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1.
Front Cell Infect Microbiol ; 14: 1308362, 2024.
Artigo em Inglês | MEDLINE | ID: mdl-38476167

RESUMO

Infectious peritonitis is a leading cause of peritoneal functional impairment and a primary factor for therapy discontinuation in peritoneal dialysis (PD) patients. Although bacterial infections are a common cause of peritonitis episodes, emerging evidence suggests a role for viral pathogens. Toll-like receptors (TLRs) specifically recognize conserved pathogen-associated molecular patterns (PAMPs) from bacteria, viruses, and fungi, thereby orchestrating the ensuing inflammatory/immune responses. Among TLRs, TLR3 recognizes viral dsRNA and triggers antiviral response cascades upon activation. Epigenetic regulation, mediated by histone deacetylase (HDAC), has been demonstrated to control several cellular functions in response to various extracellular stimuli. Employing epigenetic target modulators, such as epidrugs, is a current therapeutic option in several cancers and holds promise in treating viral diseases. This study aims to elucidate the impact of TLR3 stimulation on the plasticity of human mesothelial cells (MCs) in PD patients and to investigate the effects of HDAC1-3 inhibition. Treatment of MCs from PD patients with the TLR3 agonist polyinosinic:polycytidylic acid (Poly(I:C)), led to the acquisition of a bona fide mesothelial-to-mesenchymal transition (MMT) characterized by the upregulation of mesenchymal genes and loss of epithelial-like features. Moreover, Poly(I:C) modulated the expression of several inflammatory cytokines and chemokines. A quantitative proteomic analysis of MCs treated with MS-275, an HDAC1-3 inhibitor, unveiled altered expression of several proteins, including inflammatory cytokines/chemokines and interferon-stimulated genes (ISGs). Treatment with MS-275 facilitated MMT reversal and inhibited the interferon signature, which was associated with reduced STAT1 phosphorylation. However, the modulation of inflammatory cytokine/chemokine production was not univocal, as IL-6 and CXCL8 were augmented while TNF-α and CXCL10 were decreased. Collectively, our findings underline the significance of viral infections in acquiring a mesenchymal-like phenotype by MCs and the potential consequences of virus-associated peritonitis episodes for PD patients. The observed promotion of MMT reversal and interferon response inhibition by an HDAC1-3 inhibitor, albeit without a general impact on inflammatory cytokine production, has translational implications deserving further analysis.


Assuntos
Benzamidas , Interferon Tipo I , Peritonite , Piridinas , Viroses , Humanos , Interferon Tipo I/metabolismo , Receptor 3 Toll-Like/metabolismo , Epigênese Genética , Proteômica , Citocinas/metabolismo , Quimiocinas/metabolismo , Poli I-C/farmacologia , Receptores Toll-Like/metabolismo , Viroses/genética , Fenótipo , Histona Desacetilase 1/genética , Histona Desacetilase 1/metabolismo
2.
J Exp Clin Cancer Res ; 43(1): 27, 2024 Jan 23.
Artigo em Inglês | MEDLINE | ID: mdl-38254102

RESUMO

BACKGROUND: Peritoneal metastasis, which accounts for 85% of all epithelial ovarian carcinoma (EOC) metastases, is a multistep process that requires the establishment of adhesive interactions between cancer cells and the peritoneal membrane. Interrelations between EOC and the mesothelial stroma are critical to facilitate the metastatic process. No data is available so far on the impact of histone acetylation/deacetylation, a potentially relevant mechanism governing EOC metastasis, on mesothelial cells (MCs)-mediated adhesion. METHODS: Static adhesion and peritoneal clearance experiments were performed pretreating mesenchymal-like MCs and platinum-sensitive/resistant EOC cell lines with MS-275-a Histone deacetylase (HDAC)1-3 pharmacological inhibitor currently used in combination trials. Results were acquired by confocal microscopy and were analyzed with an automated Opera software. The role of HDAC1/2 was validated by genetic silencing. The role of α4-, α5-α1 Integrins and Fibronectin-1 was validated using specific monoclonal antibodies. Quantitative proteomic analysis was performed on primary MCs pretreated with MS-275. Decellularized matrices were generated from either MS-275-exposed or untreated cells to study Fibronectin-1 extracellular secretion. The effect of MS-275 on ß1 integrin activity was assessed using specific monoclonal antibodies. The role of Talin-1 in MCs/EOC adhesion was analyzed by genetic silencing. Talin-1 ectopic expression was validated as a rescue tool from MS-275-induced phenotype. The in vivo effect of MS-275-induced MC remodeling was validated in a mouse model of peritoneal EOC dissemination. RESULTS: Treatment of MCs with non-cytotoxic concentrations of MS-275 caused a consistent reduction of EOC adhesion. Proteomic analysis revealed several pathways altered upon MC treatment with MS-275, including ECM deposition/remodeling, adhesion receptors and actin cytoskeleton regulators. HDAC1/2 inhibition hampered actin cytoskeleton polymerization by downregulating actin regulators including Talin-1, impairing ß1 integrin activation, and leading to abnormal extracellular secretion and distribution of Fibronectin-1. Talin-1 ectopic expression rescued EOC adhesion to MS-275-treated MCs. In an experimental mouse model of metastatic EOC, MS-275 limited tumor invasion, Fibronectin-1 secretion and the sub-mesothelial accumulation of MC-derived carcinoma-associated fibroblasts. CONCLUSION: Our study unveils a direct impact of HDAC-1/2 in the regulation of MC/EOC adhesion and highlights the regulation of MC plasticity by epigenetic inhibition as a potential target for therapeutic intervention in EOC peritoneal metastasis.


