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1.
J Hepatol ; 78(2): 343-355, 2023 02.
Artículo en Inglés | MEDLINE | ID: mdl-36309131

RESUMEN

BACKGROUND & AIMS: Despite recent approvals, the response to treatment and prognosis of patients with advanced hepatocellular carcinoma (HCC) remain poor. Claudin-1 (CLDN1) is a membrane protein that is expressed at tight junctions, but it can also be exposed non-junctionally, such as on the basolateral membrane of the human hepatocyte. While CLDN1 within tight junctions is well characterized, the role of non-junctional CLDN1 and its role as a therapeutic target in HCC remains unexplored. METHODS: Using humanized monoclonal antibodies (mAbs) specifically targeting the extracellular loop of human non-junctional CLDN1 and a large series of patient-derived cell-based and animal model systems we aimed to investigate the role of CLDN1 as a therapeutic target for HCC. RESULTS: Targeting non-junctional CLDN1 markedly suppressed tumor growth and invasion in cell line-based models of HCC and patient-derived 3D ex vivo models. Moreover, the robust effect on tumor growth was confirmed in vivo in a large series of cell line-derived xenograft and patient-derived xenograft mouse models. Mechanistic studies, including single-cell RNA sequencing of multicellular patient HCC tumorspheres, suggested that CLDN1 regulates tumor stemness, metabolism, oncogenic signaling and perturbs the tumor immune microenvironment. CONCLUSIONS: Our results provide the rationale for targeting CLDN1 in HCC and pave the way for the clinical development of CLDN1-specific mAbs for the treatment of advanced HCC. IMPACT AND IMPLICATIONS: Hepatocellular carcinoma (HCC) is associated with high mortality and unsatisfactory treatment options. Herein, we identified the cell surface protein Claudin-1 as a treatment target for advanced HCC. Monoclonal antibodies targeting Claudin-1 inhibit tumor growth in patient-derived ex vivo and in vivo models by modulating signaling, cell stemness and the tumor immune microenvironment. Given the differentiated mechanism of action, the identification of Claudin-1 as a novel therapeutic target for HCC provides an opportunity to break the plateau of limited treatment response. The results of this preclinical study pave the way for the clinical development of Claudin-1-specific antibodies for the treatment of advanced HCC. It is therefore of key impact for physicians, scientists and drug developers in the field of liver cancer and gastrointestinal oncology.


Asunto(s)
Carcinoma Hepatocelular , Neoplasias Hepáticas , Humanos , Animales , Ratones , Carcinoma Hepatocelular/genética , Claudina-1/genética , Neoplasias Hepáticas/genética , Carcinógenos , Microambiente Tumoral , Anticuerpos Monoclonales/farmacología , Anticuerpos Monoclonales/uso terapéutico , Línea Celular Tumoral
2.
JCI Insight ; 7(13)2022 07 08.
Artículo en Inglés | MEDLINE | ID: mdl-35801591

RESUMEN

Hepatocellular carcinoma (HCC) is a leading cause of death among cirrhotic patients, for which chemopreventive strategies are lacking. Recently, we developed a simple human cell-based system modeling a clinical prognostic liver signature (PLS) predicting liver disease progression and HCC risk. In a previous study, we applied our cell-based system for drug discovery and identified captopril, an approved angiotensin converting enzyme (ACE) inhibitor, as a candidate compound for HCC chemoprevention. Here, we explored ACE as a therapeutic target for HCC chemoprevention. Captopril reduced liver fibrosis and effectively prevented liver disease progression toward HCC development in a diethylnitrosamine (DEN) rat cirrhosis model and a diet-based rat model for nonalcoholic steatohepatitis-induced (NASH-induced) hepatocarcinogenesis. RNA-Seq analysis of cirrhotic rat liver tissues uncovered that captopril suppressed the expression of pathways mediating fibrogenesis, inflammation, and carcinogenesis, including epidermal growth factor receptor (EGFR) signaling. Mechanistic data in liver disease models uncovered a cross-activation of the EGFR pathway by angiotensin. Corroborating the clinical translatability of the approach, captopril significantly reversed the HCC high-risk status of the PLS in liver tissues of patients with advanced fibrosis. Captopril effectively prevents fibrotic liver disease progression toward HCC development in preclinical models and is a generic and safe candidate drug for HCC chemoprevention.


