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1.
Theranostics ; 14(12): 4894-4915, 2024.
Artigo em Inglês | MEDLINE | ID: mdl-39239522

RESUMO

Rationale: Regulatory processes of transcription factors (TFs) shape heart development and influence the adult heart's response to stress, contributing to cardiac disorders. Despite their significance, the precise mechanisms underpinning TF-mediated regulation remain elusive. Here, we identify that EBF1, as a TF, is highly expressed in human heart tissues. EBF1 is reported to be associated with human cardiovascular disease, but its roles are unclear in heart. In this study, we investigated EBF1 function in cardiac system. Methods: RNA-seq was utilized to profile EBF1 expression patterns. CRISPR/Cas9 was utilized to knock out EBF1 to investigate its effects. Human pluripotent stem cells (hPSCs) differentiated into cardiac lineages were used to mimic cardiac development. Cardiac function was evaluated on mouse model with Ebf1 knockout by using techniques such as echocardiography. RNA-seq was conducted to analyze transcriptional perturbations. ChIP-seq was employed to elucidate EBF1-bound genes and the underlying regulatory mechanisms. Results: EBF1 was expressed in some human and mouse cardiomyocyte. Knockout of EBF1 inhibited cardiac development. ChIP-seq indicated EBF1's binding on promoters of cardiogenic TFs pivotal to cardiac development, facilitating their transcriptional expression and promoting cardiac development. In mouse, Ebf1 depletion triggered transcriptional perturbations of genes, resulting in cardiac remodeling. Mechanistically, we found that EBF1 directly bound to upstream chromatin regions of cardiac hypertrophy-inducing genes, contributing to cardiac hypertrophy. Conclusions: We uncover the mechanisms underlying EBF1-mediated regulatory processes, shedding light on cardiac development, and the pathogenesis of cardiac remodeling. These findings emphasize EBF1's critical role in orchestrating diverse aspects of cardiac processes and provide a promising therapeutic intervention for cardiomyopathy.


Assuntos
Perfilação da Expressão Gênica , Miócitos Cardíacos , Transativadores , Animais , Humanos , Camundongos , Transativadores/genética , Transativadores/metabolismo , Miócitos Cardíacos/metabolismo , Diferenciação Celular/genética , Coração/fisiopatologia , Camundongos Knockout , Células-Tronco Pluripotentes/metabolismo , Transcriptoma/genética , Sistemas CRISPR-Cas/genética
2.
Development ; 151(17)2024 Sep 01.
Artigo em Inglês | MEDLINE | ID: mdl-39254648

RESUMO

During embryonic development, Wnt signaling influences both proliferation and sensory formation in the cochlea. How this dual nature of Wnt signaling is coordinated is unknown. In this study, we define a novel role for a Wnt-regulated gene, Mybl2, which was already known to be important for proliferation, in determining the size and patterning of the sensory epithelium in the murine cochlea. Using a quantitative spatial analysis approach and analyzing Mybl2 loss-of-function, we show that Mybl2 promoted proliferation in the inner sulcus domain but limited the size of the sensory domain by influencing their adjoining boundary position via Jag1 regulation during development. Mybl2 loss-of-function simultaneously decreased proliferation in the inner sulcus and increased the size of the sensory domain, resulting in a wider sensory epithelium with ectopic inner hair cell formation during late embryonic stages. These data suggest that progenitor cells in the inner sulcus determine boundary formation and pattern the sensory epithelium via MYBL2.


Assuntos
Proliferação de Células , Cóclea , Proteína Jagged-1 , Células-Tronco , Animais , Cóclea/embriologia , Cóclea/citologia , Cóclea/metabolismo , Camundongos , Epitélio/embriologia , Epitélio/metabolismo , Células-Tronco/citologia , Células-Tronco/metabolismo , Proteína Jagged-1/metabolismo , Proteína Jagged-1/genética , Regulação da Expressão Gênica no Desenvolvimento , Via de Sinalização Wnt , Padronização Corporal/genética , Transativadores/metabolismo , Transativadores/genética , Células Ciliadas Auditivas Internas/metabolismo , Células Ciliadas Auditivas Internas/citologia , Proteínas de Ciclo Celular/metabolismo , Proteínas de Ciclo Celular/genética
3.
Nat Commun ; 15(1): 7856, 2024 Sep 09.
Artigo em Inglês | MEDLINE | ID: mdl-39251623

RESUMO

Despite recent advances in systemic therapy for hepatocellular carcinoma (HCC), the prognosis of hepatitis B virus (HBV)-induced HCC patients remains poor. By screening a sgRNA library targeting human deubiquitinases, we find that ubiquitin-specific peptidase 26 (USP26) deficiency impairs HBV-positive HCC cell proliferation. Genetically engineered murine models with Usp26 knockout confirm that Usp26 drives HCC tumorigenesis. Mechanistically, we find that the HBV-encoded protein HBx binds to the promoter and induces the production of USP26, which is an X-linked gene exclusively expressed in the testis. HBx consequently promotes the association of USP26 with SIRT1 to synergistically stabilize SIRT1 by deubiquitination, which promotes cell proliferation and impedes cell apoptosis to accelerate HCC tumorigenesis. In patients with HBV-positive HCC, USP26 is robustly induced, and its levels correlate with SIRT1 levels and poor prognosis. Collectively, our study highlights a causative link between HBV infection, deubiquitinase induction and development of HCC, identifying a druggable target, USP26.