Assuntos
Benzamidas , Carcinoma Epitelial do Ovário , Adesão Celular , Histona Desacetilase 1 , Histona Desacetilase 2 , Neoplasias Ovarianas , Neoplasias Peritoneais , Animais , Feminino , Humanos , Camundongos , Citoesqueleto de Actina/metabolismo , Anticorpos Monoclonais , Carcinoma Epitelial do Ovário/metabolismo , Epitélio , Proteínas da Matriz Extracelular/metabolismo , Fibronectinas , Histona Desacetilase 1/metabolismo , Integrina alfa5 , Integrina beta1/genética , Neoplasias Ovarianas/genética , Neoplasias Ovarianas/metabolismo , Neoplasias Peritoneais/genética , Neoplasias Peritoneais/metabolismo , Proteômica , Piridinas , Talina/genética , Talina/metabolismo , Histona Desacetilase 2/metabolismo , Adesão Celular/genética
3.
Front Immunol ; 14: 1287656, 2023.
Artigo em Inglês | MEDLINE | ID: mdl-37965335

RESUMO

Introduction: Peripheral monocytes in humans are conventionally divided into classical (CL, CD14++CD16-), intermediate (INT, CD14++CD16+) and non-classical (NC, CD14dim/-CD16++) cells, based on their expression levels of CD14 and CD16. A major fraction of the NC-monocytes has been shown to express the 6-sulfo LacNAc (slan) antigen, but whether these slan+/NC-monocytes represent the prototypical non-classical monocytes or whether they are simply a sub-fraction with identical features as the remainder of NC monocytes is still unclear. Methods: We analyzed transcriptome (by bulk and single cell RNA-seq), proteome, cell surface markers and production of discrete cytokines by peripheral slan+/NC- and slan-/NC-monocytes, in comparison to total NC-, CL- and INT- monocytes. Results: By bulk RNA-seq and proteomic analysis, we found that slan+/NC-monocytes express higher levels of genes and proteins specific of NC-monocytes than slan-/NC-monocytes do. Unsupervised clustering of scRNA-seq data generated one cluster of NC- and one of INT-monocytes, where all slan+/NC-monocytes were allocated to the NC-monocyte cluster, while slan-/NC-monocytes were found, in part (13.4%), within the INT-monocyte cluster. In addition, total NC- and slan-/NC-monocytes, but not slan+/NC-monocytes, were found by both bulk RNA-seq and scRNA-seq to contain a small percentage of natural killer cells. Conclusion: In addition to comparatively characterize total NC-, slan-/NC- and slan+/NC-monocyte transcriptomes and proteomes, our data prove that slan+/NC-, but not slan-/NC-, monocytes are more representative of prototypical NC-monocytes.


Assuntos
Monócitos , Proteômica , Humanos , Leucócitos Mononucleares
4.
Front Cell Infect Microbiol ; 13: 1257683, 2023.
Artigo em Inglês | MEDLINE | ID: mdl-38162580

RESUMO

Background: Despite the significant progress achieved in understanding the pathology and clinical management of SARS-CoV-2 infection, still pathogenic and clinical issues need to be clarified. Treatment with modulators of epigenetic targets, i.e., epidrugs, is a current therapeutic option in several cancers and could represent an approach in the therapy of viral diseases. Results: Aim of this study was the analysis of the role of histone deacetylase (HDAC) inhibition in the modulation of SARS-CoV-2 infection of mesothelial cells (MCs).MeT5A cells, a pleura MC line, were pre-treated with different specific class I and IIb HDAC inhibitors. Unexpectedly, treatment with HDAC1-3 inhibitors significantly increased ACE2/TMPRSS2 expression, suggesting a role in favoring SARS-CoV-2 infection. We focused our analysis on the most potent ACE2/TMPRSS2 inducer among the inhibitors analysed, MS-275, a HDAC1-3 inhibitor. ACE2/TMPRSS2 expression was validated by Western Blot (WB) and immunofluorescence. The involvement of HDAC inhibition in receptor induction was confirmed by HDAC1/HDAC2 silencing. In accordance to the ACE2/TMPRSS2 expression data, MS-275 increased SARS-CoV-2 replication and virus propagation in Vero E6 cells.Notably, MS-275 was able to increase ACE2/TMPRSS2 expression and SARS-CoV-2 production, although to a lesser extent, also in the lung adenocarcinoma cell line Calu-3 cells.Mechanistically, treatment with MS-275 increased H3 and H4 histone acetylation at ACE2/TMPRSS2 promoters, increasing their transcription. Conclusion: This study highlights a previously unrecognized effect of HDAC1-3 inhibition in increasing SARS-CoV-2 cell entry, replication and productive infection correlating with increased expression of ACE2 and TMPRSS2. These data, while adding basic insight into COVID-19 pathogenesis, warn for the use of HDAC inhibitors in SARS-CoV-2 patients.


