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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.
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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
The intracellular delivery of messenger (m)RNA holds great potential for the discovery and development of vaccines and therapeutics. Yet, in many applications, a major obstacle to clinical translation of mRNA therapy is the lack of efficient strategy to precisely deliver RNA sequence to liver tissues and cells. In this study, we synthesized virus-like mesoporous silica (V-SiO2) nanoparticles for effectively deliver the therapeutic RNA. Then, the cationic polymer polyethylenimine (PEI) was included for the further silica surface modification (V-SiO2-P). Negatively charged mRNA motifs were successfully linked on the surface of V-SiO2 through electrostatic interactions with PEI (m@V-SiO2-P). Finally, the supported lipid bilayer (LB) was completely wrapped on the bionic inspired surface of the nanoparticles (m@V-SiO2-P/LB). Importantly, we found that, compared with traditional liposomes with mRNA loading (m@LNPs), the V-SiO2-P/LB bionic-like morphology effectively enhanced mRNA delivery effect to hepatocytes both in vitro and in vivo, and PEI modification concurrently promoted mRNA binding and intracellular lysosomal escape. Furthermore, m@V-SiO2-P increased the blood circulation time (t1/2 = 7 h) to be much longer than that of the m@LNPs (4.2 h). Understanding intracellular delivery mediated by the V-SiO2-P/LB nanosystem will inspire the next-generation of highly efficient and effective mRNA therapies. In addition, the nanosystem can also be applied to the oral cavity, forehead, face and other orthotopic injections.
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Circular RNAs (circRNAs) have been shown to have a vital effect on hepatoma progression. The purpose of this study was to explore the function and mechanism of circRNA testis expressed 2 (circ_TEX2, circ_0004913) in hepatoma pathogenesis. Real-time quantitative polymerase chain reaction (RT-qPCR) was used to detect circ_TEX2, miR-96-5p, and sprouty-related EVH1 domain containing 1 (SPRED1) expression. Western blot analyzed the proliferating cell nuclear antigen (PCNA), SPRED1, and the apoptosis-related protein levels. 3-(4, 5-Dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT), 5-ethynyl-2'-deoxyuridine (EdU), and colony formation assays were used to test cell proliferation. Cell migration and invasion were analyzed by transwell assay, and cell apoptosis was detected by flow cytometry. Dual-luciferase reporter assay was done to analyze the target relationship between miR-96-5p and circ_TEX2 or SPRED1. The effects of circ_TEX2 on tumor growth in vivo were verified by xenograft model experiment and immunohistochemistry assay. The levels of circ_TEX2 and SPRED1 were down-regulated in hepatoma tissues and cells, and miR-96-5p expression was up-regulated. Overexpression of circ_TEX2 could inhibit the proliferation, migration, and invasion and boost cell apoptosis of hepatoma cells. Circ_TEX2 affected SPRED1 expression by sponging miR-96-5p. The overexpression of miR-96-5p could overturn the influence of circ_TEX2 up-regulation on malignant behaviors of hepatoma cells, and reduced SPRED1 expression could reverse the function of miR-96-5p knockdown on hepatoma cell malignant behaviors. Circ_TEX2 could suppress the growth of xenograft tumors in vivo. Our study demonstrates the tumor-suppressive role of circ_TEX2 in hepatoma through miR-96-5p/SPRED1 axis, suggesting that strategies directed toward restoring the production of circ_TEX2 might have a therapeutic value for hepatoma treatment.