Assuntos
Carcinoma Hepatocelular , Proliferação de Células , Epigênese Genética , Vírus da Hepatite B , Neoplasias Hepáticas , Sirtuína 1 , Transativadores , Proteínas Virais Reguladoras e Acessórias , Carcinoma Hepatocelular/virologia , Carcinoma Hepatocelular/genética , Carcinoma Hepatocelular/metabolismo , Carcinoma Hepatocelular/patologia , Humanos , Animais , Neoplasias Hepáticas/virologia , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/metabolismo , Neoplasias Hepáticas/patologia , Vírus da Hepatite B/genética , Camundongos , Sirtuína 1/metabolismo , Sirtuína 1/genética , Transativadores/metabolismo , Transativadores/genética , Masculino , Proliferação de Células/genética , Proteínas Virais Reguladoras e Acessórias/metabolismo , Carcinogênese/genética , Hepatite B/virologia , Hepatite B/complicações , Hepatite B/genética , Hepatite B/metabolismo , Linhagem Celular Tumoral , Camundongos Knockout , Regulação Neoplásica da Expressão Gênica , Enzimas Desubiquitinantes/metabolismo , Enzimas Desubiquitinantes/genética , Apoptose/genética , Cisteína Endopeptidases/metabolismo , Cisteína Endopeptidases/genética , Regiões Promotoras Genéticas/genética
4.
Arch Microbiol ; 206(10): 401, 2024 Sep 11.
Artigo em Inglês | MEDLINE | ID: mdl-39261350

RESUMO

Staphylococcus aureus is a notorious pathogen predominantly involved in skin and soft tissue infections, exhibiting a distinct innate sex bias. This study explores the influence of testosterone on the virulence of S. aureus and elucidates its underlying mechanisms. Utilizing a skin abscess model in intact and castrated male mice, we assessed the effects of testosterone on S. aureus pathogenicity. Compared to controls, castrated mice showed significantly reduced abscess sizes and decreased bacterial loads, highlighting the role of testosterone in modulating the severity of S. aureus infections. In vitro experiments revealed that testosterone enhances the hemolytic activity, cytotoxicity, and oxidative stress resistance of S. aureus. Real-time quantitative PCR analysis showed a significant upregulation of the genes encoding α-hemolysin (hla) and phenol-soluble modulin (psmα). Importantly, testosterone treatment significantly enhanced the expression of the accessory gene regulator (Agr) quorum-sensing system components (agrC, agrA, agrB, agrD), while the SaeRS system (saeR, saeS, and sbi) exhibited only slight changes. Gene knockout experiments revealed that deletion of agrC, rather than saeRS and agrBD, abolishes the testosterone-induced enhancement of hemolysis and gene expression, underscoring the key role of AgrC. Molecular docking simulations indicated a direct interaction between testosterone and AgrC protein, with a strong binding affinity at the active site residue SER201. This study provides new insights into the mechanistic basis of how testosterone enhances the pathogenicity of S. aureus, potentially contributing to increased male susceptibility to S. aureus infections and offering a targeted approach for therapeutic interventions.


Assuntos
Proteínas de Bactérias , Infecções Estafilocócicas , Staphylococcus aureus , Testosterona , Masculino , Testosterona/farmacologia , Testosterona/metabolismo , Animais , Staphylococcus aureus/genética , Staphylococcus aureus/patogenicidade , Staphylococcus aureus/efeitos dos fármacos , Staphylococcus aureus/metabolismo , Camundongos , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Virulência , Infecções Estafilocócicas/microbiologia , Transativadores/genética , Transativadores/metabolismo , Regulação Bacteriana da Expressão Gênica , Percepção de Quorum , Simulação de Acoplamento Molecular , Toxinas Bacterianas/metabolismo , Toxinas Bacterianas/genética , Abscesso/microbiologia , Hemólise , Proteínas Hemolisinas/metabolismo , Proteínas Hemolisinas/genética
5.
Nat Commun ; 15(1): 7956, 2024 Sep 11.
Artigo em Inglês | MEDLINE | ID: mdl-39261466

RESUMO

Paracrine IL-2 signalling drives the CD8 + T cell expansion and differentiation that allow protection against viral infections, but the underlying molecular events are incompletely understood. Here we show that the transcription factor SRF, a master regulator of cytoskeletal gene expression, is required for effective IL-2 signalling during L. monocytogenes infection. Acting cell-autonomously with its actin-regulated cofactors MRTF-A and MRTF-B, SRF is dispensible for initial TCR-mediated CD8+ T cell proliferation, but is required for sustained IL-2 dependent CD8+ effector T cell expansion, and persistence of memory cells. Following TCR activation, Mrtfab-null CD8+ T cells produce IL-2 normally, but homotypic clustering is impaired both in vitro and in vivo. Expression of cytoskeletal structural and regulatory genes, most notably actins, is defective in Mrtfab-null CD8+ T cells. Activation-induced cell clustering in vitro requires F-actin assembly, and Mrtfab-null cell clusters are small, contain less F-actin, and defective in IL-2 retention. Clustering of Mrtfab-null cells can be partially restored by exogenous actin expression. IL-2 mediated CD8+ T cell proliferation during infection thus depends on the control of cytoskeletal dynamics and actin gene expression by MRTF-SRF signalling.


Assuntos
Linfócitos T CD8-Positivos , Citoesqueleto , Interleucina-2 , Camundongos Endogâmicos C57BL , Fator de Resposta Sérica , Transativadores , Linfócitos T CD8-Positivos/imunologia , Linfócitos T CD8-Positivos/metabolismo , Interleucina-2/metabolismo , Interleucina-2/genética , Animais , Transativadores/metabolismo , Transativadores/genética , Citoesqueleto/metabolismo , Camundongos , Fator de Resposta Sérica/metabolismo , Fator de Resposta Sérica/genética , Listeria monocytogenes/imunologia , Listeriose/imunologia , Listeriose/genética , Listeriose/microbiologia , Actinas/metabolismo , Regulação da Expressão Gênica , Transdução de Sinais , Camundongos Knockout , Proliferação de Células , Ativação Linfocitária
6.
Virulence ; 15(1): 2399798, 2024 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-39229975