Asunto(s)
Captopril , Carcinoma Hepatocelular , Neoplasias Hepáticas , Inhibidores de la Enzima Convertidora de Angiotensina/farmacología , Inhibidores de la Enzima Convertidora de Angiotensina/uso terapéutico , Animales , Captopril/farmacología , Captopril/uso terapéutico , Carcinogénesis , Carcinoma Hepatocelular/tratamiento farmacológico , Carcinoma Hepatocelular/prevención & control , Quimioprevención , Progresión de la Enfermedad , Receptores ErbB/metabolismo , Cirrosis Hepática/prevención & control , Neoplasias Hepáticas/tratamiento farmacológico , Neoplasias Hepáticas/prevención & control , Peptidil-Dipeptidasa A/metabolismo , Ratas , Activación Transcripcional
3.
Nat Commun ; 12(1): 5525, 2021 09 17.
Artículo en Inglés | MEDLINE | ID: mdl-34535664

RESUMEN

Chronic liver disease and hepatocellular carcinoma (HCC) are life-threatening diseases with limited treatment options. The lack of clinically relevant/tractable experimental models hampers therapeutic discovery. Here, we develop a simple and robust human liver cell-based system modeling a clinical prognostic liver signature (PLS) predicting long-term liver disease progression toward HCC. Using the PLS as a readout, followed by validation in nonalcoholic steatohepatitis/fibrosis/HCC animal models and patient-derived liver spheroids, we identify nizatidine, a histamine receptor H2 (HRH2) blocker, for treatment of advanced liver disease and HCC chemoprevention. Moreover, perturbation studies combined with single cell RNA-Seq analyses of patient liver tissues uncover hepatocytes and HRH2+, CLEC5Ahigh, MARCOlow liver macrophages as potential nizatidine targets. The PLS model combined with single cell RNA-Seq of patient tissues enables discovery of urgently needed targets and therapeutics for treatment of advanced liver disease and cancer prevention.


Asunto(s)
Descubrimiento de Drogas , Hígado/patología , Modelos Biológicos , Animales , Carcinogénesis/patología , Carcinoma Hepatocelular/patología , Línea Celular Tumoral , Quimioprevención , Estudios de Cohortes , AMP Cíclico/metabolismo , Proteína de Unión a Elemento de Respuesta al AMP Cíclico/metabolismo , Modelos Animales de Enfermedad , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Células HEK293 , Hepacivirus/fisiología , Hepatitis C/genética , Hepatocitos/efectos de los fármacos , Hepatocitos/metabolismo , Hepatocitos/patología , Humanos , Vigilancia Inmunológica/efectos de los fármacos , Inflamación/patología , Hígado/efectos de los fármacos , Hígado/metabolismo , Cirrosis Hepática/patología , Neoplasias Hepáticas/patología , Macrófagos/efectos de los fármacos , Macrófagos/metabolismo , Macrófagos/patología , Masculino , Ratones Noqueados , Nizatidina/farmacología , Pronóstico , Transducción de Señal/efectos de los fármacos , Transcriptoma/genética
4.
Gut ; 69(2): 380-392, 2020 02.
Artículo en Inglés | MEDLINE | ID: mdl-31076402

RESUMEN

OBJECTIVE: Infection of human hepatocytes by the hepatitis C virus (HCV) is a multistep process involving both viral and host factors. microRNAs (miRNAs) are small non-coding RNAs that post-transcriptionally regulate gene expression. Given that miRNAs were indicated to regulate between 30% and 75% of all human genes, we aimed to investigate the functional and regulatory role of miRNAs for the HCV life cycle. DESIGN: To systematically reveal human miRNAs affecting the HCV life cycle, we performed a two-step functional high-throughput miRNA mimic screen in Huh7.5.1 cells infected with recombinant cell culture-derived HCV. miRNA targeting was then assessed using a combination of computational and functional approaches. RESULTS: We uncovered miR-501-3p and miR-619-3p as novel modulators of HCV assembly/release. We discovered that these miRNAs regulate O-linked N-acetylglucosamine (O-GlcNAc) transferase (OGT) protein expression and identified OGT and O-GlcNAcylation as regulators of HCV morphogenesis and infectivity. Furthermore, increased OGT expression in patient-derived liver tissue was associated with HCV-induced liver disease and cancer. CONCLUSION: miR-501-3p and miR-619-3p and their target OGT are previously undiscovered regulatory host factors for HCV assembly and infectivity. In addition to its effect on HCV morphogenesis, OGT may play a role in HCV-induced liver disease and hepatocarcinogenesis.


