RESUMO
Liver fibrosis (LF) is a major cause of morbidity and mortality worldwide. Hepatic stellate cells (HSCs) are the primary source of extracellular matrix in the liver and their activation is a central event in LF development. Extracellular vesicles (EVs) are intercellular communication agents, which play important roles in physiological processes in chronic liver diseases. The aim of this study was to examine the crosstalk between hepatocytes and HSCs mediated by hepatocyte-secreted EVs. EVs were purified from primary mouse hepatocytes, HepG2 cell lines, under normal or stressed conditions. The effect of EVs on primary HSCs (pHSCs) differentiation was evaluated by measuring of differentiation markers. In addition, their impact on the carbon tetrachloride (CCl4)-induced fibrosis mouse model was evaluated. The results demonstrated that HepG2-EVs regulate HSC differentiation and that under stress conditions, promoted pHSCs differentiation into the myofibroblast phenotype. The evaluation of miRNA sequences in the HepG2 secreted EVs demonstrated high levels of miR-423-5p. The examination of EV cargo following stress conditions identified a significant reduction of miR-423-5p in HepG2-EVs relative to HepG2-EVs under normal conditions. In addition, pHSCs transfected with miR-423-5p mimic and exhibit lower mRNA levels of alpha smooth muscle actin and Collagen type 1 alpha, and the mRNA expression level of genes targeted the family with sequence-similarity-3 (FAM3) and Monoacylglycerol lipase (Mgll). This study strengthened the hypothesis that EVs are involved in LF and that their cargo changes in stress conditions. In addition, miR-423-5p was shown to be involved in HSCs differentiation and hence, fibrosis development.
Assuntos
Vesículas Extracelulares , MicroRNAs , Animais , Humanos , Camundongos , Vesículas Extracelulares/metabolismo , Células Hep G2 , Células Estreladas do Fígado/metabolismo , Cirrose Hepática/metabolismo , MicroRNAs/genética , MicroRNAs/metabolismo , RNA Mensageiro/metabolismoRESUMO
Non-alcoholic fatty liver disease (NAFLD) and non-alcoholic steatohepatitis (NASH) are prevalent liver conditions that underlie the development of life-threatening cirrhosis, liver failure and liver cancer. Chronic necro-inflammation is a critical factor in development of NASH, yet the cellular and molecular mechanisms of immune dysregulation in this disease are poorly understood. Here, using single-cell transcriptomic analysis, we comprehensively profiled the immune composition of the mouse liver during NASH. We identified a significant pathology-associated increase in hepatic conventional dendritic cells (cDCs) and further defined their source as NASH-induced boost in cycling of cDC progenitors in the bone marrow. Analysis of blood and liver from patients on the NAFLD/NASH spectrum showed that type 1 cDCs (cDC1) were more abundant and activated in disease. Sequencing of physically interacting cDC-T cell pairs from liver-draining lymph nodes revealed that cDCs in NASH promote inflammatory T cell reprogramming, previously associated with NASH worsening. Finally, depletion of cDC1 in XCR1DTA mice or using anti-XCL1-blocking antibody attenuated liver pathology in NASH mouse models. Overall, our study provides a comprehensive characterization of cDC biology in NASH and identifies XCR1+ cDC1 as an important driver of liver pathology.
Assuntos
Células Dendríticas/imunologia , Fígado Gorduroso/imunologia , Hepatopatia Gordurosa não Alcoólica/imunologia , Receptores de Quimiocinas/genética , Animais , Células da Medula Óssea/imunologia , Células da Medula Óssea/patologia , Reprogramação Celular/genética , Reprogramação Celular/imunologia , Células Dendríticas/patologia , Dieta Hiperlipídica/efeitos adversos , Modelos Animais de Doenças , Fígado Gorduroso/genética , Fígado Gorduroso/patologia , Feminino , Humanos , Fígado/imunologia , Fígado/patologia , Linfonodos/imunologia , Linfonodos/patologia , Masculino , Camundongos , Hepatopatia Gordurosa não Alcoólica/genética , Hepatopatia Gordurosa não Alcoólica/patologia , Receptores de Quimiocinas/imunologia , Linfócitos T/imunologia , Linfócitos T/patologiaRESUMO
BACKGROUND: Nonalcoholic fatty liver disease (NAFLD) is common in bariatric surgery candidates. We evaluated the effect of sleeve gastrectomy (SG) on NAFLD using validated non-invasive measures. METHODS: Patients with morbid obesity and NAFLD, planned for SG, were evaluated before and after surgery. Data collected included anthropometrics, biochemistry, adiponectin, SteatoTest™, NashTest™, FibroTest™, OWLiver® test and real-time ShearWave™ elastography (SWE). RESULTS: Twenty-six subjects were included in the study, mean age 44.1 ± 4.8 years, 69.2% males. One year following SG, body mass index decreased significantly from 41.7 ± 4.8 kg/m2 to 29.6 ± 4.5 kg/m2. Concomitantly, significant improvements in triglycerides, ALT, diabetes markers and adiponectin were observed. Mean steatosis, as measured by SteatoTest™, was significantly improved. Steatohepatitis score measured by NashTest™ and OWLiver® significantly decreased. Mean fibrosis, as measured by SWE liver stiffness and FibroTest™, did not change over time. CONCLUSION: Steatosis and steatohepatitis are significantly improved by SG as measured by non-invasive measures.