RESUMO

Staphylococcus aureus is the most common cause of skin and soft tissue infections (SSTIs) with Methicillin-Resistant S. aureus (MRSA) strains being a major contributor in both community and hospital settings. S. aureus relies on metabolic diversity and a large repertoire of virulence factors to cause disease. This includes α-hemolysin (Hla), an integral player in tissue damage found in various models, including SSTIs. Previously, we identified a role for the Spx adapter protein, YjbH, in the regulation of several virulence factors and as an inhibitor of pathogenesis in a sepsis model. In this study, we found that YjbH is critical for tissue damage during SSTI, and its absence leads to decreased proinflammatory chemokines and cytokines in the skin. We identified no contribution of YjbI, encoded on the same transcript as YjbH. Using a combination of reporters and quantitative hemolysis assays, we demonstrated that YjbH impacts Hla expression and activity both in vitro and in vivo. Additionally, expression of Hla from a non-native promoter reversed the tissue damage phenotype of the ΔyjbIH mutant. Lastly, we identified reduced Agr activity as the likely cause for reduced Hla production in the ΔyjbH mutant. This work continues to define the importance of YjbH in the pathogenesis of S. aureus infection as well as identify a new pathway important for Hla production.


Assuntos
Proteínas de Bactérias , Toxinas Bacterianas , Regulação Bacteriana da Expressão Gênica , Proteínas Hemolisinas , Staphylococcus aureus , Transativadores , Proteínas Hemolisinas/metabolismo , Proteínas Hemolisinas/genética , Toxinas Bacterianas/metabolismo , Toxinas Bacterianas/imunologia , Toxinas Bacterianas/genética , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Staphylococcus aureus/patogenicidade , Staphylococcus aureus/imunologia , Staphylococcus aureus/genética , Camundongos , Animais , Transativadores/genética , Transativadores/metabolismo , Infecções Cutâneas Estafilocócicas/microbiologia , Infecções Cutâneas Estafilocócicas/imunologia , Infecções Cutâneas Estafilocócicas/patologia , Staphylococcus aureus Resistente à Meticilina/patogenicidade , Staphylococcus aureus Resistente à Meticilina/genética , Staphylococcus aureus Resistente à Meticilina/imunologia , Pele/microbiologia , Pele/patologia , Pele/imunologia , Fatores de Virulência/genética , Humanos , Infecções dos Tecidos Moles/microbiologia , Infecções dos Tecidos Moles/imunologia , Infecções Estafilocócicas/imunologia , Infecções Estafilocócicas/microbiologia , Proteínas Adaptadoras de Transdução de Sinal/genética , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Citocinas/metabolismo , Citocinas/imunologia , Citocinas/genética
7.
Life Sci ; 356: 123036, 2024 Nov 01.
Artigo em Inglês | MEDLINE | ID: mdl-39222836

RESUMO

AIMS: Cardiac fibrosis is characterized by aberrant collagen deposition in the heart. Macrophage polarization or infiltration is the main reason to accelerate the collagen deposition. We attempted to investigate the involvement of MKL1 in macrophages during the development of cardiac fibrosis. MATERIALS AND METHODS: Cardiac fibrosis is induced by myocardial infarction (MI). The MKL1f/f mice were crossed to the Lyz2-cre mice to generate macrophage conditional MKL1 knockout mice (MKL1ΔMφ). In addition, macrophage conditional MKL1 overexpression mice (MKL1Mϕ-OE) were constructed by crossing Lyz2-cre mice to MKL1ΔN200-Rosa26 mice. KEY FINDINGS: MKL1 expression was significantly increased in macrophages of both ischemic cardiomyopathy (ICM) patients and mice induced to develop myocardial infarction. Deletion of MKL1 in macrophages improved the heart function after MI-induced cardiac fibrosis. Consistently, MKL1Mϕ-OE mice displayed more severe cardiac fibrosis and worsened heart function than the control mice after MI. Moreover, administration of a small-molecule MKL1 inhibitor CCG-1423 also decreased the collagen deposition after MI. SIGNIFICANCE: Our data demonstrate that MKL1 in macrophages contributes to cardiac fibrosis pathogenesis and reinforce the notion that targeting MKL1 may yield effective antifibrotic therapeutics in the heart.


Assuntos
Modelos Animais de Doenças , Fibrose , Macrófagos , Infarto do Miocárdio , Transativadores , Animais , Humanos , Masculino , Camundongos , Colágeno/metabolismo , Fibrose/metabolismo , Macrófagos/metabolismo , Macrófagos/patologia , Camundongos Endogâmicos C57BL , Camundongos Knockout , Infarto do Miocárdio/patologia , Infarto do Miocárdio/metabolismo , Miocárdio/patologia , Miocárdio/metabolismo , Transativadores/metabolismo , Transativadores/genética
8.
Life Sci Alliance ; 7(11)2024 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-39256052

RESUMO

Eukaryotic gene expression is regulated at the transcriptional and post-transcriptional levels, with disruption of regulation contributing significantly to human diseases. The 5' m7G mRNA cap is a central node in post-transcriptional regulation, participating in both mRNA stabilization and translation efficiency. In mammals, DCP1a and DCP1b are paralogous cofactor proteins of the mRNA cap hydrolase DCP2. As lower eukaryotes have a single DCP1 cofactor, the functional advantages gained by this evolutionary divergence remain unclear. We report the first functional dissection of DCP1a and DCP1b, demonstrating that they are non-redundant cofactors of DCP2 with unique roles in decapping complex integrity and specificity. DCP1a is essential for decapping complex assembly and interactions between the decapping complex and mRNA cap-binding proteins. DCP1b is essential for decapping complex interactions with protein degradation and translational machinery. DCP1a and DCP1b impact the turnover of distinct mRNAs. The observation that different ontological groups of mRNA molecules are regulated by DCP1a and DCP1b, along with their non-redundant roles in decapping complex integrity, provides the first evidence that these paralogs have qualitatively distinct functions.


