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1.
Stem Cells ; 40(1): 35-48, 2022 03 03.
Artigo em Inglês | MEDLINE | ID: mdl-35511867

RESUMO

DNA damage repair (DDR) is a safeguard for genome integrity maintenance. Increasing DDR efficiency could increase the yield of induced pluripotent stem cells (iPSC) upon reprogramming from somatic cells. The epigenetic mechanisms governing DDR during iPSC reprogramming are not completely understood. Our goal was to evaluate the splicing isoforms of histone variant macroH2A1, macroH2A1.1, and macroH2A1.2, as potential regulators of DDR during iPSC reprogramming. GFP-Trap one-step isolation of mtagGFP-macroH2A1.1 or mtagGFP-macroH2A1.2 fusion proteins from overexpressing human cell lines, followed by liquid chromatography-tandem mass spectrometry analysis, uncovered macroH2A1.1 exclusive interaction with Poly-ADP Ribose Polymerase 1 (PARP1) and X-ray cross-complementing protein 1 (XRCC1). MacroH2A1.1 overexpression in U2OS-GFP reporter cells enhanced specifically nonhomologous end joining (NHEJ) repair pathway, while macroH2A1.1 knock-out (KO) mice showed an impaired DDR capacity. The exclusive interaction of macroH2A1.1, but not macroH2A1.2, with PARP1/XRCC1, was confirmed in human umbilical vein endothelial cells (HUVEC) undergoing reprogramming into iPSC through episomal vectors. In HUVEC, macroH2A1.1 overexpression activated transcriptional programs that enhanced DDR and reprogramming. Consistently, macroH2A1.1 but not macroH2A1.2 overexpression improved iPSC reprogramming. We propose the macroH2A1 splicing isoform macroH2A1.1 as a promising epigenetic target to improve iPSC genome stability and therapeutic potential.


Assuntos
Histonas , Células-Tronco Pluripotentes Induzidas , Animais , DNA , Reparo do DNA , Células Endoteliais/metabolismo , Histonas/metabolismo , Humanos , Células-Tronco Pluripotentes Induzidas/metabolismo , Camundongos , Proteína 1 Complementadora Cruzada de Reparo de Raio-X/genética , Proteína 1 Complementadora Cruzada de Reparo de Raio-X/metabolismo
2.
Adv Exp Med Biol ; 1396: 275-298, 2023.
Artigo em Inglês | MEDLINE | ID: mdl-36454473

RESUMO

The incidence and the burden of cardiovascular disease (CVD), coronary heart disease (CHD), type 2 diabetes mellitus (T2DM), and the metabolic syndrome are greatly increasing in our societies. Together, they account for 31% of all deaths worldwide. This chapter focuses on the role of two revolutionary discoveries that are changing the future of medicine, induced pluripotent stem cells (iPSCs) and CRISPR/Cas9 technology, in the study, and the cure of cardiovascular and metabolic diseases.We summarize the state-of-the-art knowledge about the possibility of editing iPSC genome for therapeutic applications without hampering their pluripotency and differentiation, using CRISPR/Cas technology, in the field of cardiovascular and metabolic diseases.


Assuntos
Sistema Cardiovascular , Diabetes Mellitus Tipo 2 , Células-Tronco Pluripotentes Induzidas , Doenças Metabólicas , Humanos , Edição de Genes , Diabetes Mellitus Tipo 2/genética , Diabetes Mellitus Tipo 2/terapia , Doenças Metabólicas/genética , Doenças Metabólicas/terapia
3.
Clin Oral Investig ; 27(8): 4107-4116, 2023 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-37199773

