RESUMO
BACKGROUND & AIMS: Hepatocellular carcinoma (HCC) is a cancer with multiple aetiologies and widespread prevalence. Largely refractory to current treatments, HCC is the fourth leading cause of cancer-related deaths worldwide. MicroRNAs (miRNAs) are important regulators in HCCs. We aimed to identify tumour suppressor miRNAs during tumour regression in a conditional c-MYC-driven mouse model (LT2/MYC) of HCC, and to evaluate their therapeutic potential for HCC treatment. METHODS: We performed miRNA expression profiling of developed and regressing LT2/MYC tumours and in-depth in vitro gain- and loss-of-function analyses. The effect of adeno-associated virus (AAV) vector-mediated miR-342-3p treatment was evaluated in 3 HCC mouse models. RESULTS: We identified miR-342-3p as a tumour suppressor miRNA in HCC, with increased expression in regressing tumours. Forced miR-342-3p expression in hepatoma cells showed significantly decreased cell proliferation, migration, and colony formation. In vivo administration of AAV-miR-342-3p led to significant attenuation of tumour development and increased overall survival. We identified monocarboxylic acid transporter 1 (MCT1) as a bona fide target of miR-342-3p in HCC. We show that the tumour suppressor role of miR-342-3p is executed partly by modulating the lactate transport function of MCT1. Importantly, we find miR-342-3p downregulated in tumours from patients with HCC compared with matched non-tumour tissues, inversely correlating with MCT1 expression. We observed similar findings in TCGA-LIHC data. CONCLUSIONS: In our study, we identified and validated miR-342-3p as a tumour suppressor miRNA in HCC. We demonstrated its therapeutic efficacy in significantly attenuating tumour development, and prolonging survival, in different HCC mouse models. Identification of miR-342-3p as an effective tumour suppressor opens a therapeutic avenue for miRNA-mediated attenuation of HCC development. LAY SUMMARY: Hepatocellular carcinoma (HCC), the most common type of liver cancer, affects diverse populations and has a global impact, being the fourth leading cause of cancer deaths worldwide. There are currently no systemic therapies for HCC that can significantly prolong long-term survival. Thus, novel effective treatment options are urgently required. To understand the molecular basis of tumour regression, we compared tumours and regressing liver tumours in mice. We show that a small non-coding miRNA, miR-342-3p, is a tumour suppressor in HCC. Expression of miR-342-3p is low in tumours and high in regressing tumours. When miR-342-3p is delivered to mouse livers with HCC, it can significantly slow down liver tumour development and improve survival. Our study highlights the promising therapeutic potential of miR-342-3p intervention in HCC.
Assuntos
Transporte Biológico/efeitos dos fármacos , Carcinoma Hepatocelular , Neoplasias Hepáticas , MicroRNAs/genética , Transportadores de Ácidos Monocarboxílicos , Simportadores , Animais , Carcinoma Hepatocelular/genética , Carcinoma Hepatocelular/metabolismo , Carcinoma Hepatocelular/terapia , Movimento Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Modelos Animais de Doenças , Regulação para Baixo , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Genes Supressores de Tumor , Humanos , Ácido Láctico/metabolismo , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/metabolismo , Neoplasias Hepáticas/terapia , Camundongos , MicroRNAs/farmacologia , Transportadores de Ácidos Monocarboxílicos/genética , Transportadores de Ácidos Monocarboxílicos/metabolismo , Simportadores/genética , Simportadores/metabolismo , Transfecção/métodos , Resultado do TratamentoRESUMO