Assuntos
Cirurgia Bariátrica , Cirrose Hepática/diagnóstico , Hepatopatia Gordurosa não Alcoólica/diagnóstico , Obesidade Mórbida/cirurgia , Adulto , Idoso , Técnicas de Imagem por Elasticidade , Estudos de Viabilidade , Feminino , Humanos , Fígado/diagnóstico por imagem , Cirrose Hepática/sangue , Cirrose Hepática/etiologia , Cirrose Hepática/cirurgia , Testes de Função Hepática , Masculino , Pessoa de Meia-Idade , Hepatopatia Gordurosa não Alcoólica/sangue , Hepatopatia Gordurosa não Alcoólica/etiologia , Hepatopatia Gordurosa não Alcoólica/cirurgia , Obesidade Mórbida/complicações , Estudos Prospectivos , Índice de Gravidade de Doença , Resultado do Tratamento , Redução de Peso , Adulto JovemRESUMO
Non-alcoholic fatty liver disease (NAFLD) comprises a spectrum of progressive liver pathologies, ranging from simple steatosis to non-alcoholic steatohepatitis (NASH), fibrosis and cirrhosis. A liver biopsy is currently required to stratify high-risk patients, and predicting the degree of liver inflammation and fibrosis using non-invasive tests remains challenging. Here, we sought to develop a novel, cost-effective screening tool for NAFLD based on thermal imaging. We used a commercially available and non-invasive thermal camera and developed a new image processing algorithm to automatically predict disease status in a small animal model of fatty liver disease. To induce liver steatosis and inflammation, we fed C57/black female mice (8 weeks old) a methionine-choline deficient diet (MCD diet) for 6 weeks. We evaluated structural and functional liver changes by serial ultrasound studies, histopathological analysis, blood tests for liver enzymes and lipids, and measured liver inflammatory cell infiltration by flow cytometry. We developed an image processing algorithm that measures relative spatial thermal variation across the skin covering the liver. Thermal parameters including temperature variance, homogeneity levels and other textural features were fed as input to a t-SNE dimensionality reduction algorithm followed by k-means clustering. During weeks 3,4, and 5 of the experiment, our algorithm demonstrated a 100% detection rate and classified all mice correctly according to their disease status. Direct thermal imaging of the liver confirmed the presence of changes in surface thermography in diseased livers. We conclude that non-invasive thermal imaging combined with advanced image processing and machine learning-based analysis successfully correlates surface thermography with liver steatosis and inflammation in mice. Future development of this screening tool may improve our ability to study, diagnose and treat liver disease.
Assuntos
Fígado Gorduroso/diagnóstico por imagem , Hepatopatia Gordurosa não Alcoólica/diagnóstico por imagem , Termografia/métodos , Algoritmos , Animais , Automação/métodos , Colina/administração & dosagem , Deficiência de Colina/metabolismo , Dieta/métodos , Modelos Animais de Doenças , Fígado Gorduroso/diagnóstico , Feminino , Humanos , Processamento de Imagem Assistida por Computador/métodos , Fígado/diagnóstico por imagem , Metionina/administração & dosagem , Metionina/deficiência , Camundongos , Camundongos Endogâmicos C57BL , Hepatopatia Gordurosa não Alcoólica/diagnóstico , UltrassonografiaRESUMO
Hepatocellular carcinoma (HCC) is the second leading cause of cancer-related mortality worldwide. While cirrhosis is the main risk factor for HCC, the factors influencing progression from cirrhosis to HCC remain largely unknown. Gut microbiota plays a key role in liver diseases; however, its association with HCC remains elusive. This study aimed to elucidate microbial differences between patients with HCC-associated cirrhosis (HCC-cirrhosis) and cirrhotic patients without HCC and healthy volunteers and to explore the associations between diet, lifestyle, and the microbiome of these patients. Fecal samples and food frequency questionnaires were collected from 95 individuals (30 HCC-cirrhosis patients, 38 cirrhotic patients without HCC, and 27 age- and body mass index [BMI]-matched healthy volunteers). 16S rRNA gene sequencing was performed. Bacterial richness in cirrhosis and HCC-cirrhosis patients was significantly lower than in healthy controls. The HCC-cirrhosis group was successfully classified with an area under the curve (AUC) value of 0.9 based on the dysbiotic fecal microbial signature. The HCC-cirrhosis group had a significant overrepresentation of Clostridium and CF231 and reduced Alphaproteobacteria abundance compared to cirrhotic patients without HCC. Patients with HCC-cirrhosis who were overweight displayed significantly decreased bacterial richness and altered microbiota composition compared to their normal-weight counterparts. There was a significant correlation in the HCC-cirrhosis group between intake of artificial sweeteners and the presence of Akkermansia muciniphila A unique microbial signature was observed in patients with HCC-cirrhosis, irrespective of cirrhosis stage, diet, or treatment. BMI, dietary sugar, and artificial sweeteners were significantly associated with alterations in the microbiome of HCC-cirrhosis patients. However, the increased abundance of Clostridium and CF231 observed in HCC-cirrhosis patients was not influenced by environmental factors, implying that this change was due to development of HCC.IMPORTANCE Development of hepatocellular carcinoma in patients with cirrhosis is associated with alterations in intestinal microbiota, including an escalation of dysbiosis and reduced bacterial richness. This study demonstrates that reduced bacterial richness and dysbiosis escalate with the progression of cirrhosis from compensated to decompensated cirrhosis and to HCC-associated cirrhosis (HCC-cirrhosis). Moreover, we report for the first time the effect of environmental factors on HCC-cirrhosis. Excess weight was associated with increased dysbiosis in patients with HCC compared to their normal-weight counterparts. Moreover, fatty liver, consumption of artificial sweeteners, and high-sugar foods were associated with altered microbial composition, including altered levels of Akkermansia muciniphila in HCC-cirrhosis. We have successfully determined that levels of Alphaproteobacteria and the two genera CF231 and Clostridium are significantly altered in cirrhotic patients who develop hepatocellular carcinoma, independently of cirrhosis severity and dietary habits.