RESUMO

OBJECTIVE: This review aimed at evaluating the possible benefits that caloric restriction (CR) may provide to periodontal disease progression and response to treatment. MATERIAL AND METHODS: Electronic search on Medline, Embase and Cochrane, and manual search were performed to identify pre-clinical and on human studies reporting the consequences of CR on clinical and inflammatory parameters related to periodontitis. Newcastle Ottawa System and SYRCLE scale were used to assess the risk of bias. RESULTS: Four thousand nine hundred eighty articles were initially screened, and a total of 6 articles were finally included, consisting of 4 animal studies and 2 studies in humans. Due to the limited number of studies and heterogeneity of the data, results were presented in descriptive analyses. All studies showed that, compared to the normal (ad libitum) diet, CR might have the potential to reduce the local and systemic hyper-inflammatory state as well as disease progression in periodontal patients. CONCLUSIONS: Within the existing limitations, this review highlights that CR showed some improvements in the periodontal condition by reducing the local and systemic inflammation related to the periodontitis and by improving clinical parameters. However, the results should be interpreted with caution since robust research such as randomized clinical trials is still missing. CLINICAL RELEVANCE: This review shows that some dietary/caloric restrictions approaches may have the potential to improve periodontal conditions and, in addition, highlights a need for human studies with a robust methodology in order to draw stronger evidence-based conclusions.


Assuntos
Doenças da Gengiva , Doenças Periodontais , Periodontite , Animais , Humanos , Doenças Periodontais/prevenção & controle , Progressão da Doença
4.
Int J Mol Sci ; 24(2)2023 Jan 04.
Artigo em Inglês | MEDLINE | ID: mdl-36674455

RESUMO

Liquid biopsies have emerged as a minimally invasive cancer detection and monitoring method, which could identify cancer-related alterations in nucleosome or histone levels and modifications in blood, saliva, and urine. Histones, the core component of the nucleosome, are essential for chromatin compaction and gene expression modulation. Increasing evidence suggests that circulating histones and histone complexes, originating from cell death or immune cell activation, could act as promising biomarkers for cancer detection and management. In this review, we provide an overview of circulating histones as a powerful liquid biopsy approach and methods for their detection. We highlight current knowledge on circulating histones in hematologic malignancies and solid cancer, with a focus on their role in cancer dissemination, monitoring, and tumorigenesis. Last, we describe recently developed strategies to identify cancer tissue-of-origin in blood plasma based on nucleosome positioning, inferred from nucleosomal DNA fragmentation footprint, which is independent of the genetic landscape.


Assuntos
Histonas , Neoplasias , Humanos , Histonas/metabolismo , Nucleossomos , Cromatina/genética , Neoplasias/diagnóstico , Neoplasias/genética
5.
FASEB J ; 35(8): e21793, 2021 08.
Artigo em Inglês | MEDLINE | ID: mdl-34320234

RESUMO

Gene expression and epigenetic processes in several brain regions regulate physiological processes such as cognitive functions and social behavior. MacroH2A1.1 is a ubiquitous variant of histone H2A that regulates cell stemness and differentiation in various organs. Whether macroH2A1.1 has a modulatory role in emotional behavior is unknown. Here, we employed macroH2A1.1 knock-out (-/- ) mice to perform a comprehensive battery of behavioral tests, and an assessment of hippocampal synaptic plasticity (long-term potentiation) accompanied by whole hippocampus RNA sequencing. MacroH2A1.1-/- mice exhibit a stunningly enhancement both of sociability and of active stress-coping behavior, reflected by the increased social behavior in social activity tests and higher mobility time in the forced swim test, respectively. They also display an increased hippocampal synaptic plasticity, accompanied by significant neurotransmission transcriptional networks changes. These results suggest that systemic depletion of histone macroH2A1.1 supports an epigenetic control necessary for hippocampal function and social behavior.


Assuntos
Comportamento Animal , Hipocampo/citologia , Histonas/classificação , Histonas/metabolismo , Plasticidade Neuronal/fisiologia , Adaptação Psicológica , Animais , Regulação da Expressão Gênica , Histonas/genética , Camundongos , Camundongos Knockout , Comportamento Social , Estresse Psicológico
7.
Cell Commun Signal ; 19(1): 44, 2021 04 08.
Artigo em Inglês | MEDLINE | ID: mdl-33832488