BACKGROUND & AIMS: Therapeutic targeting of injuries that require transient restoration of proteins by mRNA delivery is an attractive approach that, until recently, has remained poorly explored. In this study, we examined the therapeutic utility of mRNA delivery for liver fibrosis and cirrhosis. Specifically, we aimed to demonstrate the therapeutic efficacy of human hepatocyte nuclear factor alpha (HNF4A) mRNA in mouse models of fibrosis and cirrhosis. METHODS: We investigated restoration of hepatocyte functions by HNF4A mRNA transfection in vitro, and analyzed the attenuation of liver fibrosis and cirrhosis in multiple mouse models, by delivering hepatocyte-targeted biodegradable lipid nanoparticles (LNPs) encapsulating HNF4A mRNA. To identify potential mechanisms of action, we performed microarray-based gene expression profiling, single-cell RNA sequencing, and chromatin immunoprecipitation. We used primary liver cells and human liver buds for additional functional validation. RESULTS: Expression of HNF4A mRNA led to restoration of the metabolic activity of fibrotic primary murine and human hepatocytes in vitro. Repeated in vivo delivery of LNP-encapsulated HNF4A mRNA induced a robust inhibition of fibrogenesis in 4 independent mouse models of hepatotoxin- and cholestasis-induced liver fibrosis. Mechanistically, we discovered that paraoxonase 1 is a direct target of HNF4A and it contributes to HNF4A-mediated attenuation of liver fibrosis via modulation of liver macrophages and hepatic stellate cells. CONCLUSION: Collectively, our findings provide the first direct preclinical evidence of the applicability of HNF4A mRNA therapeutics for the treatment of fibrosis in the liver. LAY SUMMARY: Liver fibrosis and cirrhosis remain unmet medical needs and contribute to high mortality worldwide. Herein, we take advantage of a promising therapeutic approach to treat liver fibrosis and cirrhosis. We demonstrate that restoration of a key gene, HNF4A, via mRNA encapsulated in lipid nanoparticles decreased injury in multiple mouse models of fibrosis and cirrhosis. Our study provides proof-of-concept that mRNA therapy is a promising strategy for reversing liver fibrosis and cirrhosis.
Assuntos
Fator 4 Nuclear de Hepatócito/farmacologia , Cirrose Hepática/tratamento farmacológico , Animais , Modelos Animais de Doenças , Fator 4 Nuclear de Hepatócito/uso terapêutico , Camundongos , RNA Mensageiro/farmacologia , RNA Mensageiro/uso terapêuticoRESUMO
OBJECTIVE: Liver fibrosis and cirrhosis resulting from chronic liver injury represent a major healthcare burden worldwide. Growth differentiation factor (GDF) 11 has been recently investigated for its role in rejuvenation of ageing organs, but its role in chronic liver diseases has remained unknown. Here, we investigated the expression and function of GDF11 in liver fibrosis, a common feature of most chronic liver diseases. DESIGN: We analysed the expression of GDF11 in patients with liver fibrosis, in a mouse model of liver fibrosis and in hepatic stellate cells (HSCs) as well as in other liver cell types. The functional relevance of GDF11 in toxin-induced and cholestasis-induced mouse models of liver fibrosis was examined by in vivo modulation of Gdf11 expression using adeno-associated virus (AAV) vectors. The effect of GDF11 on leucine-rich repeat-containing G-protein-coupled receptor 5 (LGR5)+ liver progenitor cells was studied in mouse and human liver organoid culture. Furthermore, in vivo depletion of LGR5+ cells was induced by injecting AAV vectors expressing diptheria toxin A under the transcriptional control of Lgr5 promoter. RESULTS: We showed that the expression of GDF11 is upregulated in patients with liver fibrosis and in experimentally induced murine liver fibrosis models. Furthermore, we found that therapeutic application of GDF11 mounts a protective response against fibrosis by increasing the number of LGR5+ progenitor cells in the liver. CONCLUSION: Collectively, our findings uncover a protective role of GDF11 during liver fibrosis and suggest a potential application of GDF11 for the treatment of chronic liver disease.