Assuntos
COVID-19 , SARS-CoV-2 , Humanos , COVID-19/metabolismo , Enzima de Conversão de Angiotensina 2/metabolismo , Inibidores de Histona Desacetilases/farmacologia , Inibidores de Histona Desacetilases/metabolismo , Pulmão/metabolismo , Células Epiteliais , Histona Desacetilase 1/metabolismo
5.
Cell Death Dis ; 13(11): 965, 2022 11 17.
Artigo em Inglês | MEDLINE | ID: mdl-36396626

RESUMO

Histone acetylation/deacetylation play an essential role in modifying chromatin structure and in regulating cell plasticity in eukaryotic cells. Therefore, histone deacetylase (HDAC) pharmacological inhibitors are promising tools in the therapy of fibrotic diseases and in cancer. Peritoneal fibrosis is a pathological process characterized by many cellular and molecular alterations, including the acquisition of invasive/pro-fibrotic abilities by mesothelial cells (MCs) through induction of mesothelial to mesenchymal transition (MMT). The aim of this study was to characterize the molecular mechanism of the antifibrotic role of HDAC1 inhibition. Specifically, treatment with MS-275, an HDAC1-3 inhibitor previously known to promote MMT reversal, induced the expression of several TGFBRI mRNA-targeting miRNAs. Among them, miR-769-5p ectopic expression was sufficient to promote MMT reversal and to limit MC migration and invasion, whereas miR-769-5p silencing further enhanced mesenchymal gene expression. These results were confirmed by HDAC1 genetic silencing. Interestingly, miR-769-5p silencing maintained mesenchymal features despite HDAC1 inhibition, thus indicating that it is necessary to drive MMT reversal induced by HDAC1 inhibition. Besides TGFBRI, miR-769-5p was demonstrated to target SMAD2/3 and PAI-1 expression directly. When analyzing molecular mechanisms underlying miR-769-5p expression, we found that the transcription factor Wilms' tumor 1 (WT1), a master gene controlling MC development, binds to the miR-769-5p promoter favoring its expression. Interestingly, both WT1 expression and binding to miR-769-5p promoter were increased by HDAC1 inhibition and attenuated by TGFß1 treatment. Finally, we explored the significance of these observations in the cell-to-cell communication: we evaluated the ability of miR-769-5p to be loaded into extracellular vesicles (EVs) and to promote MMT reversal in recipient mesenchymal-like MCs. Treatment of fibrotic MCs with EVs isolated from miR-769-5p over-expressing MCs promoted the down-regulation of specific mesenchymal targets and the reacquisition of an epithelial-like morphology. In conclusion, we highlighted an HDAC1-WT1-miR-769-5p axis potentially relevant for therapies aimed at counteracting organ fibrosis.


Assuntos
Transição Epitelial-Mesenquimal , MicroRNAs , Movimento Celular/genética , Transição Epitelial-Mesenquimal/genética , Epitélio/metabolismo , MicroRNAs/metabolismo
6.
Int J Mol Sci ; 23(3)2022 Feb 08.
Artigo em Inglês | MEDLINE | ID: mdl-35163819

RESUMO

While blue LED (b-LED) light is increasingly being studied for its cytotoxic activity towards bacteria in therapy of skin-related infections, its effects on eukaryotic cells plasticity are less well characterized. Moreover, since different protocols are often used, comparing the effect of b-LED towards both microorganisms and epithelial surfaces may be difficult. The aim of this study was to analyze, in the same experimental setting, both the bactericidal activity and the effects on human keratinocytes. Exposure to b-LED induced an intense cytocidal activity against Gram-positive (i.e, Staphylococcus aureus) and Gram-negative (i.e., Pseudomonas aeruginosa) bacteria associated with catheter-related infections. Treatment with b-LED of a human keratinocyte cell line induced a transient cell cycle arrest. At the molecular level, exposure to b-LED induced a transient downregulation of Cyclin D1 and an upregulation of p21, but not signs of apoptosis. Interestingly, a transient induction of phosphor-histone γ-H2Ax, which is associated with genotoxic damages, was observed. At the same time, keratinocytes underwent a transient epithelial to mesenchymal transition (EMT)-like phenotype, characterized by E-cadherin downregulation and SNAIL/SLUG induction. As a functional readout of EMT induction, a scratch assay was performed. Surprisingly, b-LED treatment provoked a delay in the scratch closure. In conclusion, we demonstrated that b-LED microbicidal activity is associated with complex responses in keratinocytes that certainly deserve further analysis.


Assuntos
Pontos de Checagem do Ciclo Celular/efeitos da radiação , Queratinócitos/citologia , Luz/efeitos adversos , Pseudomonas aeruginosa/crescimento & desenvolvimento , Staphylococcus aureus/crescimento & desenvolvimento , Antígenos CD/metabolismo , Caderinas/metabolismo , Proliferação de Células , Ciclina D1/metabolismo , Inibidor de Quinase Dependente de Ciclina p21/metabolismo , Síndrome de Down , Transição Epitelial-Mesenquimal/efeitos da radiação , Regulação da Expressão Gênica/efeitos dos fármacos , Células HaCaT , Humanos , Queratinócitos/metabolismo , Queratinócitos/efeitos da radiação , Viabilidade Microbiana/efeitos da radiação , Pseudomonas aeruginosa/efeitos da radiação , Fatores de Transcrição da Família Snail/metabolismo , Staphylococcus aureus/efeitos da radiação
7.
Int J Mol Sci ; 23(2)2022 Jan 14.
Artigo em Inglês | MEDLINE | ID: mdl-35055098