RESUMO
BACKGROUND: Vitamin D is a key immune-modulator that plays a role in the innate and adaptive immune systems. Certain pathogens impair the immune defense by downregulating the vitamin D receptor (VDR) pathway. Low serum levels of vitamin D are associated with increased hepatitis B virus (HBV) replication. Our study aimed to assess the in-vitro relationship between HBV production and Vitamin D signaling pathway and to explore the associated mechanism(s). METHODS: HBV transcription and replication was evaluated by qRT-PCR of the HBV-RNA and covalently closed circular DNA (cccDNA). Furthermore, we have transfected the 1.3 X HBV-Luc plasmid to the cells and measured the Luciferase activity using Luminometer. Vitamin D signaling pathway activation was evaluated by measuring the expression levels of VDR, CYP24A1, Tumor necrosis factor α (TNFα) and cathelicidin (CAMP) by qRT-PCR. All assays were performed on HepG2.2.15, HepG2, and HepAD38 cells treated with or without Vitamin D active metabolite: calcitriol. RESULTS: Calcitriol did not suppress HBV transcription, cccDNA expression or HBV RNA levels in HepG2.2.15 cells. However, VDR transcript levels in HepG2.215 cells were significantly lower compared to HepG2 cells. Similar results were obtained in HepAD38 cell where VDR expression was down-regulated when HBV transcript level was up-regulated. In addition, calcitriol induced VDR-associated signaling, resulting in upregulation of CYP24A1, TNFα and CAMP expression level in HepG2 cells but not in the HepG2.2.15 cells. CONCLUSIONS: These findings indicate that VDR expression is downregulated in HBV-transfected cells, thereby preventing vitamin D from inhibiting transcription and translation of HBV in vitro. HBV might use this mechanism to avoid the immunological defense system by affecting both TNFα and CAMP signaling pathways.
Assuntos
Carcinoma Hepatocelular/genética , Vírus da Hepatite B/fisiologia , Neoplasias Hepáticas/genética , Receptores de Calcitriol/genética , Peptídeos Catiônicos Antimicrobianos/genética , Calcitriol/farmacologia , Carcinoma Hepatocelular/metabolismo , Linhagem Celular Tumoral , Regulação para Baixo , Vírus da Hepatite B/efeitos dos fármacos , Humanos , Neoplasias Hepáticas/metabolismo , RNA Viral , Receptores de Calcitriol/metabolismo , Fator de Necrose Tumoral alfa/genética , Replicação Viral/efeitos dos fármacos , CatelicidinasRESUMO
BACKGROUND: During liver injury, hepatic stellate cells (HSCs) can undergo activation and transform into alpha-smooth muscle actin (αSMA)-expressing contractile myofibroblast-like cells, leading to deposition of excessive scar matrix. We have recently demonstrated that depletion of adenosine deaminase acting on double-stranded RNA (ADAR1) from mouse hepatocytes leads to HSC activation and induction of inflammation and hepatic fibrosis that is mediated by interleukin 6 (IL-6). Our aim was to identify and characterize the molecular pathways involved in the direct, inflammation-independent activation of HSCs by IL-6. METHODS: Primary HSCs were isolated from mouse livers. mRNA levels of αSMA and Col1a were analyzed using qRT-PCR. Protein levels of αSMA, MAPK, p-MAPK, p38, p-p38, STAT3 and p-STAT3 were assessed by Western Blot analysis. The effect of specific signal transduction pathway inhibitors (i.e., SB203580 (P-38 inhibitor), U0126 (MAPK inhibitor), S3I-201 (STAT3 inhibitor) and Ruxolitinib (Jak1/2 inhibitor)) was also studied. RESULTS: Primary HSCs treated with IL-6 demonstrated upregulation of αSMA and Col1a mRNA levels as well as increased αSMA protein levels. Moreover, the phenotypic transition of quiescent HSCs toward myofibroblast-like cells was noted upon administration of IL-6 and not in untreated samples. In addition, the phosphorylation levels of p38, MAPK and STAT3 increased 30 minutes after treatment, and was followed by a decline in the phosphorylation levels 2-4 hours post-treatment. However, addition of specific signal transduction pathway inhibitors curbed this effect, and resulted in αSMA and Col1a expression levels similar to those measured in untreated control samples. CONCLUSION: IL-6 can directly induce the transition of HSCs toward myofibroblast-like cells. The effect is mediated by the activation of both MAPK and JAK/STAT signaling pathways. Elimination of either MAPK or JAK/STAT signaling pathways inhibits HSC stimulation. These results might pave the road toward the development of potential therapeutic interventions for hepatic fibrosis.