Assuntos
Proteínas Morfogenéticas Ósseas/metabolismo , Fatores de Diferenciação de Crescimento/metabolismo , Cirrose Hepática/metabolismo , Fígado/metabolismo , Células-Tronco/metabolismo , Animais , Modelos Animais de Doenças , Imunofluorescência , Fluxo Gênico , Humanos , Hibridização In Situ , Fígado/citologia , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Regulação para CimaRESUMO
BACKGROUND & AIMS: Fibrosis, a cardinal feature of a dysfunctional liver, significantly contributes to the ever-increasing mortality due to end-stage chronic liver diseases. The crosstalk between hepatocytes and hepatic stellate cells (HSCs) plays a key role in the progression of fibrosis. Although ample efforts have been devoted to elucidate the functions of HSCs during liver fibrosis, the regulatory functions of hepatocytes remain elusive. METHODS: Using an unbiased functional microRNA (miRNA) screening, we investigated the ability of hepatocytes to regulate fibrosis by fine-tuning gene expression via miRNA modulation. The in vivo functional analyses were performed by inhibiting miRNA in hepatocytes using adeno-associated virus in carbon-tetrachloride- and 3,5-di-diethoxycarbonyl-1,4-dihydrocollidine-induced liver fibrosis. RESULTS: Blocking miRNA-221-3p function in hepatocytes during chronic liver injury facilitated recovery of the liver and faster resolution of the deposited extracellular matrix. Furthermore, we demonstrate that reduced secretion of C-C motif chemokine ligand 2, as a result of post-transcriptional regulation of GNAI2 (G protein alpha inhibiting activity polypeptide 2) by miRNA-221-3p, mitigates liver fibrosis. CONCLUSIONS: Collectively, miRNA modulation in hepatocytes, an easy-to-target cell type in the liver, may serve as a potential therapeutic approach for liver fibrosis. LAY SUMMARY: Liver fibrosis majorly contributes to mortality resulting from various liver diseases. We discovered a small RNA known as miRNA-221-3p, whose downregulation in hepatocytes results in reduced liver fibrosis. Thus, inhibition of miRNA-221-3p may serve as one of the therapeutic approaches for treatment of liver fibrosis.
Assuntos
Hepatócitos/metabolismo , Cirrose Hepática Experimental/metabolismo , MicroRNAs/genética , MicroRNAs/metabolismo , Animais , Tetracloreto de Carbono/farmacologia , Dependovirus/genética , Regulação para Baixo/genética , Matriz Extracelular/metabolismo , Feminino , Regulação da Expressão Gênica , Células HEK293 , Células Estreladas do Fígado/metabolismo , Humanos , Cirrose Hepática Experimental/induzido quimicamente , Cirrose Hepática Experimental/patologia , Camundongos , Camundongos Endogâmicos BALB C , TransfecçãoRESUMO
How MYC reprograms metabolism in primary tumors remains poorly understood. Using integrated gene expression and metabolite profiling, we identify six pathways that are coordinately deregulated in primary MYC-driven liver tumors: glutathione metabolism; glycine, serine, and threonine metabolism; aminoacyl-tRNA biosynthesis; cysteine and methionine metabolism; ABC transporters; and mineral absorption. We then focus our attention on glutathione (GSH) and glutathione disulfide (GSSG), as they are markedly decreased in MYC-driven tumors. We find that fewer glutamine-derived carbons are incorporated into GSH in tumor tissue relative to non-tumor tissue. Expression of GCLC, the rate-limiting enzyme of GSH synthesis, is attenuated by the MYC-induced microRNA miR-18a. Inhibition of miR-18a in vivo leads to increased GCLC protein expression and GSH abundance in tumor tissue. Finally, MYC-driven liver tumors exhibit increased sensitivity to acute oxidative stress. In summary, MYC-dependent attenuation of GCLC by miR-18a contributes to GSH depletion in vivo, and low GSH corresponds with increased sensitivity to oxidative stress in tumors. Our results identify new metabolic pathways deregulated in primary MYC tumors and implicate a role for MYC in regulating a major antioxidant pathway downstream of glutamine.