Asunto(s)
Hepacivirus/patogenicidad , Hepatitis C Crónica/genética , N-Acetilglucosaminiltransferasas/fisiología , Regulación de la Expresión Génica/fisiología , Técnicas de Silenciamiento del Gen/métodos , Estudio de Asociación del Genoma Completo/métodos , Hepacivirus/fisiología , Hepatitis C Crónica/virología , Hepatocitos/virología , Interacciones Huésped-Patógeno/genética , Humanos , Estadios del Ciclo de Vida/genética , MicroARNs/genética , Morfogénesis/fisiología , N-Acetilglucosaminiltransferasas/genética , Regulación hacia Arriba , Virulencia/genética
5.
Gastroenterology ; 157(2): 537-551.e9, 2019 08.
Artículo en Inglés | MEDLINE | ID: mdl-30978357

RESUMEN

BACKGROUND & AIMS: The mechanisms of hepatitis C virus (HCV) infection, liver disease progression, and hepatocarcinogenesis are only partially understood. We performed genomic, proteomic, and metabolomic analyses of HCV-infected cells and chimeric mice to learn more about these processes. METHODS: Huh7.5.1dif (hepatocyte-like cells) were infected with culture-derived HCV and used in RNA sequencing, proteomic, metabolomic, and integrative genomic analyses. uPA/SCID (urokinase-type plasminogen activator/severe combined immunodeficiency) mice were injected with serum from HCV-infected patients; 8 weeks later, liver tissues were collected and analyzed by RNA sequencing and proteomics. Using differential expression, gene set enrichment analyses, and protein interaction mapping, we identified pathways that changed in response to HCV infection. We validated our findings in studies of liver tissues from 216 patients with HCV infection and early-stage cirrhosis and paired biopsy specimens from 99 patients with hepatocellular carcinoma, including 17 patients with histologic features of steatohepatitis. Cirrhotic liver tissues from patients with HCV infection were classified into 2 groups based on relative peroxisome function; outcomes assessed included Child-Pugh class, development of hepatocellular carcinoma, survival, and steatohepatitis. Hepatocellular carcinomas were classified according to steatohepatitis; the outcome was relative peroxisomal function. RESULTS: We quantified 21,950 messenger RNAs (mRNAs) and 8297 proteins in HCV-infected cells. Upon HCV infection of hepatocyte-like cells and chimeric mice, we observed significant changes in levels of mRNAs and proteins involved in metabolism and hepatocarcinogenesis. HCV infection of hepatocyte-like cells significantly increased levels of the mRNAs, but not proteins, that regulate the innate immune response; we believe this was due to the inhibition of translation in these cells. HCV infection of hepatocyte-like cells increased glucose consumption and metabolism and the STAT3 signaling pathway and reduced peroxisome function. Peroxisomes mediate ß-oxidation of very long-chain fatty acids; we found intracellular accumulation of very long-chain fatty acids in HCV-infected cells, which is also observed in patients with fatty liver disease. Cells in livers from HCV-infected mice had significant reductions in levels of the mRNAs and proteins associated with peroxisome function, indicating perturbation of peroxisomes. We found that defects in peroxisome function were associated with outcomes and features of HCV-associated cirrhosis, fatty liver disease, and hepatocellular carcinoma in patients. CONCLUSIONS: We performed combined transcriptome, proteome, and metabolome analyses of liver tissues from HCV-infected hepatocyte-like cells and HCV-infected mice. We found that HCV infection increases glucose metabolism and the STAT3 signaling pathway and thereby reduces peroxisome function; alterations in the expression levels of peroxisome genes were associated with outcomes of patients with liver diseases. These findings provide insights into liver disease pathogenesis and might be used to identify new therapeutic targets.


