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1.
Genes Dev ; 31(13): 1325-1338, 2017 07 01.
Artigo em Inglês | MEDLINE | ID: mdl-28794185

RESUMO

Deciphering the fundamental mechanisms controlling cardiac specification is critical for our understanding of how heart formation is initiated during embryonic development and for applying stem cell biology to regenerative medicine and disease modeling. Using systematic and unbiased functional screening approaches, we discovered that the Id family of helix-loop-helix proteins is both necessary and sufficient to direct cardiac mesoderm formation in frog embryos and human embryonic stem cells. Mechanistically, Id proteins specify cardiac cell fate by repressing two inhibitors of cardiogenic mesoderm formation-Tcf3 and Foxa2-and activating inducers Evx1, Grrp1, and Mesp1. Most importantly, CRISPR/Cas9-mediated ablation of the entire Id (Id1-4) family in mouse embryos leads to failure of anterior cardiac progenitor specification and the development of heartless embryos. Thus, Id proteins play a central and evolutionarily conserved role during heart formation and provide a novel means to efficiently produce cardiovascular progenitors for regenerative medicine and drug discovery applications.


Assuntos
Linhagem da Célula/genética , Coração/embriologia , Proteínas Inibidoras de Diferenciação/genética , Proteínas Inibidoras de Diferenciação/metabolismo , Organogênese/genética , Animais , Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Diferenciação Celular/genética , Linhagem Celular , Embrião de Mamíferos/citologia , Embrião de Mamíferos/embriologia , Embrião não Mamífero/citologia , Embrião não Mamífero/embriologia , Células-Tronco Embrionárias/citologia , Células-Tronco Embrionárias/fisiologia , Edição de Genes , Regulação da Expressão Gênica no Desenvolvimento/genética , Cardiopatias Congênitas/genética , Humanos , Mesoderma/citologia , Mesoderma/fisiologia , Camundongos , Mutação , Sementes , Xenopus laevis/embriologia
2.
EMBO J ; 37(23)2018 12 03.
Artigo em Inglês | MEDLINE | ID: mdl-30389666

RESUMO

While mucosal inflammation is a major source of stress during enteropathogen infection, it remains to be fully elucidated how the host benefits from this environment to clear the pathogen. Here, we show that host stress induced by different stimuli mimicking inflammatory conditions strongly reduces the binding of Shigella flexneri to epithelial cells. Mechanistically, stress activates acid sphingomyelinase leading to host membrane remodeling. Consequently, knockdown or pharmacological inhibition of the acid sphingomyelinase blunts the stress-dependent inhibition of Shigella binding to host cells. Interestingly, stress caused by intracellular Shigella replication also results in remodeling of the host cell membrane, in vitro and in vivo, which precludes re-infection by this and other non-motile pathogens. In contrast, Salmonella Typhimurium overcomes the shortage of permissive entry sites by gathering effectively at the remaining platforms through its flagellar motility. Overall, our findings reveal host membrane remodeling as a novel stress-responsive cell-autonomous defense mechanism that protects epithelial cells from infection by non-motile bacterial pathogens.


Assuntos
Membrana Celular/imunologia , Disenteria Bacilar/imunologia , Células Epiteliais/imunologia , Imunidade Inata , Infecções por Salmonella/imunologia , Salmonella typhimurium/imunologia , Shigella flexneri/imunologia , Estresse Fisiológico/imunologia , Animais , Membrana Celular/patologia , Disenteria Bacilar/patologia , Células Epiteliais/patologia , Cobaias , Infecções por Salmonella/patologia
3.
Nucleic Acids Res ; 48(11): 5891-5906, 2020 06 19.
Artigo em Inglês | MEDLINE | ID: mdl-32421830

RESUMO

Originally identified as an RNA polymerase II interactor, Che-1/AATF (Che-1) has now been recognized as a multifunctional protein involved in cell-cycle regulation and cancer progression, as well as apoptosis inhibition and response to stress. This protein displays a peculiar nucleolar localization and it has recently been implicated in pre-rRNA processing and ribosome biogenesis. Here, we report the identification of a novel function of Che-1 in the regulation of ribosomal RNA (rRNA) synthesis, in both cancer and normal cells. We demonstrate that Che-1 interacts with RNA polymerase I and nucleolar upstream binding factor (UBF) and promotes RNA polymerase I-dependent transcription. Furthermore, this protein binds to the rRNA gene (rDNA) promoter and modulates its epigenetic state by contrasting the recruitment of HDAC1. Che-1 downregulation affects RNA polymerase I and UBF recruitment on rDNA and leads to reducing rDNA promoter activity and 47S pre-rRNA production. Interestingly, Che-1 depletion induces abnormal nucleolar morphology associated with re-distribution of nucleolar proteins. Finally, we show that upon DNA damage Che-1 re-localizes from rDNA to TP53 gene promoter to induce cell-cycle arrest. This previously uncharacterized function of Che-1 confirms the important role of this protein in the regulation of ribosome biogenesis, cellular proliferation and response to stress.