Assuntos
Glutamato-Cisteína Ligase/antagonistas & inibidores , Glutationa/metabolismo , Neoplasias Hepáticas/metabolismo , Proteínas Proto-Oncogênicas c-myc/metabolismo , Animais , Linhagem Celular Tumoral , Análise por Conglomerados , Modelos Animais de Doenças , Perfilação da Expressão Gênica , Regulação Neoplásica da Expressão Gênica , Glutamato-Cisteína Ligase/genética , Glutamato-Cisteína Ligase/metabolismo , Glutamina/metabolismo , Humanos , Neoplasias Hepáticas/genética , Redes e Vias Metabólicas/genética , Metaboloma , Metabolômica/métodos , Camundongos , Camundongos Transgênicos , MicroRNAs/genética , Estresse Oxidativo , Proteínas Proto-Oncogênicas c-myc/genética , Interferência de RNARESUMO
Conjugates based on nanostructured, superparamagnetic particles, a thermolabile linker and a cytotoxic maytansinoid were developed to serve as a model for tumour-selective drug delivery and release. It combines chemo- with thermal therapy. The linker-modified toxin was prepared by a combination of biotechnology and semisynthesis. Drug release was achieved by hyperthermia through an external oscillating electromagnetic field that induces heat inside the particles. Efficacy of this release concept was demonstrated both for cancer cell proliferation in vitro, and for tumour growth in vivo, in a xenograft mouse model. Biocompatibility studies for these magnetic-nanoparticle/ansamitocin conjugates complement this work.
Assuntos
Portadores de Fármacos/química , Nanopartículas de Magnetita/química , Maitansina/análogos & derivados , Animais , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Reação de Cicloadição , Liberação Controlada de Fármacos , Humanos , Hipertermia Induzida , Antígeno Ki-67/metabolismo , Espectroscopia de Ressonância Magnética , Maitansina/química , Maitansina/uso terapêutico , Maitansina/toxicidade , Camundongos , Camundongos Nus , Neoplasias/tratamento farmacológico , Neoplasias/patologia , Transplante HeterólogoRESUMO
A combination of mutasynthesis using a mutant strain of A. pretiosum blocked in the biosynthesis of amino-hydroxybenzoic acid (AHBA) and semisynthesis relying on a Stille cross-coupling step provided access to new ansamitocin derivatives of which one was attached by a thermolabile linker to nanostructured iron oxide particles. When exposed to an oscillating electromagnetic field the resulting iron oxide/ansamitocin conjugate 19 heats up in an aqueous suspension and the ansamitocin derivative 16 is released by means of a retro-Diels-Alder reaction. It exerts strong antiproliferative activity (IC50 =4.8â ng mg-1 ) in mouse fibroblasts. These new types of conjugates have the potential for combating cancer through hyperthermia and chemotherapy using an electromagnetic external trigger.
Assuntos
Compostos Férricos/química , Nanopartículas de Magnetita/química , Maitansina/análogos & derivados , Animais , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Reação de Cicloadição , Hidroxibenzoatos/síntese química , Hidroxibenzoatos/química , Hidroxibenzoatos/toxicidade , Nanopartículas de Magnetita/toxicidade , Maitansina/química , CamundongosRESUMO
BACKGROUND & AIMS: Current hepatic differentiation protocols for human embryonic stem cells (ESCs) require substantial improvements. MicroRNAs (miRNAs) have been reported to regulate hepatocyte cell fate during liver development, but their utility to improve hepatocyte differentiation from ESCs remains to be investigated. Therefore, our aim was to identify and to analyse hepatogenic miRNAs for their potential to improve hepatocyte differentiation from ESCs. METHODS: By miRNA profiling and in vitro screening, we identified miR-199a-5p among several potential hepatogenic miRNAs. Transplantation studies of miR-199a-5p-inhibited hepatocyte-like cells (HLCs) in the liver of immunodeficient fumarylacetoacetate hydrolase knockout mice (Fah(-/-)/Rag2(-/-)/Il2rg(-/-)) were performed to assess their in vivo liver repopulation potential. For target determination, western blot and luciferase reporter assay were carried out. RESULTS: miRNA profiling revealed 20 conserved candidate hepatogenic miRNAs. By miRNA screening, only miR-199a-5p inhibition in HLCs was found to be able to enhance the in vitro hepatic differentiation of mouse as well as human ESCs. miR-199a-5p inhibition in human ESCs-derived HLCs enhanced their engraftment and repopulation capacity in the liver of Fah(-/-)/Rag2(-/-)/Il2rg(-/-) mice. Furthermore, we identified SMARCA4 and MST1 as novel targets of miR-199a-5p that may contribute to the improved hepatocyte generation and in vivo liver repopulation. CONCLUSIONS: Our findings demonstrate that miR-199a-5p inhibition in ES-derived HLCs leads to improved hepatocyte differentiation. Upon transplantation, HLCs were able to engraft and repopulate the liver of Fah(-/-)/Rag2(-/-)/Il2rg(-/-) mice. Thus, our findings suggest that miRNA modulation may serve as a promising approach to generate more mature HLCs from stem cell sources for the treatment of liver diseases.