Assuntos
Células Estreladas do Fígado/metabolismo , Interleucina-6/metabolismo , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Fator de Transcrição STAT3/metabolismo , Transdução de Sinais , Animais , Camundongos , Camundongos Endogâmicos ICRRESUMO
Adenosine deaminase acting on RNA (ADAR) 1 binds and edits double-stranded (ds) RNA secondary structures found mainly within untranslated regions of many transcripts. In the current research, our aim was to study the role of ADAR1 in liver homeostasis. As previous studies show a conserved immunoregulatory function for ADAR1 in mammalians, we focused on its role in preventing chronic hepatic inflammation and the associated activation of hepatic stellate cells to produce extracellular matrix and promote fibrosis. We show that hepatocytes specific ADAR1 knock out (KO) mice display massive liver damage with multifocal inflammation and fibrogenesis. The bioinformatics analysis of the microarray gene-expression datasets of ADAR1 KO livers reveled a type-I interferons signature and an enrichment for immune response genes compared to control littermate livers. Furthermore, we found that in vitro silencing of ADAR1 expression in HepG2 cells leads to enhanced transcription of NFκB target genes, foremost of the pro-inflammatory cytokines IL6 and IL8. We also discovered immune cell-independent paracrine signaling among ADAR1-depleted HepG2 cells and hepatic stellate cells, leading to the activation of the latter cell type to adopt a profibrogenic phenotype. This paracrine communication dependent mainly on the production and secretion of the cytokine IL6 induced by ADAR1 silencing in hepatocytes. Thus, our findings shed a new light on the vital regulatory role of ADAR1 in hepatic immune homeostasis, chiefly its inhibitory function on the crosstalk between the NFκB and type-I interferons signaling cascades, restraining the development of liver inflammation and fibrosis.
Assuntos
Adenosina Desaminase/genética , Hepatite/genética , Interferon Tipo I/biossíntese , Cirrose Hepática/genética , Fígado/imunologia , NF-kappa B/metabolismo , Animais , Matriz Extracelular/metabolismo , Expressão Gênica/imunologia , Células Hep G2 , Células Estreladas do Fígado/imunologia , Células Estreladas do Fígado/metabolismo , Hepatite/imunologia , Hepatócitos/imunologia , Hepatócitos/metabolismo , Humanos , Imunidade Inata/genética , Interleucina-6/biossíntese , Interleucina-8/biossíntese , Fígado/patologia , Cirrose Hepática/imunologia , Camundongos , Camundongos Knockout , Comunicação Parácrina/imunologia , RNA de Cadeia Dupla/metabolismo , Transdução de SinaisRESUMO
About 80% of hepatocellular carcinoma (HCC) is caused by hepatitis B virus (HBV) and/or hepatitis C virus (HCV) infections especially in the setting of established cirrhosis or advanced fibrosis, making HCC prevention a major goal of antiviral therapy. HCC tumors are highly complex and heterogeneous resulting from the aberrant function of multiple molecular pathways. The roles of HCV or HBV in promoting HCC development are still either directly or indirectly are still speculative, but the evidence for both effects is compelling. In patients with chronic hepatitis viral infection, cirrhosis is not a prerequisite for tumorigenesis.
Assuntos
Carcinoma Hepatocelular/virologia , DNA Viral , Hepacivirus/fisiologia , Vírus da Hepatite B/fisiologia , Hepatite B Crônica/complicações , Hepatite C Crônica/complicações , Neoplasias Hepáticas/virologia , Animais , Antivirais/uso terapêutico , Carcinoma Hepatocelular/prevenção & controle , Hepacivirus/genética , Vírus da Hepatite B/genética , Hepatite B Crônica/tratamento farmacológico , Hepatite C Crônica/tratamento farmacológico , Humanos , Cirrose Hepática/complicações , Cirrose Hepática/virologia , Neoplasias Hepáticas/prevenção & controle , Vírus Oncogênicos , Integração ViralRESUMO
Adenosine to inosine (A-to-I) RNA editing is a base recoding process within precursor messenger RNA, catalyzed by members of the adenosine deaminase acting on RNA (ADAR) family. A notable example occurs at the Q/R site of the α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid glutamate receptor subunit GluA2. Abnormally, low editing at this site leads to excessive calcium influx and cell death. We studied hippocampus and caudate samples from Alzheimer's disease (AD) patients and age-matched healthy controls, using direct sequencing and a high accuracy primer-extension technique to assess RNA editing at the Q/R GluA2 site. Both techniques revealed lower, more variable RNA editing in AD, specific to the hippocampus and the GluA2 site. Deficient editing also characterized the hippocampus of apolipoprotein ε4 allele carriers, regardless of clinical diagnosis. In AD, messenger RNA expression of neuronal markers was decreased in the hippocampus, and expression of the Q/R-site editing enzyme ADAR2 was decreased in caudate. These findings provide a link between neurodegenerative processes and deficient RNA editing of the GluA2 Q/R site, and may contribute to both diagnosis and treatment of AD.