Assuntos
Proteínas Reguladoras de Apoptose/metabolismo , DNA Ribossômico/genética , Genes de RNAr/genética , RNA Polimerase I/metabolismo , Proteínas Repressoras/metabolismo , Transcrição Gênica , Proteínas Reguladoras de Apoptose/deficiência , Proteínas Reguladoras de Apoptose/genética , Proteínas Mutadas de Ataxia Telangiectasia/metabolismo , Pontos de Checagem do Ciclo Celular , Linhagem Celular , Nucléolo Celular/metabolismo , Nucléolo Celular/patologia , Dano ao DNA , DNA Ribossômico/metabolismo , Homeostase , Humanos , Fosforilação , Proteínas Pol1 do Complexo de Iniciação de Transcrição/metabolismo , Regiões Promotoras Genéticas , Proteínas Repressoras/deficiência , Proteínas Repressoras/genética , Ribossomos/metabolismo
4.
Methods ; 152: 55-64, 2019 01 01.
Artigo em Inglês | MEDLINE | ID: mdl-30292796

RESUMO

MicroRNAs (miRNAs) are small non-coding RNAs that post-transcriptionally modulate gene expression and orchestrate a wide range of biological and pathological processes. The use of high-throughput screening technologies, in particular microscopy-based screenings (also known as high-content screenings), coupled with genome-wide libraries for modulation of miRNA levels, allow for comprehensive functional analysis of each member of the miRNome in different phenotypic cell-based assays. The wealth of information obtained from such screenings spans across various fields of research, including cancer, cardiovascular, cell reprogramming, and infection biology. Here, we provide an overview of the rationale for performing screenings using synthetic libraries of miRNA mimics and inhibitors, and of the microscopy-based miRNA screenings performed to date. Moreover, a list of resources available for such endeavor is provided. Finally, we describe a detailed procedure for a case study where microscopy-based screening using a library of miRNA mimics was performed to identify miRNAs that control infection of epithelial cells by the bacterial pathogen Salmonella. The methodologies described here can be easily adapted for screenings addressing other biological questions.


Assuntos
MicroRNAs/fisiologia , Infecções por Salmonella/genética , Células Epiteliais/microbiologia , Regulação da Expressão Gênica , Humanos , Salmonella , Transfecção/métodos
5.
Hum Mol Genet ; 26(22): 4375-4387, 2017 11 15.
Artigo em Inglês | MEDLINE | ID: mdl-28973155

RESUMO

Glioblastoma (GBM) is a deadly and therapy resistant malignant brain tumour, characterized by an aggressive and diffuse growth pattern, which prevents complete surgical resection. Despite advances in the identification of genomic and molecular alterations that fuel the tumour, average patient survival post-diagnosis remains very low (∼14.6-months). In addition to being highly heterogeneous, GBM tumour cells exhibit high adaptive capacity to targeted molecular therapies owing to an established network of signalling cascades with functional redundancy, which provides them with robust compensatory survival mechanisms. Here, we investigated whether a multimodal strategy combining multitargeted tyrosine kinase inhibitors (MTKIs) and microRNA (miRNA) modulation could overcome the signalling pathway redundancy in GBM and, hence, promote tumour cell death. By performing a high-throughput screening, we identified a myriad of miRNAs, including those belonging to the miR-302-3p/372-3p/373-3p/520-3p family, which coordinately act with the MTKI sunitinib to decrease GBM cell viability. Two members of this family, hsa-miRNA-302a-3p and hsa-miRNA-520 b, were found to modulate the expression of receptor tyrosine kinase mediators (including AKT1, PIK3CA and SOS1) in U87 and DBTRG human GBM cells. Importantly, administration of mimics of these miRNAs with sunitinib or axitinib resulted in decreased tumour cell proliferation and enhanced cell death, whereas no significant effect was observed when coupling miRNA modulation with temozolomide, the first-line drug for GBM therapy. Overall, our results provide evidence that combining the 'horizontal' inhibition of signalling pathways promoted by MTKIs with the 'vertical' inhibition of the downstream signalling cascade promoted by hsa-miR-302a-3p and hsa-miR-520 b constitutes a promising approach towards GBM treatment.