Assuntos
Regulação da Expressão Gênica , Hepatócitos/metabolismo , Células-Tronco Embrionárias Humanas/metabolismo , Transplante de Fígado , MicroRNAs/genética , RNA/genética , Animais , Western Blotting , Diferenciação Celular , Células Cultivadas , Hepatócitos/citologia , Células-Tronco Embrionárias Humanas/citologia , Humanos , Camundongos , Camundongos Knockout , MicroRNAs/antagonistas & inibidores , MicroRNAs/biossíntese , Reação em Cadeia da Polimerase em Tempo RealRESUMO
UNLABELLED: Hepatocellular carcinoma (HCC) is associated with poor survival for patients and few effective treatment options, raising the need for novel therapeutic strategies. MicroRNAs (miRNAs) play important roles in tumor development and show deregulated patterns of expression in HCC. Because of the liver's unique affinity for small nucleic acids, miRNA-based therapy has been proposed in the treatment of liver disease. Thus, there is an urgent need to identify and characterize aberrantly expressed miRNAs in HCC. In our study, we profiled miRNA expression changes in de novo liver tumors driven by MYC and/or RAS, two canonical oncogenes activated in a majority of human HCCs. We identified an up-regulated miRNA megacluster comprised of 53 miRNAs on mouse chromosome 12qF1 (human homolog 14q32). This miRNA megacluster is up-regulated in all three transgenic liver models and in a subset of human HCCs. An unbiased functional analysis of all miRNAs within this cluster was performed. We found that miR-494 is overexpressed in human HCC and aids in transformation by regulating the G1 /S cell cycle transition through targeting of the Mutated in Colorectal Cancer tumor suppressor. miR-494 inhibition in human HCC cell lines decreases cellular transformation, and anti-miR-494 treatment of primary MYC-driven liver tumor formation significantly diminishes tumor size. CONCLUSION: Our findings identify a new therapeutic target (miR-494) for the treatment of HCC.