Assuntos
Carcinoma Hepatocelular , Neoplasias Hepáticas , MicroRNAs , Masculino , Humanos , Carcinoma Hepatocelular/genética , Neoplasias Hepáticas/genética , Apoptose/genética , Bioensaio , Proliferação de Células , MicroRNAs/genética , Proteínas Adaptadoras de Transdução de SinalRESUMO
BACKGROUND AND AIMS: Current liver-directed gene therapies look for adeno-associated virus (AAV) vectors with improved efficacy. With this background, capsid engineering is explored. Whereas shuffled capsid library screenings have resulted in potent liver targeting variants with one first vector in human clinical trials, modifying natural serotypes by peptide insertion has so far been less successful. Here, we now report on two capsid variants, MLIV.K and MLIV.A, both derived from a high-throughput in vivo AAV peptide display selection screen in mice. APPROACH AND RESULTS: The variants transduce primary murine and human hepatocytes at comparable efficiencies, a valuable feature in clinical development, and show significantly improved liver transduction efficacy, thereby allowing a dose reduction, and outperform parental AAV2 and AAV8 in targeting human hepatocytes in humanized mice. The natural heparan sulfate proteoglycan binding ability is markedly reduced, a feature that correlates with improved hepatocyte transduction. A further property that might contribute to the improved transduction efficacy is the lower capsid melting temperature. Peptide insertion also caused a moderate change in sensitivity to human sera containing anti-AAV2 neutralizing antibodies, revealing the impact of epitopes located at the basis of the AAV capsid protrusions. CONCLUSIONS: In conclusion, MLIV.K and MLIV.A are AAV peptide display variants selected in immunocompetent mice with improved hepatocyte tropism and transduction efficiency. Because these features are maintained across species, MLIV variants provide remarkable potential for translation of therapeutic approaches from mice to men.
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Capsídeo , Dependovirus , Animais , Camundongos , Humanos , Capsídeo/química , Capsídeo/metabolismo , Sorogrupo , Dependovirus/genética , Transdução Genética , Vetores Genéticos , Fígado/metabolismo , Peptídeos/análise , Peptídeos/genética , Peptídeos/metabolismo , Terapia Genética/métodosRESUMO
Hemochromatosis is one of the most common inherited metabolic diseases among white populations and predominantly originates from a homozygous C282Y mutation in the HFE gene. The G > A transition at position c.845 of the gene causes misfolding of the HFE protein, ultimately resulting in its absence at the cell membrane. Consequently, the lack of interaction with the transferrin receptors 1 and 2 leads to systemic iron overload. We screened potential gRNAs in a highly precise cell culture assay and applied an AAV8 split-vector expressing the adenine base editor ABE7.10 and our candidate gRNA in 129-Hfetm.1.1Nca mice. Here we show that a single injection of our therapeutic vector leads to a gene correction rate of >10% and improved iron metabolism in the liver. Our study presents a proof-of-concept for a targeted gene correction therapy for one of the most frequent hereditary diseases affecting humans.
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Adenina , Proteína da Hemocromatose , Hemocromatose , Adenina/metabolismo , Animais , Ferritinas/genética , Hemocromatose/genética , Hemocromatose/metabolismo , Hemocromatose/terapia , Proteína da Hemocromatose/genética , Proteína da Hemocromatose/metabolismo , Antígenos de Histocompatibilidade Classe I/metabolismo , Homozigoto , Ferro/metabolismo , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Camundongos , Mutação , Transferrina/metabolismoRESUMO
Family with sequence similarity 60A (FAM60A) has been reported as a new cancer-related protein that affects the malignant progression of some cancers. However, whether FAM60A plays a part in pancreatic carcinoma is undetermined. This work was designed to examine the impact of FAM60A in pancreatic carcinoma. Abundant expression of FAM60A was observed in the primary tumor tissue of pancreatic carcinoma. Moreover, a high FAM60A level was related to a poor overall survival in pancreatic carcinoma patients. Malignant behaviors of pancreatic carcinoma cells, such as proliferation and invasiveness, were markedly affected by FAM60A depletion. In addition, FAM60A depletion enhanced the drug sensitivity of pancreatic carcinoma cells to gemcitabine. Further study revealed that FAM60A depletion impaired the activities of Akt and ß-catenin. Inhibiting the activity of Akt abolished FAM60A-mediated ß-catenin activation. Re-expression of ß-catenin partially diminished the FAM60A-depletion-mediated cancer suppressive effect in pancreatic carcinoma cells. In vivo experiments demonstrated that FAM60A depletion prohibited the xenograft formation of pancreatic carcinoma cells, with concurrent reductions of Akt and ß-catenin activities. Collectively, our findings indicate that FAM60A exerts a cancer-promoting role in pancreatic carcinoma through affection of the Akt/ß-catenin pathway. This work indicates that FAM60A acts as a tumor promoter in pancreatic carcinoma and can be utilized as a potential target for anti-pancreatic carcinoma therapy development.