Asunto(s)
Hepacivirus/patogenicidad , Hepatitis C Crónica/patología , Hepatocitos/patología , Hígado/patología , Animales , Línea Celular Tumoral , Conjuntos de Datos como Asunto , Modelos Animales de Enfermedad , Perfilación de la Expresión Génica , Glucosa/metabolismo , Hepatitis C Crónica/metabolismo , Hepatitis C Crónica/virología , Hepatocitos/trasplante , Hepatocitos/virología , Humanos , Hígado/citología , Hígado/virología , Metabolómica , Ratones , Peroxisomas/metabolismo , Peroxisomas/patología , Proteómica , Factor de Transcripción STAT3/metabolismo , Quimera por Trasplante
6.
Gastroenterology ; 156(8): 2313-2329.e7, 2019 06.
Artículo en Inglés | MEDLINE | ID: mdl-30836093

RESUMEN

BACKGROUND & AIMS: Chronic hepatitis C virus (HCV) infection is an important risk factor for hepatocellular carcinoma (HCC). Despite effective antiviral therapies, the risk for HCC is decreased but not eliminated after a sustained virologic response (SVR) to direct-acting antiviral (DAA) agents, and the risk is higher in patients with advanced fibrosis. We investigated HCV-induced epigenetic alterations that might affect risk for HCC after DAA treatment in patients and mice with humanized livers. METHODS: We performed genome-wide ChIPmentation-based ChIP-Seq and RNA-seq analyses of liver tissues from 6 patients without HCV infection (controls), 18 patients with chronic HCV infection, 8 patients with chronic HCV infection cured by DAA treatment, 13 patients with chronic HCV infection cured by interferon therapy, 4 patients with chronic hepatitis B virus infection, and 7 patients with nonalcoholic steatohepatitis in Europe and Japan. HCV-induced epigenetic modifications were mapped by comparative analyses with modifications associated with other liver disease etiologies. uPA/SCID mice were engrafted with human hepatocytes to create mice with humanized livers and given injections of HCV-infected serum samples from patients; mice were given DAAs to eradicate the virus. Pathways associated with HCC risk were identified by integrative pathway analyses and validated in analyses of paired HCC tissues from 8 patients with an SVR to DAA treatment of HCV infection. RESULTS: We found chronic HCV infection to induce specific genome-wide changes in H3K27ac, which correlated with changes in expression of mRNAs and proteins. These changes persisted after an SVR to DAAs or interferon-based therapies. Integrative pathway analyses of liver tissues from patients and mice with humanized livers demonstrated that HCV-induced epigenetic alterations were associated with liver cancer risk. Computational analyses associated increased expression of SPHK1 with HCC risk. We validated these findings in an independent cohort of patients with HCV-related cirrhosis (n = 216), a subset of which (n = 21) achieved viral clearance. CONCLUSIONS: In an analysis of liver tissues from patients with and without an SVR to DAA therapy, we identified epigenetic and gene expression alterations associated with risk for HCC. These alterations might be targeted to prevent liver cancer in patients treated for HCV infection.


Asunto(s)
Antivirales/uso terapéutico , Carcinoma Hepatocelular/virología , Hepatitis C Crónica/patología , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/virología , Adulto , Animales , Carcinoma Hepatocelular/genética , Estudios de Casos y Controles , Estudios de Cohortes , Modelos Animales de Enfermedad , Epigénesis Genética , Europa (Continente) , Femenino , Regulación Neoplásica de la Expresión Génica , Hepatitis C Crónica/complicaciones , Hepatitis C Crónica/tratamiento farmacológico , Humanos , Japón , Neoplasias Hepáticas/patología , Masculino , Ratones , Ratones SCID , Distribución Aleatoria , Respuesta Virológica Sostenida
7.
Gut ; 67(5): 953-962, 2018 05.
Artículo en Inglés | MEDLINE | ID: mdl-28159835