Assuntos
Endorribonucleases , Capuzes de RNA , Estabilidade de RNA , RNA Mensageiro , Humanos , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Endorribonucleases/metabolismo , Endorribonucleases/genética , Capuzes de RNA/metabolismo , Capuzes de RNA/genética , Estabilidade de RNA/genética , Proteínas de Ligação ao Cap de RNA/metabolismo , Proteínas de Ligação ao Cap de RNA/genética , Células HEK293 , Biossíntese de Proteínas , Ligação Proteica , Regulação da Expressão Gênica , Transativadores
9.
Int J Mol Sci ; 25(17)2024 Aug 23.
Artigo em Inglês | MEDLINE | ID: mdl-39273114

RESUMO

Staphylococcus aureus acts both as a colonizing commensal bacterium and invasive pathogen. Nasal colonization is associated with an increased risk of infection caused by the identical strain. In patients with atopic dermatitis (AD), the degree of S. aureus colonization is associated with the severity of the disease. Here, we comparatively analyzed the in vivo transcriptional profile of S. aureus colonizing the nose and non-diseased skin (non-lesional skin) as opposed to the diseased skin (lesional skin-defined here as infection) of 12 patients with AD. The transcriptional profile during the asymptomatic colonization of the nose closely resembled that of the lesional skin samples for many of the genes studied, with an elevated expression of the genes encoding adhesion-related proteins and proteases. In addition, the genes that modify and remodel the cell wall and encode proteins that facilitate immune evasion showed increased transcriptional activity. Notably, in a subgroup of patients, the global virulence regulator Agr (accessory gene regulator) and downstream target genes were inactive during nasal colonization but upregulated in the lesional and non-lesional skin samples. Taken together, our results demonstrate a colonization-like transcriptional profile on diseased skin and suggest a role for the peptide quorum sensing system Agr during the transition from asymptomatic nasal colonization to skin colonization/infection.


Assuntos
Dermatite Atópica , Perfilação da Expressão Gênica , Regulação Bacteriana da Expressão Gênica , Pele , Infecções Estafilocócicas , Staphylococcus aureus , Dermatite Atópica/microbiologia , Dermatite Atópica/genética , Humanos , Staphylococcus aureus/genética , Pele/microbiologia , Pele/metabolismo , Infecções Estafilocócicas/microbiologia , Infecções Estafilocócicas/genética , Feminino , Masculino , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Adulto , Transcriptoma , Infecções Cutâneas Estafilocócicas/microbiologia , Infecções Cutâneas Estafilocócicas/genética , Mucosa Nasal/microbiologia , Transativadores
10.
Int J Mol Sci ; 25(17)2024 Sep 06.
Artigo em Inglês | MEDLINE | ID: mdl-39273600

RESUMO

Diabetes mellitus, a chronic and non-transmissible disease, triggers a wide range of micro- and macrovascular complications. The differentiation of pancreatic ß-like cells (PßLCs) from induced pluripotent stem cells (iPSCs) offers a promising avenue for regenerative medicine aimed at treating diabetes. Current differentiation protocols strive to emulate pancreatic embryonic development by utilizing cytokines and small molecules at specific doses to activate and inhibit distinct molecular signaling pathways, directing the differentiation of iPSCs into pancreatic ß cells. Despite significant progress and improved protocols, the full spectrum of molecular signaling pathways governing pancreatic development and the physiological characteristics of the differentiated cells are not yet fully understood. Here, we report a specific combination of cofactors and small molecules that successfully differentiate iPSCs into PßLCs. Our protocol has shown to be effective, with the resulting cells exhibiting key functional properties of pancreatic ß cells, including the expression of crucial molecular markers (pdx1, nkx6.1, ngn3) and the capability to secrete insulin in response to glucose. Furthermore, the addition of vitamin C and retinoic acid in the final stages of differentiation led to the overexpression of specific ß cell genes.


Assuntos
Ácido Ascórbico , Diferenciação Celular , Diabetes Mellitus , Células-Tronco Pluripotentes Induzidas , Células Secretoras de Insulina , Tretinoína , Células Secretoras de Insulina/metabolismo , Células Secretoras de Insulina/efeitos dos fármacos , Células Secretoras de Insulina/citologia , Ácido Ascórbico/farmacologia , Células-Tronco Pluripotentes Induzidas/metabolismo , Células-Tronco Pluripotentes Induzidas/citologia , Células-Tronco Pluripotentes Induzidas/efeitos dos fármacos , Tretinoína/farmacologia , Diferenciação Celular/efeitos dos fármacos , Humanos , Diabetes Mellitus/metabolismo , Proteínas de Homeodomínio/metabolismo , Proteínas de Homeodomínio/genética , Transdução de Sinais/efeitos dos fármacos , Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Transativadores/metabolismo , Transativadores/genética , Insulina/metabolismo , Proteínas do Tecido Nervoso
11.
Proc Natl Acad Sci U S A ; 121(39): e2319666121, 2024 Sep 24.
Artigo em Inglês | MEDLINE | ID: mdl-39288176