Assuntos
Doença de Alzheimer/genética , Hipocampo/metabolismo , Hipocampo/patologia , Edição de RNA/genética , RNA Mensageiro/genética , Receptores de AMPA/genética , Adenosina Desaminase/metabolismo , Idoso , Idoso de 80 Anos ou mais , Alelos , Doença de Alzheimer/diagnóstico , Doença de Alzheimer/terapia , Apolipoproteína E4/genética , Cálcio/metabolismo , Núcleo Caudado/enzimologia , Morte Celular , Feminino , Humanos , Masculino , Proteínas de Ligação a RNA/metabolismo , Análise de Sequência de DNA/métodosRESUMO
Alternative mRNA splicing is a major mechanism for gene regulation and transcriptome diversity. Despite the extent of the phenomenon, the regulation and specificity of the splicing machinery are only partially understood. Adenosine-to-inosine (A-to-I) RNA editing of pre-mRNA by ADAR enzymes has been linked to splicing regulation in several cases. Here we used bioinformatics approaches, RNA-seq and exon-specific microarray of ADAR knockdown cells to globally examine how ADAR and its A-to-I RNA editing activity influence alternative mRNA splicing. Although A-to-I RNA editing only rarely targets canonical splicing acceptor, donor, and branch sites, it was found to affect splicing regulatory elements (SREs) within exons. Cassette exons were found to be significantly enriched with A-to-I RNA editing sites compared with constitutive exons. RNA-seq and exon-specific microarray revealed that ADAR knockdown in hepatocarcinoma and myelogenous leukemia cell lines leads to global changes in gene expression, with hundreds of genes changing their splicing patterns in both cell lines. This global change in splicing pattern cannot be explained by putative editing sites alone. Genes showing significant changes in their splicing pattern are frequently involved in RNA processing and splicing activity. Analysis of recently published RNA-seq data from glioblastoma cell lines showed similar results. Our global analysis reveals that ADAR plays a major role in splicing regulation. Although direct editing of the splicing motifs does occur, we suggest it is not likely to be the primary mechanism for ADAR-mediated regulation of alternative splicing. Rather, this regulation is achieved by modulating trans-acting factors involved in the splicing machinery.
Assuntos
Adenosina Desaminase/genética , Processamento Alternativo/genética , Precursores de RNA/genética , RNA Mensageiro/genética , Adenosina/genética , Adenosina/metabolismo , Adenosina Desaminase/metabolismo , Sequência de Bases , Linhagem Celular Tumoral , Éxons/genética , Regulação da Expressão Gênica , Humanos , Inosina/genética , Edição de RNA/genética , Proteínas de Ligação a RNARESUMO
Post-transcriptional events play an important role in human development. The question arises as to whether Adenosine to Inosine RNA editing, catalyzed by the ADAR (Adenosine Deaminase acting on RNA) enzymes, differs in human embryogenesis and in adulthood. We tested the editing of various target genes in coding (FLNA, BLCAP, CYFIP2) and non-coding sequences at their Alu elements (BRCA1, CARD11, RBBP9, MDM4, FNACC), as well as the transcriptional levels of the ADAR1 enzymes. This analysis was performed on five fetal and adult human tissues: brain, heart, liver, kidney, and spleen, as well as on human embryonic stem cells (hESCs), which represent the blastocyst stage in early human development. Our results show substantially greater editing activity for most adult tissue samples relative to fetal ones, in six of the eight genes tested. To test the effect of reduced A-to-I RNA editing activity in early human development we used human embryonic stem cells (hESCs) as a model and tried to generate hESC clones that overexpress the ADAR1-p110 isoform. We were unable to achieve overexpression of ADAR1-p110 by either transfection or lentiviral infection, though we easily generated hESC clones that expressed the GFP transgene and overexpressed ADAR1-p110 in 293T cells and in primary human foreskin fibroblast (HFF) cells. Moreover, in contrast to the expected overexpression of ADAR1-p110 protein following its introduction into hESCs, the expression levels of this protein decreased dramatically 24-48 hr post infection. Similar results were obtained when we tried to overexpress ADAR1-p110 in pluripotent embryonal carcinoma cells. This suggests that ADAR1 protein is substantially regulated in undifferentiated pluripotent hESCs. Overall, our data suggest that A-to-I RNA editing plays a critical role during early human development.