RESUMEN

BACKGROUND AND AIMS: HCV infection is a leading risk factor of hepatocellular carcinoma (HCC). However, even after viral clearance, HCC risk remains elevated. HCV perturbs host cell signalling to maintain infection, and derailed signalling circuitry is a key driver of carcinogenesis. Since protein phosphatases are regulators of signalling events, we aimed to identify phosphatases that respond to HCV infection with relevance for hepatocarcinogenesis. METHODS: We assessed mRNA and microRNA (miRNA) expression profiles in primary human hepatocytes, liver biopsies and resections of patients with HCC, and analysed microarray and RNA-seq data from paired liver biopsies of patients with HCC. We revealed changes in transcriptional networks through gene set enrichment analysis and correlated phosphatase expression levels to patient survival and tumour recurrence. RESULTS: We demonstrate that tumour suppressor protein tyrosine phosphatase receptor delta (PTPRD) is impaired by HCV infection in vivo and in HCC lesions of paired liver biopsies independent from tissue inflammation or fibrosis. In liver tissue adjacent to tumour, high PTPRD levels are associated with a dampened transcriptional activity of STAT3, an increase of patient survival from HCC and reduced tumour recurrence after surgical resection. We identified miR-135a-5p as a mechanistic regulator of hepatic PTPRD expression in patients with HCV. CONCLUSIONS: We previously demonstrated that STAT3 is required for HCV infection. We conclude that HCV promotes a STAT3 transcriptional programme in the liver of patients by suppressing its regulator PTPRD via upregulation of miR-135a-5p. Our results show the existence of a perturbed PTPRD-STAT3 axis potentially driving malignant progression of HCV-associated liver disease.


Asunto(s)
Carcinoma Hepatocelular/metabolismo , Hepacivirus/patogenicidad , Hepatitis C/complicaciones , Neoplasias Hepáticas/metabolismo , MicroARNs/metabolismo , Proteínas Tirosina Fosfatasas Clase 2 Similares a Receptores/metabolismo , Adulto , Anciano , Anciano de 80 o más Años , Western Blotting , Carcinogénesis/metabolismo , Carcinoma Hepatocelular/virología , Regulación hacia Abajo , Femenino , Hepatocitos/metabolismo , Humanos , Hibridación Fluorescente in Situ , Hígado/patología , Neoplasias Hepáticas/virología , Masculino , Persona de Mediana Edad , ARN Mensajero/metabolismo , Factor de Transcripción STAT3/metabolismo , Transducción de Señal
8.
Curr Opin Virol ; 20: 99-105, 2016 10.
Artículo en Inglés | MEDLINE | ID: mdl-27741441

RESUMEN

Hepatitis C virus (HCV) infection is one of the major causes of advanced liver disease and hepatocellular carcinoma (HCC) worldwide. While the knowledge about the molecular virology of HCV infection has markedly advanced, the molecular mechanisms of disease progression leading to fibrosis, cirrhosis and HCC are still unclear. Accumulating experimental and clinical studies indicate that HCV may drive hepatocarcinogenesis directly via its proteins or transcripts, and/or indirectly through induction of chronic liver inflammation. Despite the possibility to eradicate HCV infection through direct-acting antiviral treatment, the risk of HCC persists although specific biomarkers to estimate this risk are still missing. Thus, a better understanding of HCV-induced HCC and more physiological liver disease models are required to prevent cancer development.


Asunto(s)
Carcinoma Hepatocelular/patología , Carcinoma Hepatocelular/fisiopatología , Hepatitis C Crónica/complicaciones , Humanos , Cirrosis Hepática/complicaciones , Modelos Biológicos
10.
J Virol ; 90(14): 6387-6400, 2016 07 15.
Artículo en Inglés | MEDLINE | ID: mdl-27147737