RESUMO

Mammalian Yes-associated protein (YAP) and transcriptional coactivator with PDZ-binding motif (TAZ) and Drosophila Yorkie (Yki) are transcription cofactors of the highly conserved Hippo signaling pathway. It has been long assumed that the YAP/TAZ/Yki signaling drives cell proliferation during organ growth. However, its instructive role in regulating developmentally programmed organ growth, if any, remains elusive. Out-of-context gain of YAP/TAZ/Yki signaling often turns oncogenic. Paradoxically, mechanically strained, and differentiated squamous epithelia display developmentally programmed constitutive nuclear YAP/TAZ/Yki signaling. The unknown, therefore, is how a growth-promoting YAP/TAZ/Yki signaling restricts proliferation in differentiated squamous epithelia. Here, we show that reminiscent of a tumor suppressor, Yki negatively regulates the cell growth-promoting PI3K/Akt/TOR signaling in the squamous epithelia of Drosophila tubular organs. Thus, downregulation of Yki signaling in the squamous epithelium of the adult male accessory gland (MAG) up-regulates PI3K/Akt/TOR signaling, inducing cell hypertrophy, exit from their cell cycle arrest, and, finally, culminating in squamous cell carcinoma (SCC). Thus, blocking PI3K/Akt/TOR signaling arrests Yki loss-induced MAG-SCC. Further, MAG-SCCs, like other lethal carcinomas, secrete a cachectin, Impl2-the Drosophila homolog of mammalian IGFBP7-inducing cachexia and shortening the lifespan of adult males. Moreover, in the squamous epithelium of other tubular organs, like the dorsal trunk of larval tracheal airways or adult Malpighian tubules, downregulation of Yki signaling triggers PI3K/Akt/TOR-induced cell hypertrophy. Our results reveal that Yki signaling plays an instructive, antiproliferative role in the squamous epithelia of tubular organs.


Assuntos
Proteínas de Drosophila , Drosophila melanogaster , Proteínas Nucleares , Proteínas Serina-Treonina Quinases , Transdução de Sinais , Transativadores , Proteínas de Sinalização YAP , Animais , Proteínas de Drosophila/metabolismo , Proteínas de Drosophila/genética , Proteínas de Sinalização YAP/metabolismo , Proteínas de Sinalização YAP/genética , Transativadores/metabolismo , Transativadores/genética , Masculino , Drosophila melanogaster/metabolismo , Proteínas Nucleares/metabolismo , Proteínas Nucleares/genética , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Serina-Treonina Quinases/genética , Epitélio/metabolismo , Proliferação de Células , Fosfatidilinositol 3-Quinases/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/genética , Proteínas Proto-Oncogênicas c-akt/metabolismo , Proteínas Supressoras de Tumor/metabolismo , Proteínas Supressoras de Tumor/genética
12.
Microbiology (Reading) ; 170(9)2024 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-39302176

RESUMO

Streptococcus thermophilus holds promise as a chassis for producing and secreting heterologous proteins. Used for thousands of years to ferment milk, this species has generally recognized as safe (GRAS) status in the USA and qualified presumption of safety (QPS) status in Europe. In addition, it can be easily genetically modified thanks to its natural competence, and it secretes very few endogenous proteins, which means less downstream processing is needed to purify target proteins, reducing costs. Extracellular degradation of heterologous proteins can be eliminated by introducing mutations that inactivate the genes encoding the bacterium's three major surface proteases. Here, we constructed an inducible expression system that utilizes a peptide pheromone (SHP1358) and a transcriptional regulator (Rgg1358) involved in quorum-sensing regulation. We explored the functionality of a complete version of the system, in which the inducer is produced by the bacterium itself, by synthesizing a luciferase reporter protein. This complete version was assessed with bacteria grown in a chemically defined medium but also in vivo, in the faeces of germ-free mice. We also tested an incomplete version, in which the inducer had to be added to the culture medium, by synthesizing luciferase and a secreted form of elafin, a human protein with therapeutic properties. Our results show that, in our system, protein production can be modulated by employing different concentrations of the SHP1358 inducer or other SHPs with closed amino acid sequences. We also constructed a genetic background in which all system leakiness was eliminated. In conclusion, with this new inducible expression system, we have added to the set of tools currently used to produce secreted proteins in S. thermophilus, whose myriad applications include the delivery of therapeutic peptides or proteins.


Assuntos
Proteínas de Bactérias , Percepção de Quorum , Proteínas Recombinantes , Streptococcus thermophilus , Percepção de Quorum/genética , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Streptococcus thermophilus/genética , Streptococcus thermophilus/metabolismo , Animais , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Camundongos , Regulação Bacteriana da Expressão Gênica , Transativadores/genética , Transativadores/metabolismo , Feromônios/metabolismo , Feromônios/genética
13.
Endocrinol Diabetes Metab ; 7(5): e515, 2024 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-39238175

RESUMO

AIM: In previous studies, the researchers observed an increase in insulin secretion in STZ-treated diabetic rats following treatment with the hydroalcoholic extract of Securigera securidaca (HESS) seeds. This study focuses on the relationship between the antioxidant properties of HESS with changes in diabetic pancreatic tissue and the gene expression of factors that impact insulin secretion. METHODS: In this controlled experimental study, three varying doses of HESS were administered to three groups of diabetic rats induced by STZ. Oxidative stress indicators like total antioxidant capacity (TAC), total oxidant status (TOS) and malondialdehyde were assessed in both pancreatic and liver tissues. Pancreatic histology was studied post-haematoxylin staining. Insulin and FGF21 levels in the blood were measured using the ELISA method. The expression of Nrf2 and FGF21 genes in the pancreas and liver, along with MafA and PDX-1 genes in the pancreas, was quantified using real-time PCR. RESULTS: The administration of HESS in varying doses led to a dose-dependent rise in blood insulin levels and a decrease in blood glucose levels and oxidative stress. By reducing oxidative stress, HESS treatment lowered the heightened levels of NRF2 and FGF21 in the liver and pancreas of diabetic rats, improving pancreatic tissue health. As oxidative stress decreased, the expression of MafA and PDX1 genes in the pancreas approached levels seen in healthy rats. CONCLUSION: HESS elicits an increase in insulin secretion through the mitigation of oxidative stress and tissue damage, as well as the modulation of gene expression related to the insulin transcription factors PDX-1 and MafA.