Assuntos
Neoplasias Encefálicas/genética , Neoplasias Encefálicas/terapia , Glioblastoma/genética , Glioblastoma/terapia , MicroRNAs/genética , Inibidores de Proteínas Quinases/farmacologia , Neoplasias Encefálicas/tratamento farmacológico , Neoplasias Encefálicas/metabolismo , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Proliferação de Células/genética , Terapia Combinada , Predisposição Genética para Doença , Glioblastoma/tratamento farmacológico , Glioblastoma/metabolismo , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Humanos , MicroRNAs/metabolismo , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/genética , Transfecção
6.
PLoS Pathog ; 13(4): e1006327, 2017 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-28394930

RESUMO

MicroRNAs play an important role in the interplay between bacterial pathogens and host cells, participating as host defense mechanisms, as well as exploited by bacteria to subvert host cellular functions. Here, we show that microRNAs modulate infection by Shigella flexneri, a major causative agent of bacillary dysentery in humans. Specifically, we characterize the dual regulatory role of miR-29b-2-5p during infection, showing that this microRNA strongly favors Shigella infection by promoting both bacterial binding to host cells and intracellular replication. Using a combination of transcriptome analysis and targeted high-content RNAi screening, we identify UNC5C as a direct target of miR-29b-2-5p and show its pivotal role in the modulation of Shigella binding to host cells. MiR-29b-2-5p, through repression of UNC5C, strongly enhances filopodia formation thus increasing Shigella capture and promoting bacterial invasion. The increase of filopodia formation mediated by miR-29b-2-5p is dependent on RhoF and Cdc42 Rho-GTPases. Interestingly, the levels of miR-29b-2-5p, but not of other mature microRNAs from the same precursor, are decreased upon Shigella replication at late times post-infection, through degradation of the mature microRNA by the exonuclease PNPT1. While the relatively high basal levels of miR-29b-2-5p at the start of infection ensure efficient Shigella capture by host cell filopodia, dampening of miR-29b-2-5p levels later during infection may constitute a bacterial strategy to favor a balanced intracellular replication to avoid premature cell death and favor dissemination to neighboring cells, or alternatively, part of the host response to counteract Shigella infection. Overall, these findings reveal a previously unappreciated role of microRNAs, and in particular miR-29b-2-5p, in the interaction of Shigella with host cells.


Assuntos
Interações Hospedeiro-Patógeno/genética , MicroRNAs/genética , Shigella/genética , Shigella/virologia , Replicação Viral/genética , Linhagem Celular , Replicação do DNA/genética , Perfilação da Expressão Gênica/métodos , Interações Hospedeiro-Patógeno/imunologia , Humanos , Pseudópodes/imunologia , Interferência de RNA/fisiologia
7.
Nature ; 492(7429): 376-81, 2012 Dec 20.
Artigo em Inglês | MEDLINE | ID: mdl-23222520

RESUMO

In mammals, enlargement of the heart during embryonic development is primarily dependent on the increase in cardiomyocyte numbers. Shortly after birth, however, cardiomyocytes stop proliferating and further growth of the myocardium occurs through hypertrophic enlargement of the existing myocytes. As a consequence of the minimal renewal of cardiomyocytes during adult life, repair of cardiac damage through myocardial regeneration is very limited. Here we show that the exogenous administration of selected microRNAs (miRNAs) markedly stimulates cardiomyocyte proliferation and promotes cardiac repair. We performed a high-content microscopy, high-throughput functional screening for human miRNAs that promoted neonatal cardiomyocyte proliferation using a whole-genome miRNA library. Forty miRNAs strongly increased both DNA synthesis and cytokinesis in neonatal mouse and rat cardiomyocytes. Two of these miRNAs (hsa-miR-590 and hsa-miR-199a) were further selected for testing and were shown to promote cell cycle re-entry of adult cardiomyocytes ex vivo and to promote cardiomyocyte proliferation in both neonatal and adult animals. After myocardial infarction in mice, these miRNAs stimulated marked cardiac regeneration and almost complete recovery of cardiac functional parameters. The miRNAs identified hold great promise for the treatment of cardiac pathologies consequent to cardiomyocyte loss.


Assuntos
MicroRNAs/análise , MicroRNAs/genética , Miocárdio/citologia , Regeneração/genética , Animais , Proliferação de Células , Citocinese , DNA/biossíntese , Regulação para Baixo , Biblioteca Gênica , Terapia Genética , Coração/crescimento & desenvolvimento , Humanos , Camundongos , MicroRNAs/uso terapêutico , Infarto do Miocárdio/genética , Infarto do Miocárdio/patologia , Infarto do Miocárdio/prevenção & controle , Infarto do Miocárdio/terapia , Miocárdio/metabolismo , Miócitos Cardíacos/citologia , Miócitos Cardíacos/metabolismo , Ratos , Ratos Wistar
8.
Proc Natl Acad Sci U S A ; 112(36): 11276-81, 2015 Sep 08.
Artigo em Inglês | MEDLINE | ID: mdl-26305933

RESUMO

Viral vectors based on the adeno-associated virus (AAV) hold great promise for in vivo gene transfer; several unknowns, however, still limit the vectors' broader and more efficient application. Here, we report the results of a high-throughput, whole-genome siRNA screening aimed at identifying cellular factors regulating AAV transduction. We identified 1,483 genes affecting vector efficiency more than 4-fold and up to 50-fold, either negatively or positively. Most of these factors have not previously been associated to AAV infection. The most effective siRNAs were independent from the virus serotype or analyzed cell type and were equally evident for single-stranded and self-complementary AAV vectors. A common characteristic of the most effective siRNAs was the induction of cellular DNA damage and activation of a cell cycle checkpoint. This information can be exploited for the development of more efficient AAV-based gene delivery procedures. Administration of the most effective siRNAs identified by the screening to the liver significantly improved in vivo AAV transduction efficiency.