Assuntos
Carcinoma Hepatocelular/metabolismo , Neoplasias Hepáticas Experimentais/metabolismo , MicroRNAs/metabolismo , Proteínas Supressoras de Tumor/metabolismo , Animais , Proliferação de Células , Transformação Celular Neoplásica , Feminino , Pontos de Checagem da Fase G1 do Ciclo Celular , Regulação Neoplásica da Expressão Gênica , Humanos , Masculino , Camundongos , Camundongos Transgênicos , MicroRNAs/antagonistas & inibidores , Proteínas Proto-Oncogênicas c-myc/metabolismo , Regulação para Cima , Proteínas ras/metabolismoRESUMO
UNLABELLED: The tightly controlled replication of hepatocytes in liver regeneration and uncontrolled proliferation of tumor cells in hepatocellular carcinoma (HCC) are often modulated by common regulatory pathways. Several microRNAs (miRNAs) are involved in HCC progression by modulating posttranscriptional expression of multiple target genes. miR-221, which is frequently up-regulated in HCCs, delays fulminant liver failure in mice by inhibiting apoptosis, indicating a pleiotropic role of miR-221 in hepatocytes. Here, we hypothesize that modulation of miR-221 targets in primary hepatocytes enhances proliferation, providing novel clues for enhanced liver regeneration. We demonstrate that miR-221 enhances proliferation of in vitro cultivated primary hepatocytes. Furthermore, applying two-thirds partial hepatectomy as a surgically induced liver regeneration model we show that adeno-associated virus-mediated overexpression of miR-221 in the mouse liver also accelerates hepatocyte proliferation in vivo. miR-221 overexpression leads to rapid S-phase entry of hepatocytes during liver regeneration. In addition to the known targets p27 and p57, we identify Aryl hydrocarbon nuclear translocator (Arnt) messenger RNA (mRNA) as a novel target of miR-221, which contributes to the pro-proliferative activity of miR-221. CONCLUSION: miR-221 overexpression accelerates hepatocyte proliferation. Pharmacological intervention targeting miR-221 may thus be therapeutically beneficial in liver failure by preventing apoptosis and by inducing liver regeneration.
Assuntos
Hepatócitos/fisiologia , Regeneração Hepática , MicroRNAs/metabolismo , Animais , Translocador Nuclear Receptor Aril Hidrocarboneto/metabolismo , Proliferação de Células , Hepatectomia , CamundongosRESUMO
Hepatic stellate cells (HSCs) and their activated derivatives, often referred to as myofibroblasts (MFs), play a key role in progression of chronic liver injuries leading to fibrosis, cirrhosis, and hepatocellular carcinoma. Until recently, MFs were considered a homogenous cell type majorly due to lack of techniques that allow complex molecular studies at a single-cell resolution. Recent technical advancements in genetic lineage-tracing models as well as the exponential growth of studies with single-cell transcriptome and proteome analyses have uncovered hidden heterogeneities among the HSC and MF populations in healthy states as well as chronic liver injuries at the various stages of tissue deformation. The identification of different phenotypes along the HSC/MF axis, which either maintain essential liver functions ("good" HSCs), emerge during fibrosis ("bad" HSCs), or even promote hepatocellular carcinoma ("ugly" HSCs), may lay the foundation for targeting a particular MF phenotype as potential treatment for chronic liver injuries.
Assuntos
Carcinoma Hepatocelular , Neoplasias Hepáticas , Humanos , Carcinoma Hepatocelular/patologia , Células Estreladas do Fígado/patologia , Cirrose Hepática/patologia , Fenótipo , Neoplasias Hepáticas/patologiaRESUMO
Hepatocellular carcinoma is a primary liver cancer, characterised by diverse etiology, late diagnoses, and poor prognosis. Hepatocellular carcinoma is mostly resistant to current treatment options, therefore, identification of more effective druggable therapeutic targets is needed. We found microRNA miR-20a-5p is upregulated during mouse liver tumor progression and in human hepatocellular carcinoma patients. In this study, we elucidated the therapeutic potential of targeting oncogenic miR-20a-5p, in vivo, in a xenograft model and in two transgenic hepatocellular carcinoma mouse models via adeno-associated virus-mediated miR-20a-Tough-Decoy treatment. In vivo knockdown of miR-20a-5p attenuates tumor burden and prolongs survival in the two independent hepatocellular carcinoma mouse models. We identified and validated cytochrome c as a novel target of miR-20a-5p. Cytochrome c plays a key role in initiation of the apoptotic cascade and in the electron transport chain. We show for the first time, that miR-20a modulation affects both these key functions of cytochrome c during HCC development. Our study thus demonstrates the promising 'two birds with one stone' approach of therapeutic in vivo targeting of an oncogenic miRNA, whereby more than one key deregulated cellular process is affected, and unequivocally leads to more effective attenuation of HCC progression and significantly longer overall survival.