Assuntos
Desenvolvimento Embrionário , Edição de RNA , RNA Mensageiro/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/genética , Adenosina/genética , Adenosina/metabolismo , Adenosina Desaminase/genética , Adenosina Desaminase/metabolismo , Elementos Alu , Proteína BRCA1/genética , Proteínas Adaptadoras de Sinalização CARD/genética , Proteínas de Ciclo Celular/genética , Células Cultivadas , Proteínas Contráteis/genética , Células-Tronco de Carcinoma Embrionário , Células-Tronco Embrionárias , Proteína do Grupo de Complementação C da Anemia de Fanconi/genética , Filaminas , Expressão Gênica , Regulação da Expressão Gênica no Desenvolvimento , Guanilato Ciclase/genética , Humanos , Inosina/genética , Inosina/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/genética , Proteínas dos Microfilamentos/genética , Proteínas de Neoplasias/genética , Proteínas Nucleares/genética , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Proteínas Proto-Oncogênicas/genética , RNA Mensageiro/genética , Proteínas de Ligação a RNARESUMO
PURPOSE: A-to-I RNA editing is essential for the development of normal cells and is involved in a wide variety of biological pathways. Currently, limited information suggests linkage between changes in RNA editing levels and the development of cancer. We aimed to explore the possible linkage between altered RNA editing levels and the development of human urinary bladder neoplasms. MATERIALS AND METHODS: Thirty-two patients underwent transurethral resection of bladder tumor. Normal and tumoral urinary bladder tissues were obtained from each patient during surgery. Total RNA was extracted from tissue cells and converted by RT-PCR reaction to cDNA molecules for further analysis. We explored known editing sites in RNA encoding for proteins (BLCAP, Cyfip2, FLNA, GluB Q/R) as well as in RNA transcribed from Alu elements in noncoding regions of the genes encoding for CARD11, FANCC, MDM4, BRCA1, and RBBP9 proteins. Editing levels were determined using Sequenom MassARRAY Compact Analyzer. RESULTS: Eleven tumoral tissues obtained were low grade TCC, 14 high grade TCC, 1 CIS, and another 5 inflammation. One sample contained only normal tissue. We got a total number of 30 normal bladder tissue samples and overall 29 paired samples (i.e., normal and tumoral tissues obtained from the same patient). Statistical analysis revealed no significant changes in editing levels between normal and tumoral tissues. CONCLUSIONS: Relying on the results obtained for 9 different editing sites, it can be determined that RNA editing is an epigenetic mechanism that does not participate in the evolution of urinary bladder cancer.
Assuntos
Carcinoma de Células de Transição/genética , Edição de RNA/genética , Neoplasias da Bexiga Urinária/genética , Adenosina/química , Adulto , Idoso , Idoso de 80 Anos ou mais , Elementos Alu/genética , Carcinoma de Células de Transição/patologia , Epigenômica , Feminino , Humanos , Inosina/química , Masculino , Pessoa de Meia-Idade , Proteínas de Neoplasias/genética , Prognóstico , Precursores de RNA/genética , RNA não Traduzido/genética , Neoplasias da Bexiga Urinária/patologiaRESUMO
BACKGROUND: Hypoxia inducible factor-1α (HIF-1α) is responsible for the majority of HIF-1-induced gene expression changes under hypoxia and for the "angiogenic switch" during tumor progression. HIF-1α is often upregulated in tumors leading to more aggressive tumor growth and chemoresistance, therefore representing an important target for antitumor intervention. We previously reported that zinc downregulated HIF-1α levels. Here, we evaluated the molecular mechanisms of zinc-induced HIF-1α downregulation and whether zinc affected HIF-1α also in vivo. METHODOLOGY/PRINCIPAL FINDINGS: Here we report that zinc downregulated HIF-1α protein levels in human prostate cancer and glioblastoma cells under hypoxia, whether induced or constitutive. Investigations into the molecular mechanisms showed that zinc induced HIF-1α proteasomal degradation that was prevented by treatment with proteasomal inhibitor MG132. HIF-1α downregulation induced by zinc was ineffective in human RCC4 VHL-null renal carcinoma cell line; likewise, the HIF-1αP402/P564A mutant was resistant to zinc treatment. Similarly to HIF-1α, zinc downregulated also hypoxia-induced HIF-2α whereas the HIF-1ß subunit remained unchanged. Zinc inhibited HIF-1α recruitment onto VEGF promoter and the zinc-induced suppression of HIF-1-dependent activation of VEGF correlated with reduction of glioblastoma and prostate cancer cell invasiveness in vitro. Finally, zinc administration downregulated HIF-1α levels in vivo, by bioluminescence imaging, and suppressed intratumoral VEGF expression. CONCLUSIONS/SIGNIFICANCE: These findings, by demonstrating that zinc induces HIF-1α proteasomal degradation, indicate that zinc could be useful as an inhibitor of HIF-1α in human tumors to repress important pathways involved in tumor progression, such as those induced by VEGF, MDR1, and Bcl2 target genes, and hopefully potentiate the anticancer therapies.