Assuntos
Dependovirus/genética , Genoma Humano/genética , Interferência de RNA , Transdução Genética , Animais , Linhagem Celular , Linhagem Celular Tumoral , Expressão Gênica , Terapia Genética/métodos , Vetores Genéticos/genética , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Células HEK293 , Células HeLa , Interações Hospedeiro-Patógeno/genética , Humanos , Fígado/metabolismo , Proteínas Luminescentes/genética , Proteínas Luminescentes/metabolismo , Camundongos , Microscopia Confocal , Microscopia de Fluorescência , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transgenes/genética
9.
J Biol Chem ; 290(49): 29652-62, 2015 Dec 04.
Artigo em Inglês | MEDLINE | ID: mdl-26468294

RESUMO

MageB2 belongs to the melanoma antigen gene (MAGE-I) family of tumor-specific antigens. Expression of this gene has been detected in human tumors of different origins. However, little is known about the protein function and how its expression affects tumor cell phenotypes. In this work, we found that human MageB2 protein promotes tumor cell proliferation in a p53-independent fashion, as observed both in cultured cells and growing tumors in mice. Gene expression analysis showed that MageB2 enhances the activity of E2F transcription factors. Mechanistically, the activation of E2Fs is related to the ability of MageB2 to interact with the E2F inhibitor HDAC1. Cellular distribution of MageB2 protein includes the nucleoli. Nevertheless, ribotoxic drugs rapidly promote its nucleolar exit. We show that MageB2 counteracts E2F inhibition by ribosomal proteins independently of Mdm2 expression. Importantly, MageB2 plays a critical role in impairing cell cycle arrest in response to Actinomycin D. The data presented here support a relevant function for human MageB2 in cancer cells both under cycling and stressed conditions, presenting a distinct functional feature with respect to other characterized MAGE-I proteins.


Assuntos
Antígenos de Neoplasias/metabolismo , Fatores de Transcrição E2F/metabolismo , Proteínas de Neoplasias/metabolismo , Proteínas Proto-Oncogênicas c-mdm2/metabolismo , Animais , Antineoplásicos/química , Ciclo Celular , Nucléolo Celular/metabolismo , Proliferação de Células , Dactinomicina/química , Fibroblastos/metabolismo , Regulação da Expressão Gênica , Proteínas de Fluorescência Verde/metabolismo , Células HCT116 , Células HEK293 , Histona Desacetilase 1/metabolismo , Histona Desacetilases/metabolismo , Humanos , Melanoma Experimental/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Ribossomos/metabolismo
10.
Stem Cells ; 32(11): 2998-3011, 2014 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-25069783

RESUMO

Mesenchymal stem/stromal cells (MSCs) are the precursors of various cell types that compose both normal and cancer tissue microenvironments. In order to support the widely diversified parenchymal cells and tissue organization, MSCs are characterized by a large degree of heterogeneity, although available analyses of molecular and transcriptional data do not provide clear evidence. We have isolated MSCs from high-grade serous ovarian cancers (HG-SOCs) and various normal tissues (N-MSCs), demonstrated their normal genotype and analyzed their transcriptional activity with respect to the large comprehensive FANTOM5 sample dataset. Our integrative analysis conducted against the extensive panel of primary cells and tissues of the FANTOM5 project allowed us to mark the HG-SOC-MSCs CAGE-seq transcriptional heterogeneity and to identify a cell-type-specific transcriptional activity showing a significant relationship with primary mesothelial cells. Our analysis shows that MSCs isolated from different tissues are highly heterogeneous. The mesothelial-related gene signature identified in this study supports the hypothesis that HG-SOC-MSCs are bona fide representatives of the ovarian district. This finding indicates that HG-SOC-MSCs could actually derive from the coelomic mesothelium, suggesting that they might be linked to the epithelial tumor through common embryological precursors.


Assuntos
Diferenciação Celular/fisiologia , Células-Tronco Mesenquimais/citologia , Neoplasias Epiteliais e Glandulares/patologia , Neoplasias Mesoteliais/metabolismo , Neoplasias Ovarianas/patologia , Microambiente Tumoral/fisiologia , Carcinoma Epitelial do Ovário , Feminino , Humanos , Gradação de Tumores/métodos , Neoplasias Epiteliais e Glandulares/metabolismo , Neoplasias Mesoteliais/patologia , Neoplasias Ovarianas/metabolismo
11.
Am J Pathol ; 183(6): 1747-1757, 2013 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-24096076

