Antineoplásicos , Diseño de Fármacos , Ensayos de Selección de Medicamentos Antitumorales , Tetrahidroisoquinolinas , Humanos , Tetrahidroisoquinolinas/farmacología , Tetrahidroisoquinolinas/química , Tetrahidroisoquinolinas/síntesis química , Antineoplásicos/farmacología , Antineoplásicos/síntesis química , Antineoplásicos/química , Relación Estructura-Actividad , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Estructura Molecular , Relación Dosis-Respuesta a DrogaRESUMEN
In pancreatic ductal adenocarcinomas (PDAC), profound hypoxia plays key roles in regulating cancer cell behavior, including proliferation, migration, and resistance to therapies. The initial part of this research highlights the important role played by long noncoding RNA (lncRNA) MKLN1-AS, which is controlled by hypoxia-inducible factor-1 alpha (HIF-1α), in the progression of PDAC. Human samples of PDAC showed a notable increase in MKLN1-AS expression, which was linked to a worse outcome. Forced expression of MKLN1-AS greatly reduced the inhibitory impact on the growth and spread of PDAC cells caused by HIF-1α depletion. Experiments on mechanisms showed that HIF-1α influences the expression of MKLN1-AS by directly attaching to a hypoxia response element in the promoter region of MKLN1-AS.MKLN1-AS acts as a competitive endogenous RNA (ceRNA) by binding to miR-185-5p, resulting in the regulation of TEAD1 expression and promoting cell proliferation, migration, and tumor growth. TEAD1 subsequently enhances the development of PDAC. Our study results suggest that MKLN1-AS could serve as a promising target for treatment and a valuable indicator for predicting outcomes in PDAC. PDAC is associated with low oxygen levels, and the long non-coding RNA MKLN1-AS interacts with TEAD1 in this context.
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Carcinoma Ductal Pancreático , Movimiento Celular , Proliferación Celular , Proteínas de Unión al ADN , Regulación Neoplásica de la Expresión Génica , Subunidad alfa del Factor 1 Inducible por Hipoxia , MicroARNs , Neoplasias Pancreáticas , ARN Largo no Codificante , Factores de Transcripción de Dominio TEA , Animales , Humanos , Ratones , Carcinoma Ductal Pancreático/genética , Carcinoma Ductal Pancreático/metabolismo , Carcinoma Ductal Pancreático/patología , Línea Celular Tumoral , Movimiento Celular/genética , Proliferación Celular/genética , Progresión de la Enfermedad , Proteínas de Unión al ADN/metabolismo , Proteínas de Unión al ADN/genética , Regulación Neoplásica de la Expresión Génica/genética , Subunidad alfa del Factor 1 Inducible por Hipoxia/metabolismo , Subunidad alfa del Factor 1 Inducible por Hipoxia/genética , Ratones Desnudos , MicroARNs/genética , MicroARNs/metabolismo , Proteínas Nucleares/metabolismo , Proteínas Nucleares/genética , Neoplasias Pancreáticas/genética , Neoplasias Pancreáticas/patología , Neoplasias Pancreáticas/metabolismo , ARN Largo no Codificante/genética , ARN Largo no Codificante/metabolismo , Transducción de Señal/genética , Factores de Transcripción de Dominio TEA/metabolismo , Factores de Transcripción/metabolismo , Factores de Transcripción/genéticaRESUMEN
Multiplexed in situ RNA imaging offers new opportunities for gene expression profiling by providing high-throughput spatial information. In this work, we present a cyclic combinatorial fluorescent in situ hybridization (combFISH) assay to achieve multiplexed detection of RNA in cell cultures and tissues. Specifically, multiplexing is achieved through cyclic interrogation of barcode sequences on the rolling circle amplicons generated from the padlock probe assay by using sets of combinatorial detection probes. Theoretically, combFISH can detect 64 genes in three hybridization cycles by combinatorial barcoding using 12 fluorescently labeled detection probes. Our method eliminates sequencing-by-ligation (SBL) chemistry in the in situ sequencing protocol and directly uses RNA as targets for ligation, making it more straightforward. We showed that our method works in fresh-frozen and formalin-fixed paraffin-embedded tissue sections. With its straightforward protocols, we expect our method to be adopted by the scientific community and extended to clinical settings.