Assuntos
Regulação para Baixo , Regulação Neoplásica da Expressão Gênica , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Zinco/farmacologia , Antineoplásicos/farmacologia , Neoplasias Encefálicas/metabolismo , Linhagem Celular , Linhagem Celular Tumoral , Progressão da Doença , Glioblastoma/metabolismo , Humanos , Técnicas In Vitro , Masculino , Neoplasias da Próstata/metabolismo , Complexo de Endopeptidases do Proteassoma/farmacologia , Fator A de Crescimento do Endotélio Vascular/biossínteseRESUMO
BACKGROUND: Adenosine to inosine (A-to-I) RNA-editing is an essential post-transcriptional mechanism that occurs in numerous sites in the human transcriptome, mainly within Alu repeats. It has been shown to have consistent levels of editing across individuals in a few targets in the human brain and altered in several human pathologies. However, the variability across human individuals of editing levels in other tissues has not been studied so far. RESULTS: Here, we analyzed 32 skin samples, looking at A-to-I editing level in three genes within coding sequences and in the Alu repeats of six different genes. We observed highly consistent editing levels across different individuals as well as across tissues, not only in coding targets but, surprisingly, also in the non evolutionary conserved Alu repeats. CONCLUSIONS: Our findings suggest that A-to-I RNA-editing of Alu elements is a tightly regulated process and, as such, might have been recruited in the course of primate evolution for post-transcriptional regulatory mechanisms.
Assuntos
Adenosina/genética , Elementos Alu/genética , Sequência Conservada/genética , Inosina/genética , Fases de Leitura Aberta/genética , Edição de RNA/genética , Linhagem Celular , Humanos , Proteínas Nucleares/genética , Especificidade de Órgãos/genética , Proteínas Proto-Oncogênicas c-fyn/genética , Pele/metabolismoRESUMO
Adenosine to Inosine (A-to-I) RNA editing is a site-specific modification of RNA transcripts, catalyzed by members of the ADAR (Adenosine Deaminase Acting on RNA) protein family. RNA editing occurs in human RNA in thousands of different sites. Some of the sites are located in protein-coding regions but the majority is found in non-coding regions, such as 3'UTRs, 5'UTRs and introns - mainly in Alu elements. While editing is found in all tissues, the highest levels of editing are found in the brain. It was shown that editing levels within protein-coding regions are increased during embryogenesis and after birth and that RNA editing is crucial for organism viability as well as for normal development. In this study we characterized the A-to-I RNA editing phenomenon during neuronal and spontaneous differentiation of human embryonic stem cells (hESCs). We identified high editing levels of Alu repetitive elements in hESCs and demonstrated a global decrease in editing levels of non-coding Alu sites when hESCs are differentiating, particularly into the neural lineage. Using RNA interference, we showed that the elevated editing levels of Alu elements in undifferentiated hESCs are highly dependent on ADAR1. DNA microarray analysis showed that ADAR1 knockdown has a global effect on gene expression in hESCs and leads to a significant increase in RNA expression levels of genes involved in differentiation and development processes, including neurogenesis. Taken together, we speculate that A-to-I editing of Alu sequences plays a role in the regulation of hESC early differentiation decisions.
Assuntos
Adenosina/metabolismo , Elementos Alu/genética , Células-Tronco Embrionárias/citologia , Células-Tronco Embrionárias/metabolismo , Inosina/metabolismo , Edição de RNA , Adenosina Desaminase/deficiência , Adenosina Desaminase/genética , Adenosina Desaminase/metabolismo , Animais , Diferenciação Celular , Regulação Enzimológica da Expressão Gênica/genética , Inativação Gênica , Humanos , Camundongos , Neurônios/citologia , Fases de Leitura Aberta/genética , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Proteínas de Ligação a RNARESUMO
Adenosine-to-inosine (A-to-I) RNA editing was recently shown to be abundant in the human transcriptome, affecting thousands of genes. Employing a bioinformatic approach, we identified significant global hypoediting of Alu repetitive elements in brain, prostate, lung, kidney, and testis tumors. Experimental validation confirmed this finding, showing significantly reduced editing in Alu sequences within MED13 transcripts in brain tissues. Looking at editing of specific recoding and noncoding sites, including in cancer-related genes, a more complex picture emerged, with a gene-specific editing pattern in tumors vs. normal tissues. Additionally, we found reduced RNA levels of all three editing mediating enzymes, ADAR, ADARB1, and ADARB2, in brain tumors. The reduction of ADARB2 correlated with the grade of malignancy of glioblastoma multiforme, the most aggressive of brain tumors, displaying a 99% decrease in ADARB2 RNA levels. Consistently, overexpression of ADAR and ADARB1 in the U87 glioblastoma multiforme cell line resulted in decreased proliferation rate, suggesting that reduced A-to-I editing in brain tumors is involved in the pathogenesis of cancer. Altered epigenetic control was recently shown to play a central role in oncogenesis. We suggest that A-to-I RNA editing may serve as an additional epigenetic mechanism relevant to cancer development and progression.