RESUMO

Oral mucositis (OM) is a serious and acute side effect in patients with cancer who receive chemotherapy or radiotherapy, often leading to the suspension of therapy and a need for opioid analgesic and enteral/parenteral nutrition, with an effect on patient survival. Among the various interventions proposed in OM management, laser therapy is becoming a recommended treatment option but has limitations due to its heterogeneous laser parameters. Here, we report on our successful clinical experience on the use of class IV laser therapy to treat OM induced by different chemotherapy regimens. To shed light on the mechanisms of action of laser therapy in improving OM resolution, we have developed an animal model of chemotherapy-induced OM, in which we compare the efficacy of the standard low-power laser therapy protocol with an innovative protocol, defined as high-power laser therapy. We show that high-power laser therapy is more effective than low-power laser therapy in improving OM lesion healing, reducing the inflammatory burden, and preserving tissue integrity. In addition, high-power laser therapy has been particularly effective in promoting the formation of new arterioles within the granulation tissue. Our results provide important insights into the mechanism of action of biostimulating laser therapy on OM in vivo and pave a way for clinical experimentation with the use of high-power laser therapy.


Assuntos
Antineoplásicos/efeitos adversos , Terapia a Laser , Neoplasias/tratamento farmacológico , Estomatite , Adulto , Idoso , Idoso de 80 Anos ou mais , Animais , Antineoplásicos/administração & dosagem , Modelos Animais de Doenças , Feminino , Humanos , Masculino , Camundongos , Pessoa de Meia-Idade , Estomatite/induzido quimicamente , Estomatite/patologia , Estomatite/cirurgia
12.
Mol Ther ; 20(11): 2087-97, 2012 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-22850678

RESUMO

Gene therapy vectors based on the adeno-associated virus (AAV) are extremely efficient for gene transfer into post-mitotic cells of heart, muscle, brain, and retina. The reason for their exquisite tropism for these cells has long remained elusive. Here, we show that upon terminal differentiation, cardiac and skeletal myocytes downregulate proteins of the DNA damage response (DDR) and that this markedly induces permissivity to AAV transduction. We observed that expression of members of the MRN complex (Mre11, Rad50, Nbs1), which bind the incoming AAV genomes, faded in cardiomyocytes at ~2 weeks after birth, as well as upon myoblast differentiation in vitro; in both cases, withdrawal of the cells from the cell cycle coincided with increased AAV permissivity. Treatment of proliferating cells with short-interfering RNAs (siRNAs) against the MRN proteins, or with microRNA-24, which is normally upregulated upon terminal differentiation and negatively controls the Nbs1 levels, significantly increased permissivity to AAV transduction. Consistently, delivery of these small RNAs to the juvenile liver concomitant with AAV markedly improved in vivo hepatocyte transduction. Collectively, these findings support the conclusion that cellular DDR proteins inhibit AAV transduction and that terminal cell differentiation relieves this restriction.


Assuntos
Diferenciação Celular , Dependovirus/genética , Regulação da Expressão Gênica no Desenvolvimento , Fibras Musculares Esqueléticas/fisiologia , Miócitos Cardíacos/fisiologia , Transdução Genética , Transportadores de Cassetes de Ligação de ATP/genética , Transportadores de Cassetes de Ligação de ATP/metabolismo , Hidrolases Anidrido Ácido , Animais , Ciclo Celular , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Proliferação de Células , Dano ao DNA , Enzimas Reparadoras do DNA/genética , Enzimas Reparadoras do DNA/metabolismo , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Regulação para Baixo , Técnicas de Silenciamento de Genes , Células HeLa , Coração/crescimento & desenvolvimento , Humanos , Fígado/metabolismo , Fígado/virologia , Proteína Homóloga a MRE11 , Camundongos , MicroRNAs/genética , MicroRNAs/metabolismo , Fibras Musculares Esqueléticas/virologia , Miócitos Cardíacos/virologia , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , RNA Interferente Pequeno/genética , Ratos , beta-Galactosidase/biossíntese , beta-Galactosidase/genética
13.
EMBO Mol Med ; 15(1): e16033, 2023 01 11.
Artigo em Inglês | MEDLINE | ID: mdl-36426578

RESUMO

The telomeric repeat-binding factor 2 (TRF2) is a telomere-capping protein that plays a key role in the maintenance of telomere structure and function. It is highly expressed in different cancer types, and it contributes to cancer progression. To date, anti-cancer strategies to target TRF2 remain a challenge. Here, we developed a miRNA-based approach to reduce TRF2 expression. By performing a high-throughput luciferase screening of 54 candidate miRNAs, we identified miR-182-3p as a specific and efficient post-transcriptional regulator of TRF2. Ectopic expression of miR-182-3p drastically reduced TRF2 protein levels in a panel of telomerase- or alternative lengthening of telomeres (ALT)-positive cancer cell lines. Moreover, miR-182-3p induced DNA damage at telomeric and pericentromeric sites, eventually leading to strong apoptosis activation. We also observed that treatment with lipid nanoparticles (LNPs) containing miR-182-3p impaired tumor growth in triple-negative breast cancer (TNBC) models, including patient-derived tumor xenografts (PDTXs), without affecting mouse survival or tissue function. Finally, LNPs-miR-182-3p were able to cross the blood-brain barrier and reduce intracranial tumors representing a possible therapeutic option for metastatic brain lesions.