Assuntos
Diabetes Mellitus Experimental , Secreção de Insulina , Insulina , Extratos Vegetais , Sementes , Regulação para Cima , Animais , Extratos Vegetais/farmacologia , Diabetes Mellitus Experimental/metabolismo , Diabetes Mellitus Experimental/tratamento farmacológico , Ratos , Sementes/química , Secreção de Insulina/efeitos dos fármacos , Insulina/metabolismo , Masculino , Securidaca , Estresse Oxidativo/efeitos dos fármacos , Ratos Wistar , Fator 2 Relacionado a NF-E2/metabolismo , Fatores de Transcrição/metabolismo , Fatores de Transcrição/genética , Fatores de Crescimento de Fibroblastos/metabolismo , Pâncreas/metabolismo , Pâncreas/patologia , Antioxidantes/farmacologia , Fígado/metabolismo , Transativadores , Proteínas de Homeodomínio
14.
Int J Biol Sci ; 20(12): 4691-4712, 2024.
Artigo em Inglês | MEDLINE | ID: mdl-39309447

RESUMO

B-Myb, also known as MYB proto-oncogene like 2 (MYBL2), is an important transcription factor implicated in transcription regulation, cell cycle and tumorigenesis. However, the molecular mechanism underlying B-Myb-controlled transactivation in different cell contexts as well as its functional implication in cancers remains elusive. In this study, we have conducted a comprehensive genome-wide analysis of B-Myb binding sites in multiple immortalized or cancer cell lines and identified its critical target genes. The results revealed that B-Myb regulates a common set of core cell cycle genes and cell type-specific genes through collaboration with other important transcription factors (e.g. NFY and MuvB complex) and binding to cell type-invariant promoters and cell type-specific enhancers and super-enhancers. KIF2C, UBE2C and MYC were further validated as B-Myb target genes. Loss-of-function analysis demonstrated that KIF2C knockdown inhibited tumor cell growth both in vitro and in vivo, suppressed cell motility and cell cycle progression, accompanied with defects in microtubule organization and mitosis, strongly suggesting that KIF2C is a critical regulator of cancer cell growth and mitosis, and maintains high cancer cell motility ability and microtubule dynamics. Pan-cancer transcriptomic analysis revealed that the overexpression of both B-Myb and KIF2C presents as independent prognostic markers in various types of cancer. Notably, B-Myb associates with NFYB, binds to target gene promoters, enhancers and super-enhancers, and provokes a cascade of oncogenic gene expression profiles in cancers. Overall, our results highly suggest the critical implication of B-Myb-mediated gene regulation in cancers, and the promising therapeutic and prognostic potentials of B-Myb and KIF2C for cancer diagnosis and treatment.


Assuntos
Ativação Transcricional , Humanos , Ativação Transcricional/genética , Linhagem Celular Tumoral , Neoplasias/genética , Neoplasias/metabolismo , Proto-Oncogene Mas , Regulação Neoplásica da Expressão Gênica , Cinesinas/metabolismo , Cinesinas/genética , Transativadores/metabolismo , Transativadores/genética , Animais , Proteínas de Ciclo Celular/metabolismo , Proteínas de Ciclo Celular/genética , Camundongos , Estudo de Associação Genômica Ampla , Regiões Promotoras Genéticas , Movimento Celular/genética
15.
Zhongguo Dang Dai Er Ke Za Zhi ; 26(9): 974-981, 2024.
Artigo em Chinês | MEDLINE | ID: mdl-39267514

RESUMO

OBJECTIVES: To investigate the effects and molecular mechanisms of inhibition of the Ras homolog gene (Rho)/Rho-associated coiled-coil forming protein kinase (ROCK) pathway on the proliferation and migration of airway smooth muscle cells involving myocardin (MYOCD). METHODS: Human airway smooth muscle cells were infected with the adenoviral vector Ad-ZsGreen-shRNA-hROCK1 in vitro. The cells were randomly divided into four groups: ROCK1 gene silencing control (shNC) group, shNC + arachidonic acid (AA, Rho/ROCK pathway activator) group, ROCK1 gene silencing (shROCK1) group, and shROCK1 + AA group (n=3 each). Quantitative real-time polymerase chain reaction and Western blot were used to detect the expression levels of ROCK1 and MYOCD mRNA and protein. ELISA was employed to measure the levels of globular actin and filamentous actin, while immunofluorescent staining and scratch assays were utilized to assess cell proliferation and migration. RESULTS: Compared to the shNC + AA group, the shROCK1 + AA group exhibited decreased levels of ROCK1 and MYOCD mRNA and protein expression, reduced expression levels of globular actin and filamentous actin, and diminished cell proliferation and migration capabilities (P<0.05). CONCLUSIONS: Inhibition of the Rho/ROCK pathway suppresses the proliferation and migration of airway smooth muscle cells, which may be associated with the downregulation of MYOCD.


Assuntos
Movimento Celular , Proliferação de Células , Miócitos de Músculo Liso , Transdução de Sinais , Transativadores , Quinases Associadas a rho , Quinases Associadas a rho/metabolismo , Quinases Associadas a rho/fisiologia , Quinases Associadas a rho/genética , Humanos , Miócitos de Músculo Liso/fisiologia , Miócitos de Músculo Liso/metabolismo , Células Cultivadas , Transativadores/genética , Transativadores/fisiologia , Transativadores/metabolismo , Proteínas Nucleares/genética , Proteínas Nucleares/fisiologia , Proteínas Nucleares/metabolismo , Proteínas rho de Ligação ao GTP/fisiologia , Proteínas rho de Ligação ao GTP/genética , Proteínas rho de Ligação ao GTP/metabolismo
16.
Int J Mol Sci ; 25(17)2024 Aug 26.
Artigo em Inglês | MEDLINE | ID: mdl-39273182

