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1.
Diabetologia ; 67(5): 908-927, 2024 May.
Artigo em Inglês | MEDLINE | ID: mdl-38409439

RESUMO

AIMS/HYPOTHESIS: The proinflammatory cytokines IFN-α, IFN-γ, IL-1ß and TNF-α may contribute to innate and adaptive immune responses during insulitis in type 1 diabetes and therefore represent attractive therapeutic targets to protect beta cells. However, the specific role of each of these cytokines individually on pancreatic beta cells remains unknown. METHODS: We used deep RNA-seq analysis, followed by extensive confirmation experiments based on reverse transcription-quantitative PCR (RT-qPCR), western blot, histology and use of siRNAs, to characterise the response of human pancreatic beta cells to each cytokine individually and compared the signatures obtained with those present in islets of individuals affected by type 1 diabetes. RESULTS: IFN-α and IFN-γ had a greater impact on the beta cell transcriptome when compared with IL-1ß and TNF-α. The IFN-induced gene signatures have a strong correlation with those observed in beta cells from individuals with type 1 diabetes, and the level of expression of specific IFN-stimulated genes is positively correlated with proteins present in islets of these individuals, regulating beta cell responses to 'danger signals' such as viral infections. Zinc finger NFX1-type containing 1 (ZNFX1), a double-stranded RNA sensor, was identified as highly induced by IFNs and shown to play a key role in the antiviral response in beta cells. CONCLUSIONS/INTERPRETATION: These data suggest that IFN-α and IFN-γ are key cytokines at the islet level in human type 1 diabetes, contributing to the triggering and amplification of autoimmunity.


Assuntos
Diabetes Mellitus Tipo 1 , Ilhotas Pancreáticas , Humanos , Citocinas/metabolismo , Diabetes Mellitus Tipo 1/metabolismo , Interferons/metabolismo , Fator de Necrose Tumoral alfa/metabolismo , Interferon gama/metabolismo , Ilhotas Pancreáticas/metabolismo
2.
Mol Cell ; 64(3): 565-579, 2016 11 03.
Artigo em Inglês | MEDLINE | ID: mdl-27871486

RESUMO

Expression changes of competing endogenous RNAs (ceRNAs) have been proposed to influence microRNA (miRNA) activity and thereby regulate other transcripts containing miRNA-binding sites. Here, we find that although miRNA levels define the extent of repression, they have little effect on the magnitude of the ceRNA expression change required to observe derepression. Canonical 6-nt sites, which typically mediate modest repression, can nonetheless compete for miRNA binding, with potency ∼20% of that observed for canonical 8-nt sites. In aggregate, low-affinity/background sites also contribute to competition. Sites with extensive additional complementarity can appear as more potent, but only because they induce miRNA degradation. Cooperative binding of proximal sites for the same or different miRNAs does increase potency. These results provide quantitative insights into the stoichiometric relationship between miRNAs and target abundance, target-site spacing, and affinity requirements for ceRNA-mediated gene regulation, and the unusual circumstances in which ceRNA-mediated gene regulation might be observed.


Assuntos
Regulação da Expressão Gênica , Redes Reguladoras de Genes , Hepatócitos/metabolismo , MicroRNAs/genética , RNA Mensageiro/genética , Adenoviridae/genética , Adenoviridae/metabolismo , Animais , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Sítios de Ligação , Ligação Competitiva , Genes Reporter , Hepatócitos/citologia , Proteínas Luminescentes/genética , Proteínas Luminescentes/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , MicroRNAs/metabolismo , Plasmídeos/química , Plasmídeos/metabolismo , Cultura Primária de Células , RNA Mensageiro/metabolismo , Transformação Genética , Proteína Vermelha Fluorescente
3.
J Biol Chem ; 290(39): 23680-91, 2015 Sep 25.
Artigo em Inglês | MEDLINE | ID: mdl-26240150

RESUMO

MicroRNAs (miRNAs) are important regulators of cell-autonomous gene expression that influence many biological processes. They are also released from cells and are present in virtually all body fluids, including blood, urine, saliva, sweat, and milk. The functional role of nutritionally obtained extracellular miRNAs is controversial, and irrefutable demonstration of exogenous miRNA uptake by cells and canonical miRNA function is still lacking. Here we show that miRNAs are present at high levels in the milk of lactating mice. To investigate intestinal uptake of miRNAs in newborn mice, we employed genetic models in which newborn miR-375 and miR-200c/141 knockout mice received milk from wild-type foster mothers. Analysis of the intestinal epithelium, blood, liver, and spleen revealed no evidence for miRNA uptake. miR-375 levels in hepatocytes were at the limit of detection and remained orders of magnitude below the threshold for target gene regulation (between 1000 and 10,000 copies/cell). Furthermore, our study revealed rapid degradation of milk miRNAs in intestinal fluid. Together, our results indicate a nutritional rather than gene-regulatory role of miRNAs in the milk of newborn mice.