Assuntos
MicroRNAs , Neoplasias de Mama Triplo Negativas , Animais , Humanos , Camundongos , Apoptose , Linhagem Celular Tumoral , Regulação Neoplásica da Expressão Gênica , MicroRNAs/genética , MicroRNAs/metabolismo , Telômero/metabolismo , Neoplasias de Mama Triplo Negativas/genética , Neoplasias de Mama Triplo Negativas/metabolismo , Neoplasias de Mama Triplo Negativas/patologia
14.
Autophagy ; 18(8): 1785-1800, 2022 08.
Artigo em Inglês | MEDLINE | ID: mdl-34781820

RESUMO

Modulation of the host cell cycle has emerged as a common theme among the pathways regulated by bacterial pathogens, arguably to promote host cell colonization. However, in most cases the exact benefit ensuing from such interference to the infection process remains unclear. Previously, we have shown that Salmonella actively induces G2/M arrest of host cells, and that infection is severely inhibited in cells arrested in G1. In this study, we demonstrate that Salmonella vacuolar replication is inhibited in host cells blocked in G1, whereas the cytosolic replication of the closely related pathogen Shigella is not affected. Mechanistically, we show that cells arrested in G1, but not cells arrested in G2, present dysregulated endolysosomal trafficking, displaying an abnormal accumulation of vesicles positive for late endosomal and lysosomal markers. In addition, the macroautophagic/autophagic flux and degradative lysosomal function are strongly impaired. This endolysosomal trafficking dysregulation results in sustained activation of the SPI-1 type III secretion system and lack of vacuole repair by the autophagy pathway, ultimately compromising the maturation and integrity of the Salmonella-containing vacuole. As such, Salmonella is released in the host cytosol. Collectively, our findings demonstrate that the modulation of the host cell cycle occurring during Salmonella infection is related to a disparity in the permissivity of cells arrested in G1 and G2/M, due to their intrinsic characteristics.Abbreviations: CDK4: cyclin dependent kinase 4; CDK6: cyclin dependent kinase 6; CDK4-CDK6i: CDK4-CDK6 inhibitor IV; cfu: colony-forming units; CHQ: chloroquine; DMSO: dimethyl sulfoxide; EEA1: early endosome antigen 1; FITC: fluorescein isothiocyanate; GFP: green fluorescent protein; hpi: hours post-infection; LAMP1: lysosomal associated membrane protein 1; MAP1LC3B/LC3B: microtubule associated protein 1 light chain 3 beta; MOI: multiplicity of infection; RAB7: RAB7, member RAS oncogene family; SCV: Salmonella-containing vacuole; SPI-1: Salmonella pathogenicity island-1; SPI-2: Salmonella pathogenicity island-2; TFEB: transcription factor EB; T3SS: type III secretion system.


Assuntos
Sistemas de Secreção Tipo III , Vacúolos , Autofagia , Proteínas de Bactérias/metabolismo , Ciclo Celular , Lisossomos/metabolismo , Salmonella/metabolismo , Sistemas de Secreção Tipo III/metabolismo , Vacúolos/metabolismo
15.
Nat Commun ; 13(1): 7174, 2022 11 22.
Artigo em Inglês | MEDLINE | ID: mdl-36418309

RESUMO

Staphylococcus aureus is increasingly recognized as a facultative intracellular pathogen, although the significance and pervasiveness of its intracellular lifestyle remain controversial. Here, we applied fluorescence microscopy-based infection assays and automated image analysis to profile the interaction of 191 S. aureus isolates from patients with bone/joint infections, bacteremia, and infective endocarditis, with four host cell types, at five times post-infection. This multiparametric analysis revealed that almost all isolates are internalized and that a large fraction replicate and persist within host cells, presenting distinct infection profiles in non-professional vs. professional phagocytes. Phenotypic clustering highlighted interesting sub-groups, including one comprising isolates exhibiting high intracellular replication and inducing delayed host death in vitro and in vivo. These isolates are deficient for the cysteine protease staphopain A. This study establishes S. aureus intracellular lifestyle as a prevalent feature of infection, with potential implications for the effective treatment of staphylococcal infections.