RESUMO

Elevated metastasis-associated in colon cancer 1 (MACC1) expression in colorectal cancer patients, and high transmembrane 4 L6 family member 5 (TM4SF5) protein expressed on various solid tumors' surface, are linked to aggressive cancer behavior and progression. In this study, adipose-derived stem cells (ASCs) were engineered to produce exosomes (Ex) that target the TM4SF5 protein on tumors. Moreover, MACC1-targeting microRNA was encapsulated within the Ex, resulting in TM4SF5-targeting Ex (MACC1-suppressing miRNA; miR-143). The anticancer effects of these Ex were investigated in vitro using the human colorectal cell line HCT116 and in vivo using colorectal cancer mouse xenograft models. In the in vivo assessment, administration of TM4SF5-targeting Ex[miR-143], referred to as tEx[miR-143] herein, resulted in the smallest tumor size, the lowest tumor growth rate, and the lightest excised tumors compared to other treatments (p < 0.05). It also led to the decreased expression of MACC-1 and anti-apoptotic markers MCL-1 and Bcl-xL while inducing the highest expression of pro-apoptotic markers BAX and BIM. These results were consistent with in vitro findings, where t Ex[miR-143] demonstrated the highest inhibition of HCT116 cell migration and invasion. These findings highlight the potential of tEx[miR-143] as an effective therapeutic strategy for colorectal cancer, demonstrating promising results in both targetability and anti-tumor effects in vitro and in vivo, warranting further investigation in clinical settings.


Assuntos
Neoplasias Colorretais , Exossomos , MicroRNAs , Animais , Humanos , MicroRNAs/genética , Neoplasias Colorretais/patologia , Neoplasias Colorretais/genética , Neoplasias Colorretais/metabolismo , Neoplasias Colorretais/terapia , Exossomos/metabolismo , Exossomos/genética , Camundongos , Ensaios Antitumorais Modelo de Xenoenxerto , Transativadores/genética , Transativadores/metabolismo , Células HCT116 , Proteínas de Membrana/metabolismo , Proteínas de Membrana/genética , Regulação Neoplásica da Expressão Gênica , Modelos Animais de Doenças , Linhagem Celular Tumoral , Apoptose , Camundongos Nus
17.
RNA Biol ; 21(1): 9-22, 2024 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-39219375

RESUMO

This study is to elucidate the effect of the LINC00663/EBF1/NR2F1 axis on inflammation and angiogenesis in bladder cancer (BC) and related molecular mechanisms. After transfection, functional experiments were conducted to test cell proliferation and invasion, tube formation ability, and content of inflammatory factors, Snail, E-cadherin, and VEGFA. Meanwhile, the relationships among LINC00663, EBF1, and NR2F1 were predicted and verified. In addition, xenograft experiments in nude mice were performed to observe the oncogenicity of 5637 BC cells in vivo. In BC tissues and cells, LINC00663 and NR2F1 were upregulated. Silencing NR2F1 or LINC00663 repressed cell proliferation and invasion, weakened vascular mimicry in vitro, decreased inflammatory factor, Snail, and VEGFA levels, and increased expression of E-cadherin. LINC00663 positively regulated NR2F1 expression through EBF1. Additionally, in vivo experiments showed that NR2F1 upregulation reversed the suppression effects of LINC00663 silencing on tumour growth, inflammation, and angiogenesis. Silencing LINC00663 decreased NR2F1 expression by mediating EBF1, thereby inhibiting BC inflammation and angiogenesis.


Assuntos
Fator I de Transcrição COUP , Proliferação de Células , Regulação Neoplásica da Expressão Gênica , Inflamação , Neovascularização Patológica , RNA Longo não Codificante , Transativadores , Neoplasias da Bexiga Urinária , Neoplasias da Bexiga Urinária/genética , Neoplasias da Bexiga Urinária/metabolismo , Neoplasias da Bexiga Urinária/patologia , Humanos , RNA Longo não Codificante/genética , RNA Longo não Codificante/metabolismo , Neovascularização Patológica/genética , Neovascularização Patológica/metabolismo , Animais , Camundongos , Linhagem Celular Tumoral , Fator I de Transcrição COUP/metabolismo , Fator I de Transcrição COUP/genética , Inflamação/metabolismo , Inflamação/genética , Inflamação/patologia , Transativadores/metabolismo , Transativadores/genética , Feminino , Masculino , Camundongos Nus , Inativação Gênica , Movimento Celular , Fator A de Crescimento do Endotélio Vascular/metabolismo , Fator A de Crescimento do Endotélio Vascular/genética , Angiogênese
18.
Pathol Res Pract ; 262: 155544, 2024 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-39197215

RESUMO

BACKGROUND: Triple-negative breast cancer (TNBC) is a subtype of breast cancer that lacks the expression of three receptors commonly targeted in breast cancer treatment. In this study, the research focused on investigating the role of centrosomal protein 55 (CEP55) in TNBC progression and its interaction with the transcription factor Spi-1 proto-oncogene (SPI1). METHODS: Various techniques including qRT-PCR, western blotting, and immunohistochemistry assays were utilized to examine gene expression patterns. Functional assays such as wound-healing assay, transwell invasion assay, 5-Ethynyl-2'-deoxyuridine assay, and metabolic assays were conducted to assess the impact of CEP55 on the behaviors of TNBC cells. CD163-positive macrophages were quantified by flow cytometry. The chromatin immunoprecipitation assay and dual-luciferase reporter assay were performed to assess the association of SPI1 with CEP55. A xenograft mouse model experiment was used to analyze the impact of SPI1 on tumor development in vivo. RESULTS: CEP55 and SPI1 expression levels were significantly upregulated in TNBC tissues and cells. The depletion of CEP55 led to decreased TNBC cell migration, invasion, proliferation, glucose metabolism, and M2 macrophage polarization, indicating its crucial role in promoting TNBC progression. Moreover, SPI1 transcriptionally activated CEP55 in TNBC cells, and its overexpression was associated with accelerated tumor growth in vivo. Further, CEP55 overexpression relieved SPI1 silencing-induced inhibitory effects on TNBC cell migration, invasion, proliferation, glucose metabolism, and M2 macrophage polarization. CONCLUSION: SPI1-mediated transcriptional activation of CEP55 plays a key role in enhancing TNBC cell migration, invasion, proliferation, glucose metabolism, and M2 macrophage polarization. These insights provide valuable information for potential targeted therapies to combat TNBC progression by modulating the SPI1-CEP55 axis.