Assuntos
MicroRNAs/metabolismo , Leite , Animais , Animais Recém-Nascidos , Feminino , Inativação Gênica , Mucosa Intestinal/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , MicroRNAs/genética , MicroRNAs/fisiologia
4.
Front Immunol ; 14: 1158905, 2023.
Artigo em Inglês | MEDLINE | ID: mdl-37313411

RESUMO

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) induces B and T cell responses, contributing to virus neutralization. In a cohort of 2,911 young adults, we identified 65 individuals who had an asymptomatic or mildly symptomatic SARS-CoV-2 infection and characterized their humoral and T cell responses to the Spike (S), Nucleocapsid (N) and Membrane (M) proteins. We found that previous infection induced CD4 T cells that vigorously responded to pools of peptides derived from the S and N proteins. By using statistical and machine learning models, we observed that the T cell response highly correlated with a compound titer of antibodies against the Receptor Binding Domain (RBD), S and N. However, while serum antibodies decayed over time, the cellular phenotype of these individuals remained stable over four months. Our computational analysis demonstrates that in young adults, asymptomatic and paucisymptomatic SARS-CoV-2 infections can induce robust and long-lasting CD4 T cell responses that exhibit slower decays than antibody titers. These observations imply that next-generation COVID-19 vaccines should be designed to induce stronger cellular responses to sustain the generation of potent neutralizing antibodies.


Assuntos
COVID-19 , Humanos , Vacinas contra COVID-19 , SARS-CoV-2 , Anticorpos Neutralizantes , Aprendizado de Máquina
5.
Front Endocrinol (Lausanne) ; 13: 854094, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-35860702

RESUMO

Restoration of ß-cell mass through the induction of proliferation represents an attractive therapeutic approach for the treatment of diabetes. However, intact and dispersed primary islets suffer from rapidly deteriorating viability and function ex vivo, posing a significant challenge for their experimental use in proliferation studies. Here, we describe a novel method for the assessment of compound effects on ß-cell proliferation and count using reaggregated primary human islets, or islet microtissues (MTs), which display homogeneous size and tissue architecture as well as robust and stable functionality and viability for 4 weeks in culture. We utilized this platform to evaluate the dose-dependent short- and long-term effects of harmine on ß-cell proliferation and function. Following compound treatment and EdU incorporation, islet MTs were stained and confocal-imaged for DAPI (nuclear marker), NKX6.1 (ß-cell marker), and EdU (proliferation marker), allowing automated 3D-analysis of number of total cells, ß-cells, and proliferating ß- and non-ß-cells per islet MT. In parallel, insulin secretion, intracellular insulin and ATP contents, and Caspase 3/7 activity were analyzed to obtain a comprehensive overview of islet MT function and viability. We observed that 4-day harmine treatment increased ß- and non-ß-cell proliferation, NKX6.1 expression, and basal and stimulated insulin secretion in a dose-dependent manner, while fold-stimulation of secretion peaked at intermediate harmine doses. Interestingly, 15-day harmine treatment led to a general reduction in harmine's proliferative effects as well as altered dose-dependent trends. The described methodology provides a unique tool for in vitro high-throughput evaluation of short- and long-term changes in human ß-cell proliferation, count and fraction along with a variety of functional parameters, in a representative 3D human islet model.


Assuntos
Harmina , Células Secretoras de Insulina , Proliferação de Células , Harmina/metabolismo , Harmina/farmacologia , Humanos , Insulina/metabolismo , Insulina/farmacologia , Secreção de Insulina , Células Secretoras de Insulina/metabolismo
6.
Mol Metab ; 53: 101267, 2021 11.
Artigo em Inglês | MEDLINE | ID: mdl-34116231