RESUMEN

UNLABELLED: Hepatitis C virus (HCV)-induced chronic liver disease is a leading cause of hepatocellular carcinoma (HCC). However, the molecular mechanisms underlying HCC development following chronic HCV infection remain poorly understood. MicroRNAs (miRNAs) play an important role in homeostasis within the liver, and deregulation of miRNAs has been associated with liver disease, including HCC. While host miRNAs are essential for HCV replication, viral infection in turn appears to induce alterations of intrahepatic miRNA networks. Although the cross talk between HCV and liver cell miRNAs most likely contributes to liver disease pathogenesis, the functional involvement of miRNAs in HCV-driven hepatocyte injury and HCC remains elusive. Here we combined a hepatocyte-like cell-based model system, high-throughput small RNA sequencing, computational analysis, and functional studies to investigate HCV-miRNA interactions that may contribute to liver disease and HCC. Profiling analyses indicated that HCV infection differentially regulated the expression of 72 miRNAs by at least 2-fold, including miRNAs that were previously described to target genes associated with inflammation, fibrosis, and cancer development. Further investigation demonstrated that the miR-146a-5p level was consistently increased in HCV-infected hepatocyte-like cells and primary human hepatocytes, as well as in liver tissue from HCV-infected patients. Genome-wide microarray and computational analyses indicated that miR-146a-5p overexpression modulates pathways that are related to liver disease and HCC development. Furthermore, we showed that miR-146a-5p has a positive impact on late steps of the viral replication cycle, thereby increasing HCV infection. Collectively, our data indicate that the HCV-induced increase in miR-146a-5p expression both promotes viral infection and is relevant for pathogenesis of liver disease. IMPORTANCE: HCV is a leading cause of chronic liver disease and cancer. However, how HCV induces liver cancer remains poorly understood. There is accumulating evidence that a viral cure does not eliminate the risk for HCC development. Thus, there is an unmet medical need to develop novel approaches to predict and prevent virus-induced HCC. miRNA expression is known to be deregulated in liver disease and cancer. Furthermore, miRNAs are essential for HCV replication, and HCV infection alters miRNA expression. However, how miRNAs contribute to HCV-driven pathogenesis remains elusive. Here we show that HCV induces miRNAs that may contribute to liver injury and carcinogenesis. The miR-146a-5p level was consistently increased in different cell-based models of HCV infection and in HCV patient-derived liver tissue. Furthermore, miR-146a-5p increased HCV infection. Collectively, our data are relevant to understanding viral pathogenesis and may open perspectives for novel biomarkers and prevention of virus-induced liver disease and HCC.


Asunto(s)
Carcinoma Hepatocelular/virología , Hepacivirus/patogenicidad , Hepatitis C/virología , Hepatocitos/metabolismo , Neoplasias Hepáticas/virología , Redes y Vías Metabólicas/genética , MicroARNs/genética , Adulto , Anciano , Biomarcadores/análisis , Carcinoma Hepatocelular/genética , Carcinoma Hepatocelular/patología , Femenino , Perfilación de la Expresión Génica , Hepatitis C/genética , Hepatitis C/patología , Hepatocitos/citología , Hepatocitos/virología , Secuenciación de Nucleótidos de Alto Rendimiento , Interacciones Huésped-Patógeno , Humanos , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/patología , Masculino , Persona de Mediana Edad , Activación Transcripcional , Regulación hacia Arriba
11.
Nat Biotechnol ; 33(5): 549-554, 2015 May.
Artículo en Inglés | MEDLINE | ID: mdl-25798937

RESUMEN

Hepatitis C virus (HCV) infection is a leading cause of liver cirrhosis and cancer. Cell entry of HCV and other pathogens is mediated by tight junction (TJ) proteins, but successful therapeutic targeting of TJ proteins has not been reported yet. Using a human liver-chimeric mouse model, we show that a monoclonal antibody specific for the TJ protein claudin-1 (ref. 7) eliminates chronic HCV infection without detectable toxicity. This antibody inhibits HCV entry, cell-cell transmission and virus-induced signaling events. Antibody treatment reduces the number of HCV-infected hepatocytes in vivo, highlighting the need for de novo infection by means of host entry factors to maintain chronic infection. In summary, we demonstrate that an antibody targeting a virus receptor can cure chronic viral infection and uncover TJ proteins as targets for antiviral therapy.