RESUMO

Heterogeneous nuclear ribonucleoproteins (hnRNPs) control gene expression by acting at multiple levels and are often deregulated in epithelial tumors; however, their roles in the fine regulation of cellular reprogramming, specifically in epithelial-mesenchymal transition (EMT), remain largely unknown. Here, we focused on the hnRNP-Q (also known as SYNCRIP), showing by molecular analysis that in hepatocytes it acts as a "mesenchymal" gene, being induced by TGFß and modulating the EMT. SYNCRIP silencing limits the induction of the mesenchymal program and maintains the epithelial phenotype. Notably, in HCC invasive cells, SYNCRIP knockdown induces a mesenchymal-epithelial transition (MET), negatively regulating their mesenchymal phenotype and significantly impairing their migratory capacity. In exploring possible molecular mechanisms underlying these observations, we identified a set of miRNAs (i.e., miR-181-a1-3p, miR-181-b1-3p, miR-122-5p, miR-200a-5p, and miR-let7g-5p), previously shown to exert pro- or anti-EMT activities, significantly impacted by SYNCRIP interference during EMT/MET dynamics and gathered insights, suggesting the possible involvement of this RNA binding protein in their transcriptional regulation.


Assuntos
Carcinoma Hepatocelular/etiologia , Transformação Celular Neoplásica/genética , Transição Epitelial-Mesenquimal/genética , Hepatócitos/metabolismo , Ribonucleoproteínas Nucleares Heterogêneas/genética , Neoplasias Hepáticas/etiologia , Animais , Biomarcadores , Carcinoma Hepatocelular/metabolismo , Carcinoma Hepatocelular/patologia , Linhagem Celular Tumoral , Movimento Celular/genética , Transformação Celular Neoplásica/metabolismo , Transformação Celular Neoplásica/patologia , Suscetibilidade a Doenças , Regulação Neoplásica da Expressão Gênica , Técnicas de Silenciamento de Genes , Hepatócitos/patologia , Ribonucleoproteínas Nucleares Heterogêneas/metabolismo , Neoplasias Hepáticas/metabolismo , Neoplasias Hepáticas/patologia , Camundongos , MicroRNAs/genética , Fenótipo , Interferência de RNA , Proteínas de Ligação a RNA
8.
Front Mol Biosci ; 8: 752616, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34901152

RESUMO

Although lung fibrosis has a major impact in COVID-19 disease, its pathogenesis is incompletely understood. In particular, no direct evidence of pleura implication in COVID-19-related fibrotic damage has been reported so far. In this study, the expression of epithelial cytokeratins and Wilms tumor 1 (WT1), specific markers of mesothelial cells (MCs), was analyzed in COVID-19 and unrelated pleura autoptic samples. SARS-CoV-2 replication was analyzed by RT-PCR and confocal microscopy in MeT5A, a pleura MC line. SARS-CoV-2 receptors were analyzed by RT-PCR and western blot. Inflammatory cytokines from the supernatants of SARS-CoV-2-infected MeT5A cells were analysed by Luminex and ELLA assays. Immunohistochemistry of COVID-19 pleura patients highlighted disruption of pleura monolayer and fibrosis of the sub-mesothelial stroma, with the presence of MCs with fibroblastoid morphology in the sub-mesothelial stroma, but no evidence of direct infection in vivo. Interestingly, we found evidence of ACE2 expression in MCs from pleura of COVID-19 patients. In vitro analysis shown that MeT5A cells expressed ACE2, TMPRSS2, ADAM17 and NRP1, plasma membrane receptors implicated in SARS-CoV-2 cell entry and infectivity. Moreover, MeT5A cells sustained SARS-CoV-2 replication and productive infection. Infected MeT5A cells produced interferons, inflammatory cytokines and metalloproteases. Overall, our data highlight the potential role of pleura MCs as promoters of the fibrotic reaction and regulators of the immune response upon SARS-CoV-2 infection.

9.
J Hepatol ; 75(6): 1301-1311, 2021 12.
Artigo em Inglês | MEDLINE | ID: mdl-34271004

RESUMO

BACKGROUND & AIMS: Patients with HCV who achieve a sustained virological response (SVR) on direct-acting antiviral (DAA) therapy still need to be monitored for signs of liver disease progression. To this end, the identification of both disease biomarkers and therapeutic targets is necessary. METHODS: Extracellular vesicles (EVs) purified from plasma of 15 healthy donors (HDs), and 16 HCV-infected patients before (T0) and after (T6) DAA treatment were utilized for functional and miRNA cargo analysis. EVs purified from plasma of 17 HDs and 23 HCV-infected patients (T0 and T6) were employed for proteomic and western blot analyses. Functional analysis in LX2 cells measured fibrotic markers (mRNAs and proteins) in response to EVs. Structural analysis was performed by qPCR, label-free liquid chromatography-mass spectrometry and western blot. RESULTS: On the basis of observations indicating functional differences (i.e. modulation of FN-1, ACTA2, Smad2/3 phosphorylation, collagen deposition) of plasma-derived EVs from HDs, T0 and T6, we performed structural analysis of EVs. We found consistent differences in terms of both miRNA and protein cargos: (i) antifibrogenic miR204-5p, miR181a-5p, miR143-3p, miR93-5p and miR122-5p were statistically underrepresented in T0 EVs compared to HD EVs, while miR204-5p and miR143-3p were statistically underrepresented in T6 EVs compared to HD EVs (p <0.05); (ii) proteomic analysis highlighted, in both T0 and T6, the modulation of several proteins with respect to HDs; among them, the fibrogenic protein DIAPH1 was upregulated (Log2 fold change of 4.4). CONCLUSIONS: Taken together, these results highlight structural EV modifications that are conceivably causal for long-term liver disease progression in patients with HCV despite DAA-mediated SVR. LAY SUMMARY: Direct-acting antivirals lead to virological cure in the majority of patients with chronic hepatitis C virus infection. However, the risk of liver disease progression or complications in patients with fibrosis and cirrhosis remains in some patients even after virological cure. Herein, we show that extracellular vesicle modifications could be linked to long-term liver disease progression in patients who have achieved virological cure; these modifications could potentially be used as biomarkers or treatment targets in such patients.