Asunto(s)
Hibridación Fluorescente in Situ , ARN , Hibridación Fluorescente in Situ/métodos , ARN/análisis , Humanos , Animales , Colorantes Fluorescentes/química , Perfilación de la Expresión Génica/métodosRESUMEN
Postnatal growth of mammalian oocytes is accompanied by a progressive gain of DNA methylation, which is predominantly mediated by DNMT3A, a de novo DNA methyltransferase1,2. Unlike the genome of sperm and most somatic cells, the oocyte genome is hypomethylated in transcriptionally inert regions2-4. However, how such a unique feature of the oocyte methylome is determined and its contribution to the developmental competence of the early embryo remains largely unknown. Here we demonstrate the importance of Stella, a factor essential for female fertility5-7, in shaping the oocyte methylome in mice. Oocytes that lack Stella acquire excessive DNA methylation at the genome-wide level, including in the promoters of inactive genes. Such aberrant hypermethylation is partially inherited by two-cell-stage embryos and impairs zygotic genome activation. Mechanistically, the loss of Stella leads to ectopic nuclear accumulation of the DNA methylation regulator UHRF18,9, which results in the mislocalization of maintenance DNA methyltransferase DNMT1 in the nucleus. Genetic analysis confirmed the primary role of UHRF1 and DNMT1 in generating the aberrant DNA methylome in Stella-deficient oocytes. Stella therefore safeguards the unique oocyte epigenome by preventing aberrant de novo DNA methylation mediated by DNMT1 and UHRF1.
Asunto(s)
ADN (Citosina-5-)-Metiltransferasa 1/metabolismo , Metilación de ADN , Epigénesis Genética , Oocitos/metabolismo , Proteínas Represoras/metabolismo , Animales , Proteínas Potenciadoras de Unión a CCAAT , Línea Celular , Núcleo Celular/metabolismo , Proteínas Cromosómicas no Histona , ADN (Citosina-5-)-Metiltransferasa 1/antagonistas & inhibidores , Desarrollo Embrionario , Femenino , Genoma/genética , Humanos , Ratones , Proteínas Nucleares/metabolismo , Regiones Promotoras Genéticas/genética , Proteínas Represoras/deficiencia , Proteínas Represoras/genética , Ubiquitina-Proteína Ligasas , Cigoto/metabolismoRESUMEN
OBJECTIVE: To investigate the association of state-level cigarette price and tobacco control expenditure with the large 2000-2019 decline in cigarette smoking among US 18-24 year-olds. METHODS: Smoking behaviour was assessed in the 24 most populous US states using the 1992-2019 Tobacco Use Supplements to the Current Population Survey; association with price and expenditure was tested using adjusted logistic regression. States were ranked by inflation-adjusted average price and tobacco control expenditure and grouped into tertiles. State-specific time trends were estimated, with slope changes in 2001/2002 and 2010/2011. RESULTS: Between 2000 and 2010, the odds of smoking among US young adults decreased by a third (adjusted OR, AOR 0.68, 95% CI 0.56 to 0.84). By 2019, these odds were one-quarter of their 2000 level (AOR 0.24, 95% CI 0.19 to 0.31). Among states in the lowest tertile of price/expenditure tobacco control activity, initially higher young adult smoking decreased by 13 percentage points from 2010 to 2018-2019, to a prevalence of 5.6% (95% CI 4.5% to 6.8%), equal to that in the highest tobacco-control tertile of states (6.5%, 95% CI 5.2% to 7.8%). Neither state tobacco control spending (AOR 1.0, 95% CI 0.999 to 1.002) nor cigarette price (AOR 0.96, 95% CI: 0.92 to 1.01) were associated with young adult smoking in statistical models. In 2019, seven states had prevalence over 3 SDs higher than the 24-state mean. CONCLUSION: National programmes may have filled a gap in state-level interventions, helping drive down the social acceptability of cigarette smoking among young adults across all states. Additional interventions are needed to assist high-prevalence states to further reduce smoking.