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Proteínas de Ligação a DNA/metabolismo , Neoplasias Pancreáticas , beta Catenina , Linhagem Celular Tumoral , Proliferação de Células , Glicogênio Sintase Quinase 3 beta/metabolismo , Humanos , Proteínas Proto-Oncogênicas c-akt/metabolismo , Transdução de Sinais , beta Catenina/metabolismo , Neoplasias PancreáticasRESUMO
BACKGROUND: Perineural invasion (PNI) and autophagy are two common features in the tumor microenvironment of pancreatic cancer (PanCa) and have a negative effect on prognosis. Potential mediator cells and the molecular mechanism underlying their relationships need to be fully elucidated. METHODS: To investigate the autophagy of Schwann cells (SCs) in PNI, we reproduced the microenvironment of PNI by collecting clinical PNI tissue, performing sciatic nerve injection of nude mice with cancer cells and establishing a Dorsal root ganglion (DRG) coculture system with cancer cell lines. Autophagy was detected by IHC, IF, transmission electron microscopy (TEM) and western blotting assays. Apoptosis was detected by IF, TEM and western blotting. NGF targeting molecular RO 08-2750(RO) and the autophagy inhibitor Chloroquine (CQ) were utilized to evaluate the effect on autophagy and apoptosis in SCs and PanCa cells in PNI samples. RESULTS: SC autophagy is activated in PNI by paracrine NGF from PanCa cells. Autophagy-activated Schwann cells promote PNI through a) enhanced migration and axon guidance toward PanCa cells and b) increased chemoattraction to PanCa cells. The NGF-targeting reagent RO and autophagy inhibitor CQ inhibited Schwann cell autophagic flux and induced Schwann cell apoptosis. Moreover, RO and CQ could induce PanCa cell apoptosis and showed good therapeutic effects in the PNI model. CONCLUSIONS: PanCa cells can induce autophagy in SCs through paracrine pathways such as the NGF/ATG7 pathway. Autophagic SCs exert a "nerve-repair like effect", induce a high level of autophagy of cancer cells, provide a "beacon" for the invasion of cancer cells to nerve fibers, and induce directional growth of cancer cells. Targeting NGF and autophagy for PNI treatment can block nerve infiltration and is expected to provide new directions and an experimental basis for the research and treatment of nerve infiltration in pancreatic cancer.
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Adenocarcinoma/patologia , Carcinoma Ductal Pancreático/patologia , Fator de Crescimento Neural/metabolismo , Comunicação Parácrina/fisiologia , Células de Schwann/metabolismo , Animais , Autofagia , Modelos Animais de Doenças , Feminino , Humanos , Camundongos , Invasividade Neoplásica/patologia , Ratos , Transfecção , Microambiente TumoralRESUMO
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.
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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
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
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.
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Hepatitis C virus (HCV) has no animal reservoir, infecting only humans. To investigate species barrier determinants limiting infection of rodents, murine liver complementary DNA library screening was performed, identifying transmembrane proteins Cd302 and Cr1l as potent restrictors of HCV propagation. Combined ectopic expression in human hepatoma cells impeded HCV uptake and cooperatively mediated transcriptional dysregulation of a noncanonical program of immunity genes. Murine hepatocyte expression of both factors was constitutive and not interferon inducible, while differences in liver expression and the ability to restrict HCV were observed between the murine orthologs and their human counterparts. Genetic ablation of endogenous Cd302 expression in human HCV entry factor transgenic mice increased hepatocyte permissiveness for an adapted HCV strain and dysregulated expression of metabolic process and host defense genes. These findings highlight human-mouse differences in liver-intrinsic antiviral immunity and facilitate the development of next-generation murine models for preclinical testing of HCV vaccine candidates.