Assuntos
Apoptose , Carcinoma Hepatocelular , Citocromos c , Neoplasias Hepáticas , MicroRNAs , Animais , Humanos , Camundongos , Apoptose/genética , Carcinoma Hepatocelular/genética , Carcinoma Hepatocelular/patologia , Carcinoma Hepatocelular/metabolismo , Linhagem Celular Tumoral , Citocromos c/metabolismo , Regulação Neoplásica da Expressão Gênica , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/patologia , Neoplasias Hepáticas/metabolismo , Camundongos Nus , MicroRNAs/metabolismo , MicroRNAs/genéticaRESUMO
In this issue, Wang et al.1 provide evidence of the pre-clinical as well as the clinical utility of in vitro-generated directly reprogrammed human hepatocytes in bioartificial liver. This approach will help offer patients a more curative surgical therapy for liver cancer and improve survival rates.
Assuntos
Neoplasias Hepáticas , Fígado Artificial , Humanos , Hepatócitos , FígadoRESUMO
Although therapeutic options are gradually improving, the overall prognosis for patients with hepatocellular carcinoma (HCC) is still poor. Gene therapy-based strategies are developed to complement the therapeutic armamentarium, both in early and late-stage disease. For efficient delivery of transgenes with antitumor activity, vectors demonstrating preferred tumor tropism are required. Here, we report on the natural tropism of adeno-associated virus (AAV) serotype 2 vectors for HCC. When applied intravenously in transgenic HCC mouse models, similar amounts of vectors were detected in the liver and liver tumor tissue. In contrast, transduction efficiency, as indicated by the level of transgene product, was moderate in the liver but was elevated up to 19-fold in mouse tumor tissue. Preferred transduction of HCC compared to hepatocytes was confirmed in precision-cut liver slices from human patient samples. Our mechanistic studies revealed that this preference is due to the improved intracellular processing of AAV2 vectors in HCC, resulting, for example, in nearly 4-fold more AAV vector episomes that serve as templates for gene transcription. Given this background, AAV2 vectors ought to be considered to strengthen current-or develop novel-strategies for treating HCC.
RESUMO
High polarization of nuclear spins in liquid state through hyperpolarized technology utilizing dynamic nuclear polarization has enabled the direct monitoring of (13)C metabolites in vivo at a high signal-to-noise ratio. Acquisition time limitations due to T(1) decay of the hyperpolarized signal require accelerated imaging methods, such as compressed sensing, for optimal speed and spatial coverage. In this paper, the design and testing of a new echo-planar (13)C three-dimensional magnetic resonance spectroscopic imaging (MRSI) compressed sensing sequence is presented. The sequence provides up to a factor of 7.53 in acceleration with minimal reconstruction artifacts. The key to the design is employing x and y gradient blips during a fly-back readout to pseudorandomly undersample k(f)-k(x)-k(y) space. The design was validated in simulations and phantom experiments where the limits of undersampling and the effects of noise on the compressed sensing nonlinear reconstruction were tested. Finally, this new pulse sequence was applied in vivo in preclinical studies involving transgenic prostate cancer and transgenic liver cancer murine models to obtain much higher spatial and temporal resolution than possible with conventional echo-planar spectroscopic imaging methods.