Asunto(s)
Anticuerpos Monoclonales/administración & dosificación , Claudina-1/inmunología , Hepatitis C/terapia , Cirrosis Hepática/inmunología , Animales , Anticuerpos Monoclonales/inmunología , Anticuerpos Monoclonales Humanizados/administración & dosificación , Anticuerpos Monoclonales Humanizados/inmunología , Claudina-1/uso terapéutico , Hepacivirus/inmunología , Hepacivirus/patogenicidad , Hepatitis C/inmunología , Hepatitis C/virología , Hepatocitos/inmunología , Humanos , Cirrosis Hepática/terapia , Cirrosis Hepática/virología , Ratones
12.
J Hepatol ; 62(2): 448-57, 2015 Feb.
Artículo en Inglés | MEDLINE | ID: mdl-25308172

RESUMEN

Being the largest internal organ of the human body with the unique ability of self-regeneration, the liver is involved in a wide variety of vital functions that require highly orchestrated and controlled biochemical processes. Increasing evidence suggests that microRNAs (miRNAs) are essential for the regulation of liver development, regeneration and metabolic functions. Hence, alterations in intrahepatic miRNA networks have been associated with liver disease including hepatitis, steatosis, cirrhosis and hepatocellular carcinoma (HCC). miR-122 is the most frequent miRNA in the adult liver, and a central player in liver biology and disease. Furthermore, miR-122 has been shown to be an essential host factor for hepatitis C virus (HCV) infection and an antiviral target, complementary to the standard of care using direct-acting antivirals or interferon-based treatment. This review summarizes our current understanding of the key role of miR-122 in liver physiology and disease, highlighting its role in HCC and viral hepatitis. We also discuss the perspectives of miRNA-based therapeutic approaches for viral hepatitis and liver disease.


Asunto(s)
Regulación de la Expresión Génica , Terapia Genética/métodos , Hepatopatías , MicroARNs/genética , ARN Mensajero/genética , Humanos , Hepatopatías/genética , Hepatopatías/metabolismo , Hepatopatías/terapia , MicroARNs/metabolismo
13.
PLoS One ; 8(1): e54791, 2013.
Artículo en Inglés | MEDLINE | ID: mdl-23382970

RESUMEN

Friedreich's ataxia (FRDA) is a severe neurodegenerative disease caused by GAA repeat expansion within the first intron of the frataxin gene. It has been suggested that the repeat is responsible for the disease severity due to impaired transcription thereby reducing expression of the protein. However, genotype-phenotype correlation is imperfect, and the influence of other gene regions of the frataxin gene is unknown. We hypothesized that FRDA patients may harbor specific regulatory variants in the 3'-UTR. We sequenced the 3'-UTR region of the frataxin gene in a cohort of 57 FRDA individuals and 58 controls. Seven single nucleotide polymorphisms (SNPs) out of 19 were polymorphic in our case-control sample. These SNPs defined several haplotypes with one reaching 89% of homozygosity in patients versus 24% in controls. In another cohort of 47 FRDA Reunionese patients, 94% patients were found to be homozygous for this haplotype. We found that this FRDA 3'-UTR conferred a 1.2-fold decrease in the expression of a reporter gene versus the alternative haplotype configuration. We established that differential targeting by miRNA could account for this functional variability. We specifically demonstrated the involvement of miR-124 (i.e hsa-mir-124-3p) in the down-regulation of FRDA-3'-UTR. Our results suggest for the first time that post-transcriptional regulation of frataxin occurs through the 3'-UTR and involves miRNA targeting. We propose that the involvement of miRNAs in a FRDA-specific regulation of frataxin may provide a rationale to increase residual levels of frataxin through miRNA-inhibitory molecules.


Asunto(s)
Regiones no Traducidas 3' , Ataxia de Friedreich/genética , Regulación de la Expresión Génica , Variación Genética , Proteínas de Unión a Hierro/genética , MicroARNs/genética , Secuencia de Bases , Estudios de Casos y Controles , Línea Celular , Biología Computacional/métodos , Frecuencia de los Genes , Orden Génico , Predisposición Genética a la Enfermedad , Haplotipos , Humanos , MicroARNs/metabolismo , Polimorfismo de Nucleótido Simple , Expansión de Repetición de Trinucleótido , Frataxina
14.
Proc Natl Acad Sci U S A ; 109(13): 4980-5, 2012 Mar 27.
Artículo en Inglés | MEDLINE | ID: mdl-22411793