RESUMO

BACKGROUND: Nonalcoholic fatty liver disease (NAFLD) is increasingly prevalent and represents a growing challenge in terms of prevention and treatment. A minority of affected patients develops inflammation, subsequently fibrosis, cirrhosis and hepatocellular carcinoma (HCC). HCC is a leading cause of cancer-related death. An increased number of senescent cells correlate with age-related tissue degeneration during NAFLD-induced HCC. Senolytics are promising agents that target selectively senescent cells. Previous studies showed that whereas a combination of the senolytic drugs dasatinib and quercetin (D + Q) reduced NAFLD in mice, D + Q lacked efficacy in removing doxorubicin-induced ß-gal-positive senescent cells in human HCC xenografted mice. Whether D + Q has an effect on the age-associated spectrum of NAFLD-inflammation-HCC remains unknown. METHODS: Here, we utilized an established model of age- and obesity-associated HCC, the low dose diethylnitrosamine (DEN)/high fat diet (HFD), a regimen promoting liver inflammation and tumorigenesis over a long period of 9 months. Four groups of mice each were created: group 1 included control untreated mice; group 2 included mice treated with D + Q; group 3 included mice undergoing the DEN/HFD protocol; group 4 included mice undergoing the DEN/HFD protocol with the administration of D + Q. At the end of the chemical/dietary regimen, we analyzed liver damage and cell senescence by histopathology, qPCR and immunoblotting approaches. RESULTS: Unexpectedly, D + Q worsened liver disease progression in the DEN/HFD mouse model, slightly increasing histological damage and tumorigenesis, while having no effect on senescent cells removal. CONCLUSIONS: In summary, using an animal model that fully recapitulates NAFLD, we demonstrate that these compounds are ineffective against age-associated NAFLD-induced HCC. Video Abstract.


Assuntos
Envelhecimento/patologia , Dasatinibe/efeitos adversos , Progressão da Doença , Hepatopatias/patologia , Obesidade/patologia , Quercetina/efeitos adversos , Senoterapia/efeitos adversos , Envelhecimento/genética , Animais , Dieta Hiperlipídica , Dietilnitrosamina , Modelos Animais de Doenças , Regulação da Expressão Gênica , Hepatopatias/sangue , Hepatopatias/genética , Masculino , Camundongos Endogâmicos C57BL , Hepatopatia Gordurosa não Alcoólica/sangue , Hepatopatia Gordurosa não Alcoólica/genética , Hepatopatia Gordurosa não Alcoólica/patologia , Obesidade/sangue , Obesidade/genética
8.
J Pediatr Gastroenterol Nutr ; 73(2): 161-168, 2021 08 01.
Artigo em Inglês | MEDLINE | ID: mdl-33720087

RESUMO

OBJECTIVES: Non-alcoholic fatty liver disease (NAFLD) is the leading cause of chronic liver disease in both adults and children. Along with obesity and metabolic syndrome, genetic predisposition influences the progression of NAFLD. Here, we investigated the effect of lifespan/healthspan-related single nucleotide polymorphisms (SNPs) on metabolically associated fatty liver disease in children. METHODS: We evaluated the impact of 10 SNPs involved in both human liver/metabolic diseases and healthspan (interleukin-6 [IL-6] rs1800795, antisense non coding RNA in the INK4 locus (ANRIL) rs1556516, SH2B3/ATXN2 rs7137828, FURIN rs17514846, TP53 rs1042522, APOC3 rs2542052, KL rs9536314, KL rs9527025, SIRT6 rs107251, FOXO3 rs2802292) on NAFLD-related metabolic and liver features in 177 pediatric patients with biopsy-proven NAFLD, by comparing them to 146 healthy controls. We then applied a multidimensional reduction (MDR) case-control analysis of SNP-SNP interactions, to identify the joint effect of analyzed SNPs in predicting NAFLD and associated features. RESULTS: Discrete SNPs were significantly associated with individual metabolic NAFLD features, but none of them significantly associated with NAFLD diagnosis. By testing potential synergies using the MDR approach, the best combination to diagnose NAFLD (P = 0.0011) resulted in the one encompassing IL-6 rs1800795 and ANRIL rs1556516. Consistently, the risk combinations suggested by SNP-SNP analysis strongly associated with a higher level of fasting plasma blood glucose level (P = 0.024). CONCLUSION: In conclusion, here we demonstrated a synergic interaction between IL-6 rs1800795 and ANRIL rs1556516 in the diagnosis of NAFLD, and NAFLD-associated hyperglycemia in children. Larger studies are required to confirm our findings and to elucidate mechanisms by which the genetic interaction between these two genes influences healthspan in pediatric NAFLD.