RESUMO

OBJECTIVE: The miR-200-Zeb1 axis regulates the epithelial-to-mesenchymal transition (EMT), differentiation, and resistance to apoptosis. A better understanding of these processes in diabetes is highly relevant, as ß-cell dedifferentiation and apoptosis contribute to the loss of functional ß-cell mass and diabetes progression. Furthermore, EMT promotes the loss of ß-cell identity in the in vitro expansion of human islets. Though the miR-200 family has previously been identified as a regulator of ß-cell apoptosis in vivo, studies focusing on Zeb1 are lacking. The aim of this study was thus to investigate the role of Zeb1 in ß-cell function and survival in vivo. METHODS: miR-200 and Zeb1 are involved in a double-negative feedback loop. We characterized a mouse model in which miR-200 binding sites in the Zeb1 3'UTR are mutated (Zeb1200), leading to a physiologically relevant upregulation of Zeb1 mRNA expression. The role of Zeb1 was investigated in this model via metabolic tests and analysis of isolated islets. Further insights into the distinct contributions of the miR-200 and Zeb1 branches of the feedback loop were obtained by crossing the Zeb1200 allele into a background of miR-141-200c overexpression. RESULTS: Mild Zeb1 derepression in vivo led to broad transcriptional changes in islets affecting ß-cell identity, EMT, insulin secretion, cell-cell junctions, the unfolded protein response (UPR), and the response to ER stress. The aggregation and insulin secretion of dissociated islets of mice homozygous for the Zeb1200 mutation (Zeb1200M) were impaired, and Zeb1200M islets were resistant to thapsigargin-induced ER stress ex vivo. Zeb1200M mice had increased circulating proinsulin levels but no overt metabolic phenotype, reflecting the strong compensatory ability of islets to maintain glucose homeostasis. CONCLUSIONS: This study signifies the importance of the miR-200-Zeb1 axis in regulating key aspects of ß-cell function and survival. A better understanding of this axis is highly relevant in developing therapeutic strategies for inducing ß-cell redifferentiation and maintaining ß-cell identity in in vitro islet expansion.


Assuntos
Células Secretoras de Insulina/metabolismo , MicroRNAs/metabolismo , Homeobox 1 de Ligação a E-box em Dedo de Zinco/metabolismo , Animais , Apoptose , Células Cultivadas , Estresse do Retículo Endoplasmático , Secreção de Insulina , Camundongos , Camundongos Knockout , MicroRNAs/genética , Homeobox 1 de Ligação a E-box em Dedo de Zinco/deficiência , Homeobox 1 de Ligação a E-box em Dedo de Zinco/genética
7.
Nat Commun ; 9(1): 4671, 2018 11 07.
Artigo em Inglês | MEDLINE | ID: mdl-30405106

RESUMO

The epithelial-to-mesenchymal transition (EMT) is an important mechanism for cancer progression and metastasis. Numerous in vitro and tumor-profiling studies point to the miR-200-Zeb1 axis as crucial in regulating this process, yet in vivo studies involving its regulation within a physiological context are lacking. Here, we show that miR-200 ablation in the Rip-Tag2 insulinoma mouse model induces beta-cell dedifferentiation, initiates an EMT expression program, and promotes tumor invasion. Strikingly, disrupting the miR-200 sites of the endogenous Zeb1 locus causes a similar phenotype. Reexpressing members of the miR-200 superfamily in vitro reveals that the miR-200c family and not the co-expressed and closely related miR-141 family is responsible for regulation of Zeb1 and EMT. Our results thus show that disrupting the in vivo regulation of Zeb1 by miR-200c is sufficient to drive EMT, thus highlighting the importance of this axis in tumor progression and invasion and its potential as a therapeutic target.


Assuntos
Diferenciação Celular , MicroRNAs/metabolismo , Neoplasias/genética , Neoplasias/patologia , Transdução de Sinais , Homeobox 1 de Ligação a E-box em Dedo de Zinco/metabolismo , Animais , Sequência de Bases , Diferenciação Celular/genética , Proliferação de Células/genética , Progressão da Doença , Transição Epitelial-Mesenquimal/genética , Regulação Neoplásica da Expressão Gênica , Redes Reguladoras de Genes , Camundongos Endogâmicos C57BL , MicroRNAs/genética , Mutação/genética , Invasividade Neoplásica , Homeobox 1 de Ligação a E-box em Dedo de Zinco/genética
8.
Oncol Lett ; 7(6): 1819-1825, 2014 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-24932239

RESUMO

MicroRNAs (miRNAs) are small non-coding RNAs that regulate the expression of their target genes at the post-transcriptional level. In cancer cells, miRNAs, depending on the biological functions of their target genes, may have a tumor-promoting or -suppressing effect. Treatment of cancer cells with inhibitors of DNA methylation and/or histone deacetylation modulates the expression level of miRNAs, which provides evidence for epigenetic regulation of miRNA expression. The consequences of inhibition of histone methyltransferase on miRNA expression, however, have not been thoroughly investigated. The present study examined the expression pattern of miRNAs in the non-small cell lung cancer cell line, H1299 with or without treatment of BIX01294, a potent chemical inhibitor of G9a methyltransferase that catalyzes the mono-and di-methylation of the lysine 9 residue of histone H3. By coupling microarray analysis with quantitative real-time polymerase chain reaction analysis, two miRNAs were identified that showed consistent downregulation following BIX01294 treatment. The results indicate that histone H3 methylation regulates miRNA expression in lung cancer cells, which may provide additional insight for future chemical treatment of lung cancer.

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