RESUMEN

The human genome is densely populated with transposons and transposon-like repetitive elements. Although the impact of these transposons and elements on human genome evolution is recognized, the significance of subtle variations in their sequence remains mostly unexplored. Here we report homozygosity mapping of an infantile neurodegenerative disease locus in a genetic isolate. Complete DNA sequencing of the 400-kb linkage locus revealed a point mutation in a primate-specific retrotransposon that was transcribed as part of a unique noncoding RNA, which was expressed in the brain. In vitro knockdown of this RNA increased neuronal apoptosis, consistent with the inappropriate dosage of this RNA in vivo and with the phenotype. Moreover, structural analysis of the sequence revealed a small RNA-like hairpin that was consistent with the putative gain of a functional site when mutated. We show here that a mutation in a unique transposable element-containing RNA is associated with lethal encephalopathy, and we suggest that RNAs that harbor evolutionarily recent repetitive elements may play important roles in human brain development.


Asunto(s)
Encefalopatías/genética , Secuencia Conservada/genética , Mutación/genética , Primates/genética , ARN no Traducido/genética , Retroelementos/genética , Animales , Anorexia/complicaciones , Anorexia/genética , Secuencia de Bases , Encefalopatías/complicaciones , Encefalopatías/patología , Mapeo Cromosómico , Segregación Cromosómica/genética , Cromosomas Humanos Par 8/genética , Progresión de la Enfermedad , Genes Recesivos/genética , Sitios Genéticos , Geografía , Humanos , Océano Índico , Lactante , Intrones/genética , Imagen por Resonancia Magnética , Datos de Secuencia Molecular , Conformación de Ácido Nucleico , Nucleótidos/genética , Fenotipo , ARN no Traducido/química , Adulto Joven
16.
PLoS One ; 6(6): e20746, 2011.
Artículo en Inglés | MEDLINE | ID: mdl-21695135

RESUMEN

MicroRNAs (miRNAs) are small non-coding RNAs that associate with Argonaute proteins to regulate gene expression at the post-transcriptional level in the cytoplasm. However, recent studies have reported that some miRNAs localize to and function in other cellular compartments. Mitochondria harbour their own genetic system that may be a potential site for miRNA mediated post-transcriptional regulation. We aimed at investigating whether nuclear-encoded miRNAs can localize to and function in human mitochondria. To enable identification of mitochondrial-enriched miRNAs, we profiled the mitochondrial and cytosolic RNA fractions from the same HeLa cells by miRNA microarray analysis. Mitochondria were purified using a combination of cell fractionation and immunoisolation, and assessed for the lack of protein and RNA contaminants. We found 57 miRNAs differentially expressed in HeLa mitochondria and cytosol. Of these 57, a signature of 13 nuclear-encoded miRNAs was reproducibly enriched in mitochondrial RNA and validated by RT-PCR for hsa-miR-494, hsa-miR-1275 and hsa-miR-1974. The significance of their mitochondrial localization was investigated by characterizing their genomic context, cross-species conservation and instrinsic features such as their size and thermodynamic parameters. Interestingly, the specificities of mitochondrial versus cytosolic miRNAs were underlined by significantly different structural and thermodynamic parameters. Computational targeting analysis of most mitochondrial miRNAs revealed not only nuclear but also mitochondrial-encoded targets. The functional relevance of miRNAs in mitochondria was supported by the finding of Argonaute 2 localization to mitochondria revealed by immunoblotting and confocal microscopy, and further validated by the co-immunoprecipitation of the mitochondrial transcript COX3. This study provides the first comprehensive view of the localization of RNA interference components to the mitochondria. Our data outline the molecular bases for a novel layer of crosstalk between nucleus and mitochondria through a specific subset of human miRNAs that we termed 'mitomiRs'.


Asunto(s)
Núcleo Celular/genética , Mitocondrias/genética , Interferencia de ARN , Animales , Proteínas Argonautas , Secuencia de Bases , Línea Celular , Cromosomas Humanos/genética , Secuencia Conservada/genética , Citosol/metabolismo , Factor 2 Eucariótico de Iniciación/metabolismo , Perfilación de la Expresión Génica , Regulación de la Expresión Génica , Genoma Mitocondrial/genética , Genómica , Humanos , Inmunoprecipitación , MicroARNs/aislamiento & purificación , Modelos Biológicos , Transporte de Proteínas , Transporte de ARN/genética , ARN Mensajero/genética , ARN Mensajero/metabolismo , Especificidad de la Especie
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