Assuntos
Adenosina/química , Inosina/química , Neoplasias/genética , Edição de RNA , Adenosina Desaminase/genética , Elementos Alu , Animais , Sequência de Bases , Linhagem Celular Tumoral , Proliferação de Células , Células Cultivadas , Biologia Computacional , Humanos , Camundongos , Dados de Sequência Molecular , Neoplasias/metabolismo , Precursores de RNA , RNA não Traduzido/metabolismo , Proteínas de Ligação a RNARESUMO
Many functions have been assigned to the von Hippel-Lindau tumor suppressor gene product (pVHL), including targeting the alpha subunits of the heterodimeric transcription factor HIF (hypoxia-inducible factor) for destruction. The binding of pVHL to HIFalpha requires that HIFalpha be hydroxylated on one of two prolyl residues. We introduced HIF1alpha and HIF2alpha variants that cannot be hydroxylated on these sites into the ubiquitously expressed ROSA26 locus along with a Lox-stop-Lox cassette that renders their expression Cre-dependent. Expression of the HIF2alpha variant in the skin and liver induced changes that were highly similar to those seen when pVHL is lost in these organs. Dual expression of the HIF1alpha and HIF2alpha variants in liver, however, more closely phenocopied the changes seen after pVHL inactivation than did the HIF2alpha variant alone. Moreover, gene expression profiling confirmed that the genes regulated by HIF1alpha and HIF2alpha in the liver are overlapping but non-identical. Therefore, the pathological changes caused by pVHL inactivation in skin and liver are due largely to dysregulation of HIF target genes.
Assuntos
Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Prolina/metabolismo , Proteína Supressora de Tumor Von Hippel-Lindau/metabolismo , Animais , Vasos Sanguíneos/citologia , Vasos Sanguíneos/patologia , Peso Corporal , Proliferação de Células , Células Epidérmicas , Epiderme/patologia , Regulação da Expressão Gênica , Células HeLa , Hepatócitos/citologia , Hepatócitos/patologia , Humanos , Hidroxilação , Fígado/citologia , Fígado/patologia , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Mutantes , Tamanho do Órgão , Fenótipo , Recombinação GenéticaRESUMO
Many human diseases are characterized by the development of tissue hypoxia. Inadequate oxygenation can cause cellular dysfunction and death. Tissues use many strategies, including induction of angiogenesis and alterations in metabolism, to survive under hypoxic conditions. The heterodimeric transcription factor hypoxia-inducible factor (HIF) is a master regulator of genes that promote adaptation to hypoxia. HIF activity is linked to oxygen availability because members of the EGLN family hydroxylate HIFalpha subunits on specific prolyl residues when oxygen is present, which marks them for ubiquitination and proteasomal degradation. We created a mouse that ubiquitously expresses a bioluminescent reporter consisting of firefly luciferase fused to a region of HIF that is sufficient for oxygen-dependent degradation. Our validation studies suggest that this mouse will be useful for monitoring hypoxic tissues and evaluating therapeutic agents that stabilize HIF. One such agent, the HIF prolyl hydroxylase inhibitor FG-4383, was active in the liver and kidney after systemic administration as determined by bioluminescence imaging, transcription profiling, and production of erythropoietin, indicating that the HIF transcriptional program can be manipulated in vivo with orally active organic small molecules.
Assuntos
Dioxigenases/antagonistas & inibidores , Dioxigenases/metabolismo , Inibidores Enzimáticos , Eritropoetina/biossíntese , Fator 1 Induzível por Hipóxia/metabolismo , Modelos Animais , Animais , Hipóxia Celular/fisiologia , Células Cultivadas , Primers do DNA , DNA Complementar/genética , Ensaio de Imunoadsorção Enzimática , Fator 1 Induzível por Hipóxia/genética , Prolina Dioxigenases do Fator Induzível por Hipóxia , Luciferases , Camundongos , Técnicas de Sonda Molecular , Plasmídeos/genética , Proteínas/genética , Proteínas/metabolismo , RNA não TraduzidoRESUMO
Many proteins and pathways of pharmaceutical interest impinge on ubiquitin ligases or their substrates. The cyclin-dependent kinase (Cdk) inhibitor p27, for example, is polyubiquitylated in a cell cycle-dependent manner by a ubiquitin ligase complex containing the F-box protein Skp2. Regulated turnover of p27 is due, at least partly, to its phosphorylation by Cdk2 on threonine 187, which generates a Skp2-binding site. We made a p27-luciferase (p27Luc) fusion protein and show here that its abundance, like that of p27, is regulated by Skp2 in a cell cycle-dependent manner. As predicted, p27Luc levels increased after blocking Cdk2 activity with inhibitory proteins, peptides or small interfering RNA (siRNA). Accumulation of p27Luc in response to Cdk2 inhibitory drugs (flavopiridol and R-roscovitine) was demonstrable in human tumor cells in vivo using noninvasive bioluminescent imaging. In theory, the approach described here could be used to develop bioluminescent reporters for any drug target that directly or indirectly affects the turnover of a ubiquitin ligase substrate.