Assuntos
Proteínas de Ciclo Celular , Proliferação de Células , Proto-Oncogene Mas , Ativação Transcricional , Neoplasias de Mama Triplo Negativas , Neoplasias de Mama Triplo Negativas/patologia , Neoplasias de Mama Triplo Negativas/genética , Neoplasias de Mama Triplo Negativas/metabolismo , Humanos , Feminino , Animais , Proteínas de Ciclo Celular/metabolismo , Proteínas de Ciclo Celular/genética , Camundongos , Movimento Celular/genética , Macrófagos/metabolismo , Transativadores/metabolismo , Transativadores/genética , Regulação Neoplásica da Expressão Gênica , Linhagem Celular Tumoral , Ativação de Macrófagos , Proteínas Proto-Oncogênicas/metabolismo , Proteínas Proto-Oncogênicas/genética , Proteínas Nucleares/metabolismo , Proteínas Nucleares/genética , Camundongos Nus
19.
J Commun Disord ; 111: 106455, 2024.
Artigo em Inglês | MEDLINE | ID: mdl-39213791

RESUMO

Smith-Magenis syndrome (SMS), a rare, genetically linked complex developmental disorder is associated with hearing loss and delays in speech-language development. Approximately 70 % of those with SMS have a genetic deletion within chromosome 17p11.2 and 10 % have a single gene mutation in the same area, known as the RAI1 variant form of SMS. Previous studies presented preliminary comparisons of the phenotype of those with deletion versus the mutation form of SMS, but none focused on hearing, speech-language, and communication profiles. The current study examined patient registry data and compared two groups of individuals with SMS to determine if genetic differences influence the communication phenotype and to determine the importance of the RAI1 gene in hearing, speech, and language abilities in children. Speech-language and hearing data from the international SMS Patient Registry for 33 subjects with SMS were analyzed: 23 with a genetic deletion (SMS (del)) and 10 with an RAI1 mutation (SMS (RAI1)). Hearing status, otopathology findings, early speech-language milestones, mode of communication, intelligibility, vocal quality, language abilities, and literacy skills were analyzed. There were small differences between the groups for hearing status, otopathological findings, mode of communication, voice quality, intelligibility, speech-language abilities, and literacy. Overlap in the speech-language phenotype between groups confirms previous hypotheses that suggest haploinsufficiency of the RAI1 gene is responsible for the SMS phenotype and that the RAI1 gene is critical for speech-language development. Future studies should include direct testing of receptive and expressive language abilities, including analyses of language samples, with larger groups of individuals to replicate and extend the current findings.


Assuntos
Síndrome de Smith-Magenis , Transativadores , Fatores de Transcrição , Humanos , Síndrome de Smith-Magenis/genética , Masculino , Feminino , Transativadores/genética , Criança , Pré-Escolar , Fatores de Transcrição/genética , Fenótipo , Perda Auditiva/genética , Adolescente , Mutação , Transtornos do Desenvolvimento da Linguagem/genética , Deleção Cromossômica
20.
Toxicol Appl Pharmacol ; 491: 117075, 2024 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-39173720

RESUMO

Artesunate (ART) is a derivative of artemisinin and has anti-inflammatory, anti-tumor, and anti-angiogenic properties. Although ART has been implicated in osteoarthritis (OA), the mechanism needs to be further dissected. Here, we explored the effects of ART on the development of OA and the underlying mechanism using destabilization of the medial meniscus (DMM) surgical instability model. Mice with OA were developed using DMM and treated with ART. The pathological morphology of knee joint tissues was examined, and the degeneration of joint cartilage was assessed. Mouse knee chondrocytes were isolated and induced with IL-1ß, followed by ART treatment. ART alleviates OA in mice by elevating ubiquitin carboxyl-terminal hydrolase 7 (USP7) expression, and USP7 inhibitor (P22077) treatment mitigated the protective effects of ART on chondrocytes. We also showed that USP7 mediated the deubiquitination of forkhead box protein O1 (FoxO1), while FoxO1 alleviated chondrocyte injury. In addition, FoxO1 promoted metastasis-associated protein MTA1 (MTA1) transcription, and downregulation of MTA1 exacerbated chondrocyte injury. Our study identifies that USP7/FoxO1/MTA1 is a key signaling cascade in the treatment of ART on OA.


Assuntos
Artesunato , Condrócitos , Proteína Forkhead Box O1 , Camundongos Endogâmicos C57BL , Osteoartrite , Peptidase 7 Específica de Ubiquitina , Animais , Artesunato/farmacologia , Artesunato/uso terapêutico , Proteína Forkhead Box O1/metabolismo , Proteína Forkhead Box O1/genética , Peptidase 7 Específica de Ubiquitina/metabolismo , Peptidase 7 Específica de Ubiquitina/genética , Camundongos , Masculino , Osteoartrite/tratamento farmacológico , Osteoartrite/metabolismo , Osteoartrite/patologia , Condrócitos/efeitos dos fármacos , Condrócitos/metabolismo , Condrócitos/patologia , Transativadores/metabolismo , Transativadores/genética , Transdução de Sinais/efeitos dos fármacos , Proteínas Repressoras/genética , Proteínas Repressoras/metabolismo , Transcrição Gênica/efeitos dos fármacos
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