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Hepacivirus , Hepatite C , Animais , Hepacivirus/genética , Camundongos , Camundongos Transgênicos , Internalização do VírusRESUMO
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.
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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
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.
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LASP2 (LIM and SH3 protein 2), a member of the LIM-protein subfamily of the nebulin group, was first identified as a splice variant of the nebulin gene. In the past, investigators mainly focused on the impact of LASP2 on cardiac diseases because of its identification in the myocardium. Recently, several studies have reported that LASP2 is associated with the progression of various cancers. However, there have been no investigations on the expression and function of LASP2 in pancreatic cancer (PC). In this study, we performed the quantitative real-time polymerase chain reaction and Western blot analysis to detect the expression of LASP2 in PC tissues and cell lines. PC cells were transfected with LASP2 overexpression plasmid or the negative control in the presence or absence of tumor growth factor-ß (TGF-ß). The transwell assays were used to measure the effects of LASP2 on PC cell migration and invasion. The protein expression of epithelial-mesenchymal transition (EMT) markers was detected using Western blot assay. Our results demonstrated that LASP2 was downregulated in PC tissues and cell lines. In addition, upregulation of LASP2 inhibited the PC cell migration and invasion. We also found that LASP2 upregulation reversed TGF-ß-induced EMT in PC cells. Taken together, we provided novel evidence supporting the tumor-suppressor role of LASP2 in PC and suggested it as a potential therapeutic target in PC treatment.
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Proteínas de Transporte/biossíntese , Movimento Celular , Proteínas do Citoesqueleto/biossíntese , Transição Epitelial-Mesenquimal , Regulação Neoplásica da Expressão Gênica , Proteínas com Domínio LIM/biossíntese , Proteínas de Neoplasias/metabolismo , Neoplasias Pancreáticas/metabolismo , Fator de Crescimento Transformador beta/metabolismo , Regulação para Cima , Proteínas de Transporte/genética , Linhagem Celular Tumoral , Proteínas do Citoesqueleto/genética , Humanos , Proteínas com Domínio LIM/genética , Invasividade Neoplásica , Proteínas de Neoplasias/genética , Neoplasias Pancreáticas/genética , Neoplasias Pancreáticas/patologia , Fator de Crescimento Transformador beta/genéticaRESUMO
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.
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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
In vivo conversion of fibroblasts into hepatocyte-like cells provides one potential approach for the treatment of liver fibrosis. In our previous study, we showed in vivo conversion of myofibroblasts into induced hepatocytes (iHeps) by forced expression of four transcription factors in genetic fate-tracing mouse model of chronic liver disease. These in vivo-generated iHeps showed similar expression profile with endogenous hepatocytes (eHeps) and also exhibited similar functional characteristics, such as albumin secretion, urea synthesis, cytochrome activity, and drug responsiveness. Furthermore, the targeted expression of our reprogramming factors in myofibroblasts attenuated liver fibrosis. Our study suggests that in vivo reprogramming may open new perspectives for the treatment of diseases such as liver fibrosis.