Assuntos
Antivirais/farmacologia , Hepacivirus/fisiologia , Hepatite C/tratamento farmacológico , Resposta Viral Sustentada , Antivirais/uso terapêutico , Comunicação Celular/efeitos dos fármacos , Comunicação Celular/fisiologia , Hepatite C/fisiopatologia , Humanos , Espectrometria de Massas/métodos , Espectrometria de Massas/estatística & dados numéricos
10.
Front Immunol ; 12: 607204, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33854496

RESUMO

Peritoneal fibrosis is characterized by abnormal production of extracellular matrix proteins leading to progressive thickening of the submesothelial compact zone of the peritoneal membrane. This process may be caused by a number of insults including pathological conditions linked to clinical practice, such as peritoneal dialysis, abdominal surgery, hemoperitoneum, and infectious peritonitis. All these events may cause acute/chronic inflammation and injury to the peritoneal membrane, which undergoes progressive fibrosis, angiogenesis, and vasculopathy. Among the cellular processes implicated in these peritoneal alterations is the generation of myofibroblasts from mesothelial cells and other cellular sources that are central in the induction of fibrosis and in the subsequent functional deterioration of the peritoneal membrane. Myofibroblast generation and activity is actually integrated in a complex network of extracellular signals generated by the various cellular types, including leukocytes, stably residing or recirculating along the peritoneal membrane. Here, the main extracellular factors and the cellular players are described with emphasis on the cross-talk between immune system and cells of the peritoneal stroma. The understanding of cellular and molecular mechanisms underlying fibrosis of the peritoneal membrane has both a basic and a translational relevance, since it may be useful for setup of therapies aimed at counteracting the deterioration as well as restoring the homeostasis of the peritoneal membrane.


Assuntos
Comunicação Celular , Suscetibilidade a Doenças , Fibrose Peritoneal/etiologia , Fibrose Peritoneal/metabolismo , Peritônio/imunologia , Peritônio/metabolismo , Células Estromais/metabolismo , Animais , Biomarcadores , Comunicação Celular/imunologia , Citocinas/metabolismo , Células Epiteliais/metabolismo , Humanos , Imunidade Inata , Mediadores da Inflamação/metabolismo , Leucócitos/imunologia , Leucócitos/metabolismo , Diálise Peritoneal/efeitos adversos , Fibrose Peritoneal/patologia , Peritônio/patologia , Peritonite/complicações , Peritonite/etiologia , Subpopulações de Linfócitos T/imunologia , Subpopulações de Linfócitos T/metabolismo
11.
Cancer Res ; 81(1): 103-113, 2021 01 01.
Artigo em Inglês | MEDLINE | ID: mdl-33158813

RESUMO

HOTAIR is a lncRNA overexpressed in several epithelial cancers and strongly correlated with invasion. This lncRNA was proven a pivotal element of the epithelial-to-mesenchymal transition (EMT), a transdifferentiation process triggering metastasis. Snail, master inducer of EMT, requires HOTAIR to recruit EZH2 on specific epithelial target genes (i.e., HNF4α, E-cadherin, and HNF1α) and cause their repression. Here, we designed a HOTAIR deletion mutant form, named HOTAIR-sbid, including the putative Snail-binding domain but depleted of the EZH2-binding domain. HOTAIR-sbid acted as a dominant negative of the endogenous HOTAIR. In both murine and human tumor cells, HOTAIR-sbid impaired the ability of HOTAIR to bind Snail and, in turn, trigger H3K27me3/EZH2-mediated repression of Snail epithelial target genes. Notably, HOTAIR-sbid expression was proven to reduce cellular motility, invasiveness, anchorage-independent growth, and responsiveness to TGFß-induced EMT. These data provide evidence on a lncRNA-based strategy to effectively impair the function of a master EMT-transcriptional factor. SIGNIFICANCE: This study defines an innovative RNA-based strategy to interfere with a pivotal function of the tumor-related lncRNA HOTAIR, comprising a dominant negative mutant that was computationally designed and that impairs epithelial-to-mesenchymal transition.