Assuntos
Bacteriemia , Infecções Estafilocócicas , Humanos , Staphylococcus aureus , Microscopia , Estilo de Vida
16.
Nat Commun ; 12(1): 3392, 2021 06 07.
Artigo em Inglês | MEDLINE | ID: mdl-34099666

RESUMO

Cells infected with pathogens can contribute to clearing infections by releasing signals that instruct neighbouring cells to mount a pro-inflammatory cytokine response, or by other mechanisms that reduce bystander cells' susceptibility to infection. Here, we show the opposite effect: epithelial cells infected with Salmonella Typhimurium secrete host factors that facilitate the infection of bystander cells. We find that the endoplasmic reticulum stress response is activated in both infected and bystander cells, and this leads to activation of JNK pathway, downregulation of transcription factor E2F1, and consequent reprogramming of microRNA expression in a time-dependent manner. These changes are not elicited by infection with other bacterial pathogens, such as Shigella flexneri or Listeria monocytogenes. Remarkably, the protein HMGB1 present in the secretome of Salmonella-infected cells is responsible for the activation of the IRE1 branch of the endoplasmic reticulum stress response in non-infected, neighbouring cells. Furthermore, E2F1 downregulation and the associated microRNA alterations promote Salmonella replication within infected cells and prime bystander cells for more efficient infection.


Assuntos
Efeito Espectador/genética , Fator de Transcrição E2F1/metabolismo , MicroRNAs/metabolismo , Infecções por Salmonella/imunologia , Salmonella typhimurium/imunologia , Animais , Efeito Espectador/imunologia , Modelos Animais de Doenças , Regulação para Baixo/imunologia , Fator de Transcrição E2F1/genética , Estresse do Retículo Endoplasmático/imunologia , Endorribonucleases/metabolismo , Proteína HMGB1/metabolismo , Células HeLa , Interações Hospedeiro-Patógeno/genética , Interações Hospedeiro-Patógeno/imunologia , Humanos , Listeria monocytogenes/imunologia , Sistema de Sinalização das MAP Quinases/genética , Sistema de Sinalização das MAP Quinases/imunologia , Proteínas Serina-Treonina Quinases/metabolismo , RNA-Seq , Infecções por Salmonella/genética , Infecções por Salmonella/microbiologia , Salmonella typhimurium/patogenicidade , Shigella flexneri/imunologia , Suínos
17.
Front Immunol ; 12: 755862, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34867992

RESUMO

Infection by the protozoan Trypanosoma cruzi causes Chagas disease cardiomyopathy (CCC) and can lead to arrhythmia, heart failure and death. Chagas disease affects 8 million people worldwide, and chronic production of the cytokines IFN-γ and TNF-α by T cells together with mitochondrial dysfunction are important players for the poor prognosis of the disease. Mitochondria occupy 40% of the cardiomyocytes volume and produce 95% of cellular ATP that sustain the life-long cycles of heart contraction. As IFN-γ and TNF-α have been described to affect mitochondrial function, we hypothesized that IFN-γ and TNF-α are involved in the myocardial mitochondrial dysfunction observed in CCC patients. In this study, we quantified markers of mitochondrial dysfunction and nitro-oxidative stress in CCC heart tissue and in IFN-γ/TNF-α-stimulated AC-16 human cardiomyocytes. We found that CCC myocardium displayed increased levels of nitro-oxidative stress and reduced mitochondrial DNA as compared with myocardial tissue from patients with dilated cardiomyopathy (DCM). IFN-γ/TNF-α treatment of AC-16 cardiomyocytes induced increased nitro-oxidative stress and decreased the mitochondrial membrane potential (ΔΨm). We found that the STAT1/NF-κB/NOS2 axis is involved in the IFN-γ/TNF-α-induced decrease of ΔΨm in AC-16 cardiomyocytes. Furthermore, treatment with mitochondria-sparing agonists of AMPK, NRF2 and SIRT1 rescues ΔΨm in IFN-γ/TNF-α-stimulated cells. Proteomic and gene expression analyses revealed that IFN-γ/TNF-α-treated cells corroborate mitochondrial dysfunction, transmembrane potential of mitochondria, altered fatty acid metabolism and cardiac necrosis/cell death. Functional assays conducted on Seahorse respirometer showed that cytokine-stimulated cells display decreased glycolytic and mitochondrial ATP production, dependency of fatty acid oxidation as well as increased proton leak and non-mitochondrial oxygen consumption. Together, our results suggest that IFN-γ and TNF-α cause direct damage to cardiomyocytes' mitochondria by promoting oxidative and nitrosative stress and impairing energy production pathways. We hypothesize that treatment with agonists of AMPK, NRF2 and SIRT1 might be an approach to ameliorate the progression of Chagas disease cardiomyopathy.