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Dependovirus/genética , Fibroblastos/citologia , Hepatócitos/citologia , Cirrose Hepática/terapia , Fatores de Transcrição/genética , Animais , Tetracloreto de Carbono/efeitos adversos , Linhagem da Célula , Reprogramação Celular , Modelos Animais de Doenças , Fibroblastos/metabolismo , Fator de Transcrição GATA4/genética , Vetores Genéticos/administração & dosagem , Fator 1-alfa Nuclear de Hepatócito/genética , Fator 3-gama Nuclear de Hepatócito/genética , Fator 4 Nuclear de Hepatócito/genética , Hepatócitos/metabolismo , Humanos , Cirrose Hepática/induzido quimicamente , Cirrose Hepática/genética , Cirrose Hepática/metabolismo , Camundongos , Piridinas/efeitos adversosRESUMO
During Coxsackievirus B3 (CVB3) infection hepatitis is a potentially life threatening complication, particularly in newborns. Studies with type I interferon (IFN-I) receptor (IFNAR)-deficient mice revealed a key role of the IFN-I axis in the protection against CVB3 infection, whereas the source of IFN-I and cell types that have to be IFNAR triggered in order to promote survival are still unknown. We found that CVB3 infected IFN-ß reporter mice showed effective reporter induction, especially in hepatocytes and only to a minor extent in liver-resident macrophages. Accordingly, upon in vitro CVB3 infection of primary hepatocytes from murine or human origin abundant IFN-ß responses were induced. To identify sites of IFNAR-triggering we performed experiments with Mx reporter mice, which upon CVB3 infection showed massive luciferase induction in the liver. Immunohistological studies revealed that during CVB3 infection MX1 expression of hepatocytes was induced primarily by IFNAR-, and not by IFN-III receptor (IFNLR)-triggering. CVB3 infection studies with primary human hepatocytes, in which either the IFN-I or the IFN-III axis was inhibited, also indicated that primarily IFNAR-, and to a lesser extent IFNLR-triggering was needed for ISG induction. Interestingly, CVB3 infected mice with a hepatocyte-specific IFNAR ablation showed severe liver cell necrosis and ubiquitous viral dissemination that resulted in lethal disease, as similarly detected in classical IFNAR-/- mice. In conclusion, we found that during CVB3 infection hepatocytes are major IFN-I producers and that the liver is also the organ that shows strong IFNAR-triggering. Importantly, hepatocytes need to be IFNAR-triggered in order to prevent virus dissemination and to assure survival. These data are compatible with the hypothesis that during CVB3 infection hepatocytes serve as important IFN-I producers and sensors not only in the murine, but also in the human system.
Assuntos
Infecções por Coxsackievirus , Enterovirus Humano B/imunologia , Hepatócitos/metabolismo , Interferon beta/genética , Fígado/patologia , Receptor de Interferon alfa e beta/metabolismo , Animais , Células Cultivadas , Chlorocebus aethiops , Infecções por Coxsackievirus/complicações , Infecções por Coxsackievirus/genética , Infecções por Coxsackievirus/imunologia , Infecções por Coxsackievirus/virologia , Enterovirus Humano B/crescimento & desenvolvimento , Humanos , Interferon beta/metabolismo , Fígado/virologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Necrose/virologia , Receptor de Interferon alfa e beta/genética , Transdução de Sinais/genética , Transdução de Sinais/imunologia , Células Vero , Carga Viral/genética , Carga Viral/imunologiaRESUMO
BACKGROUND/AIMS: It has been described that cells in culture with very low oxidative metabolism possess a low CO2 membrane permeability, PCO2, of â¼ 0.01 cm/s. On the other hand, cardiomyocytes and mitochondria with extremely high rates of O2 consumption exhibit very high CO2 membrane permeabilities of 0.1 and 0.3 cm/s, repectively. To ascertain that this represents a systematic relationship, we determine here PCO2 of hepatocytes, which exhibit an intermediate rate of O2 consumption. METHODS: We isolated intact hepatocytes with vitalities of â¼ 70% from rat liver and measured their CO2 permeability by the previously published mass spectrometric 18O exchange technique. RESULTS: We find a PCO2 of hepatocytes of 0.03 cm/s in the presence of FC5-208A and verapamil. FC5-208A was necessary to inhibt extracellular carbonic anhydrase, and verapamil was necessary to inhibit intracellular uptake of FC5-208A by the organic cation transporter OCT1 of hepatocytes. CONCLUSION: Rat hepatocytes with their intermediate rate of oxygen consumption also possess an intermediate CO2 permeability. From pairs of data for five types of cells/organelles, we find an excellent positive linear correlation between PCO2 and metabolic rate, suggesting an adaptation of PCO2 to the rate of O2 consumption.