Assuntos
Biomarcadores Tumorais/metabolismo , Carcinoma Hepatocelular/patologia , Transição Epitelial-Mesenquimal , Regulação Neoplásica da Expressão Gênica , Hepatócitos/patologia , RNA Longo não Codificante/genética , Fatores de Transcrição da Família Snail/metabolismo , Animais , Apoptose , Biomarcadores Tumorais/genética , Carcinoma Hepatocelular/genética , Carcinoma Hepatocelular/metabolismo , Movimento Celular , Proliferação de Células , Células Cultivadas , Hepatócitos/metabolismo , Humanos , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/metabolismo , Neoplasias Hepáticas/patologia , Camundongos , Mutação , RNA Longo não Codificante/antagonistas & inibidores , Fatores de Transcrição da Família Snail/genética
12.
Front Pharmacol ; 10: 942, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31543815

RESUMO

The cytokine transforming growth factor ß (TGFß) plays a crucial role in the induction of both epithelial-to-mesenchymal transition (EMT) program and fibro-cirrhotic process in the liver, where it contributes also to organ inflammation following several chronic injuries. All these pathological situations greatly increase the risk of hepatocellular carcinoma (HCC) and contribute to tumor progression. In particular, late-stage HCCs are characterized by constitutive activation of TGFß pathway and by an EMT molecular signature leading to the acquisition of invasive and metastatic properties. In these pathological conditions, the cytokine has been shown to induce the transcriptional downregulation of HNF1α, a master regulator of the epithelial/hepatocyte differentiation and of the EMT reverse process, the mesenchymal-to-epithelial transition (MET). Therefore, the restoration of HNF1α expression/activity has been proposed as targeted therapeutic strategy for liver fibro-cirrhosis and late-stage HCCs. In this study, TGFß is found to trigger an early functional inactivation of HNF1α during EMT process that anticipates the effects of the transcriptional downregulation of its own gene. Mechanistically, the cytokine, while not affecting the HNF1α DNA-binding capacity, impaired its ability to recruit CBP/p300 acetyltransferases on target gene promoters and, consequently, its transactivating function. The loss of HNF1α capacity to bind to CBP/p300 and HNF1α functional inactivation have been found to correlate with a change of its posttranslational modification profile. Collectively, the results obtained in this work unveil a new level of HNF1α functional inactivation by TGFß and contribute to shed light on the early events triggering EMT in hepatocytes. Moreover, these data suggest that the use of HNF1α as anti-EMT tool in a TGFß-containing microenvironment may require the design of new therapeutic strategies overcoming the TGFß-induced HNF1α inactivation.

13.
Cell Death Differ ; 26(5): 890-901, 2019 05.
Artigo em Inglês | MEDLINE | ID: mdl-30154449

RESUMO

The expression of the long noncoding RNA HOTAIR (HOX Transcript Antisense Intergenic RNA) is largely deregulated in epithelial cancers and positively correlates with poor prognosis and progression of hepatocellular carcinoma and gastrointestinal cancers. Furthermore, functional studies revealed a pivotal role for HOTAIR in the epithelial-to-mesenchymal transition, as this RNA is causal for the repressive activity of the master factor SNAIL on epithelial genes. Despite the proven oncogenic role of HOTAIR, its transcriptional regulation is still poorly understood. Here hepatocyte nuclear factor 4-α (HNF4α), as inducer of epithelial differentiation, was demonstrated to directly repress HOTAIR transcription in the mesenchymal-to epithelial transition. Mechanistically, HNF4α was found to cause the release of a chromatin loop on HOTAIR regulatory elements thus exerting an enhancer-blocking activity.


Assuntos
Cromatina/genética , Fator 4 Nuclear de Hepatócito/genética , RNA Longo não Codificante/genética , Fatores de Transcrição da Família Snail/genética , Animais , Carcinoma Hepatocelular/genética , Carcinoma Hepatocelular/patologia , Diferenciação Celular/genética , Transição Epitelial-Mesenquimal/genética , Regulação da Expressão Gênica/genética , Regulação Neoplásica da Expressão Gênica/genética , Hepatócitos/metabolismo , Hepatócitos/patologia , Humanos , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/patologia , Camundongos
14.
Liver Int ; 38(10): 1741-1750, 2018 10.
Artigo em Inglês | MEDLINE | ID: mdl-29359389

RESUMO

BACKGROUND & AIMS: Hepatitis C virus (HCV) infection is known to cause major alterations in the cross-talk between hepatic and immune cells thus contributing to the liver disease pathogenesis. Extracellular vesicles have been proved to act as major players in cell-cell communication, and their cargo changes in relation to pathophysiological states. The aim of this study was to evaluate the effects of chronic HCV infection and direct-acting antivirals (DAA) on exosome-delivered microRNAs and on their ability to modulate the innate immune response. METHODS: Exosomes isolated from the plasma of healthy donors and naïve, viremic HCV patients before and after DAA treatment have been compared for their microRNAs cargo by quantitative polymerase chain reaction. Functional assays with peripheral blood cells from healthy donors were performed to assess exosome-mediated immune responses. RESULTS: MicroRNAs associated with HCV-related immunopathogenesis which were found to be enriched in exosomes of HCV viremic patients (in particular, miR-122-5p, miR-222-3p, miR-146a, miR-150-5p, miR-30c, miR-378a-3p and miR-20a-5p) were markedly reduced by DAA therapy. This exosome-microRNA cargo modulation parallels changes in their immunomodulatory properties in ex vivo experiments. Exosomes from HCV patients inhibit NK degranulation activity and this effect correlates with miR-122-5p or miR-222-3p levels. CONCLUSIONS: Enrichment of immunomodulatory microRNAs in exosomes of HCV patients was correlated with their inhibitory activity on innate immune cells function. Direct-acting antivirals (DAA) treatment was observed to revert both microRNA content and functional profiles of systemic exosomes towards those of healthy donors. Exosome-associated microRNAs may provide valuable biomarkers to monitor immune response recovery.