Assuntos
Cardiomiopatia Chagásica/metabolismo , Interferon gama/metabolismo , Mitocôndrias/metabolismo , Miócitos Cardíacos/metabolismo , Estresse Oxidativo/fisiologia , Fator de Necrose Tumoral alfa/metabolismo , Adolescente , Adulto , Idoso , Cardiomiopatia Chagásica/patologia , Cardiomiopatia Chagásica/fisiopatologia , Criança , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Mitocôndrias/patologia , Miócitos Cardíacos/patologia , Adulto Jovem
18.
Bioconjug Chem ; 21(4): 774-83, 2010 Apr 21.
Artigo em Inglês | MEDLINE | ID: mdl-20205419

RESUMO

Despite increasing interest in cell-penetrating peptides (CPPs) as carriers for drugs and in gene therapy, the current understanding of their exact internalization mechanism is still far from complete. The cellular translocation of CPPs and their payloads has been mostly described by fluorescence- and activity-based methods, leaving the more detailed characterization at the ultrastructural level almost out of attention. Herein, we used transmission electron microscopy to characterize the membrane interaction and internalization of a cell-penetrating peptide S4(13)-PV. We demonstrate that S4(13)-PV peptide forms spherical nanoparticle-like regular structures upon association with cell surface glycosaminoglycans on the plasma membrane. Insertion of S4(13)-PV particles into plasma membrane induces disturbances and leads to the vesicular uptake of peptides by cells. We propose that for efficient cellular translocation S4(13)-PV peptides have to assemble into particles of specific size and shape. The spherical peptide particles are not dissociated in intracellular vesicles but often retain their organization and remain associated with the membrane of vesicles, destabilizing them and promoting the escape of peptides into cytosol. Lowering the temperature and inhibition of dynamins' activity reduce the internalization of S4(13)-PV peptides, but do not block it completely. Our results provide an ultrastructural insight into the interaction mode of CPPs with the plasma membrane and the distribution in cells, which might help to better understand how CPPs cross the biological membranes and gain access into cells.


Assuntos
Membrana Celular/metabolismo , Nanopartículas/química , Peptídeos/metabolismo , Glicosaminoglicanos/química , Glicosaminoglicanos/metabolismo , Células HeLa , Humanos , Microscopia Eletrônica de Transmissão , Tamanho da Partícula , Peptídeos/química , Conformação Proteica , Temperatura , Distribuição Tecidual
19.
Nat Microbiol ; 5(1): 192-205, 2020 01.
Artigo em Inglês | MEDLINE | ID: mdl-31792428

RESUMO

MicroRNAs (miRNAs) are increasingly recognized for their role in infection by bacterial pathogens, although the effect of each individual miRNA remains largely unknown. Here, we used a comparative genome-wide microscopy-based functional screening approach to identify miRNAs controlling infection by two bacterial pathogens-Salmonella enterica serovar Typhimurium and Shigella flexneri. Despite the similarities between these pathogens, we found infections to be controlled by largely non-overlapping subsets of miRNAs, seemingly reflecting different requirements prompted by their distinct intracellular lifestyles. By characterizing a small subset of miRNAs chosen among the strongest inhibitors of Shigella infection, we discovered that miR-3668, miR-4732-5p and miR-6073 exert a selective effect on Shigella infection by impairing bacterial actin-based motility by downregulating N-WASP. Additionally, by identifying let-7i-3p miRNA as a strong inhibitor of Salmonella replication and performing in-depth analysis of its mechanisms of action, we showed that this miRNA specifically inhibits Salmonella infection via modulation of endolysosomal trafficking and the vacuolar environment by targeting the host RGS2 protein. These findings illustrate two paradigms underlying miRNA-mediated regulation of bacterial infection, acting as part of the host response to infection, or as part of bacterial strategies to modulate the host environment and favour pathogenesis.


Assuntos
Infecções por Enterobacteriaceae/genética , Infecções por Enterobacteriaceae/microbiologia , MicroRNAs/genética , Salmonella typhimurium/fisiologia , Shigella flexneri/fisiologia , Animais , Regulação da Expressão Gênica , Genômica , Células HeLa , Interações Hospedeiro-Patógeno , Humanos , MicroRNAs/metabolismo , Especificidade da Espécie , Suínos
20.
Microbiol Spectr ; 7(3)2019 05.
Artigo em Inglês | MEDLINE | ID: mdl-31152522

RESUMO

MicroRNAs (miRNAs) are a well-characterized class of small noncoding RNAs that act as major posttranscriptional regulators of gene expression. Accordingly, miRNAs have been associated with a wide range of fundamental biological processes and implicated in human diseases. During the past decade, miRNAs have also been recognized for their role in the complex interplay between the host and bacterial pathogens, either as part of the host response to counteract infection or as a molecular strategy employed by bacteria to subvert host pathways for their own benefit. Importantly, the characterization of downstream miRNA targets and their underlying mechanisms of action has uncovered novel molecular factors and pathways relevant to infection. In this article, we review the current knowledge of the miRNA response to bacterial infection, focusing on different bacterial pathogens, including Salmonella enterica, Listeria monocytogenes, Mycobacterium spp., and Helicobacter pylori, among others.


Assuntos
Infecções Bacterianas/metabolismo , Fenômenos Fisiológicos Bacterianos , Interações Hospedeiro-Patógeno/fisiologia , MicroRNAs/fisiologia , Animais , Bactérias/patogenicidade , Regulação da Expressão Gênica , Helicobacter pylori , Interações Hospedeiro-Patógeno/genética , Humanos , Listeria monocytogenes , MicroRNAs/genética , Mycobacterium , Salmonella enterica
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