Assuntos
Biomarcadores Tumorais/análise , Modelos Animais de Doenças , Neoplasias Hepáticas/diagnóstico , Neoplasias Hepáticas/metabolismo , Imageamento por Ressonância Magnética/métodos , Espectroscopia de Ressonância Magnética/métodos , Neoplasias da Próstata/diagnóstico , Neoplasias da Próstata/metabolismo , Animais , Isótopos de Carbono/análise , Humanos , Masculino , Camundongos , Camundongos TransgênicosRESUMO
The last decade has witnessed significant advancements in our understanding of how small noncoding RNAs, such as microRNAs (miRNAs), regulate disease progression. One such miRNA, miR-221, has been shown to play a key role in the progression of liver fibrosis, a common feature of most liver diseases. Many reports have demonstrated the upregulation of miR-221 in liver fibrosis caused by multiple etiologies such as viral infections and nonalcoholic steatohepatitis. Inhibition of miR-221 via different strategies has shown promising results in terms of the suppression of fibrogenic gene signatures in vitro, as well as in vivo, in independent mouse models of liver fibrosis. In addition, miR-221 has also been suggested as a noninvasive serum biomarker for liver fibrosis and cirrhosis. In this review, we discuss the biology of miR-221, its significance and use as a biomarker during progression of liver fibrosis, and finally, potential and robust approaches that can be utilized to suppress liver fibrosis via inhibition of miR-221.
Assuntos
Biomarcadores/sangue , Cirrose Hepática/genética , Fígado/lesões , MicroRNAs/metabolismo , Hepatopatia Gordurosa não Alcoólica/genética , Animais , Doença Crônica , Modelos Animais de Doenças , Progressão da Doença , Humanos , Cirrose Hepática/metabolismo , Hepatopatia Gordurosa não Alcoólica/metabolismoRESUMO
The ability of the liver to regenerate and restore mass limits the increasing mortality rate due to life-threatening liver diseases. Successful liver regeneration is accomplished in multiple stages, of which the priming and proliferation phases are well studied. However, the regulatory pathways, specifically microRNA (miRNA)-mediated posttranscriptional regulation, which prevent uncontrolled proliferation and mediate the termination of liver regeneration, are not well understood. We identified differentially regulated miRNAs during the termination phase after 2/3 partial hepatectomy (PH) in mice, which is a well-established mouse model of liver regeneration. We further evaluated the function of differentially regulated miRNAs in primary mouse hepatocytes by using mimics and inhibitors and in vivo by using adeno-associated virus (AAV) serotype 8. A candidate miRNA target was identified by messenger RNA array in silico analyses and validated in primary mouse and human hepatocytes. Using miRNA profiling, we discovered miR-125b-5p as a novel regulator of hepatocyte proliferation in the late phase of liver regeneration. AAV-mediated miR-125b-5p delivery in mice enhanced the endogenous regenerative capacity and resulted in improved restoration of liver mass after 2/3 PH. Further, we found that ankyrin repeat and BTB/POZ domain containing protein 1 (Abtb1) is a direct target of miR-125b-5p in primary mouse and human hepatocytes and contributes to the pro-proliferative activity of miR-125b-5p by forkhead box G1 (FOXG1) and the cyclin-dependent kinase inhibitor 1A (p21) pathway. Conclusion: miR-125b-5p has an important role in regulating hepatocyte proliferation in the termination phase of liver regeneration and may serve as a potential therapeutic target in various liver diseases that often exhibit deregulated hepatocyte proliferation.
RESUMO
Adeno-associated virus (AAV)-based vectors are considered efficient and safe gene delivery systems in gene therapy. We combined two guide RNA genes, Cas9, and a self-linearizing repair template in one vector (AIO-SL) to correct fumarylacetoacetate hydrolase (FAH) deficiency in mice. The vector genome of 5.73 kb was packaged into VP2-depleted AAV particles (AAV2/8ΔVP2), which, however, did not improve cargo capacity. Reprogrammed hepatocytes were treated with AIO-SL.AAV2ΔVP2 and subsequently transplanted, resulting in large clusters of FAH-positive hepatocytes. Direct injection of AIO-SL.AAV8ΔVP2 likewise led to FAH expression and long-term survival. The AIO-SL vector achieved an â¼6-fold higher degree of template integration than vectors without template self-linearization. Subsequent analysis revealed that AAV8 particles, in contrast to AAV2, incorporate oversized genomes distinctly greater than 5.2 kb. Finally, our AAV8-based vector represents a promising tool for gene editing strategies to correct monogenic liver diseases requiring (large) fragment removal and/or simultaneous sequence replacement.