Assuntos
Antivirais/farmacologia , Exossomos/imunologia , Hepatite C Crônica/tratamento farmacológico , MicroRNAs/imunologia , Adulto , Idoso , Biomarcadores , Estudos de Casos e Controles , Comunicação Celular , Feminino , Perfilação da Expressão Gênica , Hepacivirus/genética , Humanos , Imunidade Inata , Masculino , Pessoa de Meia-Idade
15.
Biomed Res Int ; 2017: 2931813, 2017.
Artigo em Inglês | MEDLINE | ID: mdl-28265569

RESUMO

Exosomes are important in intercellular communication. They assure the horizontal transfer of specific functional contents (i.e., proteins, lipids, RNA molecules, and circulating DNA) from donor to recipient cells. Notably, tumor-derived exosomes (TDEs) appear to be an important vehicle of specific signals in cancer, impacting on tumor growth and metastasis. Recent researches point to the characterization of exosomes in Hepatocellular Carcinoma (HCC), the major adult liver malignancy. In this review, we summarize current findings on HCC exosomes, focusing on the identification of noncoding RNAs as exosome-enriched functional regulators and new potential biomarkers. The great potential of exosomes in future HCC diagnostic and therapeutic approaches is underlined.


Assuntos
Carcinoma Hepatocelular/metabolismo , Carcinoma Hepatocelular/terapia , Exossomos , Neoplasias Hepáticas/metabolismo , Neoplasias Hepáticas/terapia , Adulto , Animais , Carcinoma Hepatocelular/patologia , Humanos , Neoplasias Hepáticas/patologia
16.
Nucleic Acids Res ; 45(1): 155-168, 2017 01 09.
Artigo em Inglês | MEDLINE | ID: mdl-27658966

RESUMO

Adenosine deaminases acting on RNA (ADARs) are involved in RNA editing that converts adenosines to inosines in double-stranded RNAs. ADAR1 was demonstrated to be functional on different viruses exerting either antiviral or proviral effects. Concerning HIV-1, several studies showed that ADAR1 favors viral replication. The aim of this study was to investigate the composition of the ADAR1 ribonucleoprotein complex during HIV-1 expression. By using a dual-tag affinity purification procedure in cells expressing HIV-1 followed by mass spectrometry analysis, we identified 14 non-ribosomal ADAR1-interacting proteins, most of which are novel. A significant fraction of these proteins were previously demonstrated to be associated to the Long INterspersed Element 1 (LINE1 or L1) ribonucleoparticles and to regulate the life cycle of L1 retrotransposons that continuously re-enter host-genome.Hence, we investigated the function of ADAR1 in the regulation of L1 activity.By using different cell-culture based retrotransposition assays in HeLa cells, we demonstrated a novel function of ADAR1 as suppressor of L1 retrotransposition. Apparently, this inhibitory mechanism does not occur through ADAR1 editing activity. Furthermore, we showed that ADAR1 binds the basal L1 RNP complex. Overall, these data support the role of ADAR1 as regulator of L1 life cycle.


Assuntos
Adenosina Desaminase/genética , HIV-1/genética , Elementos Nucleotídeos Longos e Dispersos , Proteínas de Ligação a RNA/genética , Retroelementos , Ribonucleoproteínas/genética , Adenosina Desaminase/metabolismo , Bioensaio , Perfilação da Expressão Gênica , Regulação da Expressão Gênica , Células HEK293 , HIV-1/metabolismo , Células HeLa , Proteínas de Choque Térmico/genética , Proteínas de Choque Térmico/metabolismo , Interações Hospedeiro-Patógeno , Humanos , Anotação de Sequência Molecular , Ligação Proteica , Proteínas de Ligação a RNA/metabolismo , Ribonucleoproteínas/metabolismo , Transdução de Sinais
17.
Cell Rep ; 17(3): 799-808, 2016 10 11.
Artigo em Inglês | MEDLINE | ID: mdl-27732855

RESUMO

Despite clear evidence that exosomal microRNAs (miRNAs) are able to modulate the cellular microenvironment and that exosomal RNA cargo selection is deregulated in pathological conditions, the mechanisms controlling specific RNA sorting into extracellular vesicles are still poorly understood. Here, we identified the RNA binding protein SYNCRIP (synaptotagmin-binding cytoplasmic RNA-interacting protein; also known as hnRNP-Q or NSAP1) as a component of the hepatocyte exosomal miRNA sorting machinery. SYNCRIP knockdown impairs sorting of miRNAs in exosomes. Furthermore, SYNCRIP directly binds to specific miRNAs enriched in exosomes sharing a common extra-seed sequence (hEXO motif). The hEXO motif has a role in the regulation of miRNA localization, since embedment of this motif into a poorly exported miRNA enhances its loading into exosomes. This evidence provides insights into the mechanisms of miRNA exosomal sorting process. Moreover, these findings open the way for the possible selective modification of the miRNAs exosomal cargo.


Assuntos
Exossomos/metabolismo , Hepatócitos/metabolismo , Ribonucleoproteínas Nucleares Heterogêneas Grupo A-B/metabolismo , Ribonucleoproteínas Nucleares Heterogêneas/metabolismo , MicroRNAs/metabolismo , Transporte de RNA , Proteínas de Ligação a RNA/metabolismo , Animais , Sequência de Bases , Camundongos , Motivos de Nucleotídeos/genética , Transporte de RNA/genética
18.
PLoS One ; 11(3): e0151736, 2016.
Artigo em Inglês | MEDLINE | ID: mdl-26998606

RESUMO

Chronic liver damage leads to pathological accumulation of ECM proteins (liver fibrosis). Comprehensive characterization of the human ECM molecular composition is essential for gaining insights into the mechanisms of liver disease. To date, studies of ECM remodeling in human liver diseases have been hampered by the unavailability of purified ECM. Here, we developed a decellularization method to purify ECM scaffolds from human liver tissues. Histological and electron microscopy analyses demonstrated that the ECM scaffolds, devoid of plasma and cellular components, preserved the three-dimensional ECM structure and zonal distribution of ECM components. This method has been then applied on 57 liver biopsies of HCV-infected patients at different stages of liver fibrosis according to METAVIR classification. Label-free nLC-MS/MS proteomics and computation biology were performed to analyze the ECM molecular composition in liver fibrosis progression, thus unveiling protein expression signatures specific for the HCV-related liver fibrotic stages. In particular, the ECM molecular composition of liver fibrosis was found to involve dynamic changes in matrix stiffness, flexibility and density related to the dysregulation of predominant collagen, elastic fibers and minor components with both structural and signaling properties. This study contributes to the understanding of the molecular bases underlying ECM remodeling in liver fibrosis and suggests new molecular targets for fibrolytic strategies.


Assuntos
Progressão da Doença , Matriz Extracelular/metabolismo , Cirrose Hepática/patologia , Animais , Hepacivirus/fisiologia , Humanos , Fígado/patologia , Fígado/ultraestrutura , Cirrose Hepática/metabolismo , Cirrose Hepática/virologia , Camundongos , Proteoma/metabolismo , Proteômica , Alicerces Teciduais/química
19.
BMC Cancer ; 15: 131, 2015 Mar 15.
Artigo em Inglês | MEDLINE | ID: mdl-25886394

RESUMO

BACKGROUND: Eukaryotic Initiation factor 6 (eIF6) is a peculiar translation initiation factor that binds to the large 60S ribosomal subunits, controlling translation initiation and participating in ribosome biogenesis. In the past, knowledge about the mechanisms adopted by the cells for controlling protein synthesis by extracellular stimuli has focused on two translation initiation factors (eIF4E and eIF2), however, recent data suggest eIF6 as a newcomer in the control of downstream of signal transduction pathways. eIF6 is over-expressed in tumors and its decreased expression renders cells less prone to tumor growth. A previous work from our laboratory has disclosed that over-expression of eIF6 in transformed cell lines markedly increased cell migration and invasion. METHODS: Here, we performed a quantitative proteomic analysis of membrane-associated proteins in A2780 ovarian cancer cells over-expressing eIF6. Differentially expressed proteins upon eIF6 overproduction were further investigated in silico by Ingenuity Pathway Analysis (IPA). RT-qPCR and Western blot were performed in order to validate the proteomic data. Furthermore, the effects of a potent and selective inhibitor ML-141 in A2780 cells were evaluated using transwell migration assay. Finally, we explored the effects of eIF6 over-expression on WM793 primary melanoma cell lines. RESULTS: We demonstrated that: (i) the genes up-regulated upon eIF6 overproduction mapped to a functional network corresponding to cellular movements in a highly significant way; (ii) cdc42 plays a pivotal role as an effector of enhanced migratory phenotype induced upon eIF6 over-expression; (iii) the variations in abundance observed for cdc42 protein occur at a post-transcriptional level; (iv) the increased cell migration/invasion upon eIF6 over-expression was generalizable to other cell line models. CONCLUSIONS: Collectively, our data confirm and further extend the role of eIF6 in enhancing cell migration/invasion. We show that a number of membrane-associated proteins indeed vary in abundance upon eIF6 over-expression, and that the up-regulated proteins can be located within a functional network controlling cell motility and tumor metastasis. Full understanding of the role eIF6 plays in the metastatic process is important, also in view of the fact that this factor is a potentially druggable target to be exploited for new anti-cancer therapies.


Assuntos
Fatores de Iniciação em Eucariotos/biossíntese , Regulação Neoplásica da Expressão Gênica , Proteínas de Membrana/biossíntese , Invasividade Neoplásica , Movimento Celular/fisiologia , Feminino , Humanos , Invasividade Neoplásica/patologia
20.
Proteome Sci ; 12(1): 15, 2014 Mar 15.
Artigo em Inglês | MEDLINE | ID: mdl-24628804

RESUMO

BACKGROUND: Despite extensive research on hepatic cells precursors and their differentiated states, much remains to be learned about the mechanism underlying the self-renewal and differentiation. RESULTS: We apply the SILAC (stable isotope labeling by amino acids in cell culture) approach to quantitatively compare the membrane proteome of the resident liver stem cells (RLSCs) and their progeny spontaneously differentiated into epithelial/hepatocyte (RLSCdH). By means of nanoLC-MALDI-TOF/TOF approach, we identified and quantified 248 membrane proteins and 57 of them were found modulated during hepatocyte differentiation. Functional clustering of differentially expressed proteins by Ingenuity Pathway Analysis revealed that the most of membrane proteins found to be modulated are involved in cell-to-cell signaling/interaction pathways. Moreover, the upstream prediction analysis of proteins involved in cell-to-cell signaling and interaction unveiled that the activation of the mesenchymal to epithelial transition (MET), by the repression of TGFB1/Slug signaling, may be causal to hepatocyte differentiation. CONCLUSIONS: Taken together, this study increases the understanding of the underlying mechanisms modulating the complex biological processes of hepatic stem cell proliferation and differentiation.

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