Assuntos
Hepatopatia Gordurosa não Alcoólica , Sirtuínas , Adulto , Criança , Predisposição Genética para Doença , Humanos , Fígado , Longevidade , Hepatopatia Gordurosa não Alcoólica/genética , Polimorfismo de Nucleotídeo Único
9.
Int J Mol Sci ; 22(8)2021 Apr 16.
Artigo em Inglês | MEDLINE | ID: mdl-33923652

RESUMO

Studies over the past 30 years have revealed that adipose tissue is the major endocrine and paracrine organ of the human body. Arguably, adiopobiology has taken its reasonable place in studying obesity and related cardiometabolic diseases (CMDs), including Alzheimer's disease (AD), which is viewed herein as a neurometabolic disorder. The pathogenesis and therapy of these diseases are multiplex at basic, clinical and translational levels. Our present goal is to describe new developments in cardiometabolic and neurometabolic adipobiology. Accordingly, we focus on adipose- and/or skeletal muscle-derived signaling proteins (adipsin, adiponectin, nerve growth factor, brain-derived neuroptrophic factor, neurotrophin-3, irisin, sirtuins, Klotho, neprilysin, follistatin-like protein-1, meteorin-like (metrnl), as well as growth differentiation factor 11) as examples of metabotrophic factors (MTFs) implicated in the pathogenesis and therapy of obesity and related CMDs. We argue that these pathologies are MTF-deficient diseases. In 1993 the "vascular hypothesis of AD" was published and in the present review we propose the "vasculometabolic hypothesis of AD." We discuss how MTFs could bridge CMDs and neurodegenerative diseases, such as AD. Greater insights on how to manage the MTF network would provide benefits to the quality of human life.


Assuntos
Adipocinas/metabolismo , Síndrome Metabólica/metabolismo , Doenças Neurodegenerativas/metabolismo , Animais , Humanos , Síndrome Metabólica/tratamento farmacológico , Terapia de Alvo Molecular/métodos , Fatores de Crescimento Neural/metabolismo , Doenças Neurodegenerativas/tratamento farmacológico , Neuropeptídeos/metabolismo , Transdução de Sinais
10.
PLoS Biol ; 15(3): e2001951, 2017 03.
Artigo em Inglês | MEDLINE | ID: mdl-28358805

RESUMO

Fasting reduces glucose levels and protects mice against chemotoxicity, yet drugs that promote hyperglycemia are widely used in cancer treatment. Here, we show that dexamethasone (Dexa) and rapamycin (Rapa), commonly administered to cancer patients, elevate glucose and sensitize cardiomyocytes and mice to the cancer drug doxorubicin (DXR). Such toxicity can be reversed by reducing circulating glucose levels by fasting or insulin. Furthermore, glucose injections alone reversed the fasting-dependent protection against DXR in mice, indicating that elevated glucose mediates, at least in part, the sensitizing effects of rapamycin and dexamethasone. In yeast, glucose activates protein kinase A (PKA) to accelerate aging by inhibiting transcription factors Msn2/4. Here, we show that fasting or glucose restriction (GR) regulate PKA and AMP-activated protein kinase (AMPK) to protect against DXR in part by activating the mammalian Msn2/4 ortholog early growth response protein 1 (EGR1). Increased expression of the EGR1-regulated cardioprotective peptides atrial natriuretic peptide (ANP) and B-type natriuretic peptide (BNP) in heart tissue may also contribute to DXR resistance. Our findings suggest the existence of a glucose-PKA pathway that inactivates conserved zinc finger stress-resistance transcription factors to sensitize cells to toxins conserved from yeast to mammals. Our findings also describe a toxic role for drugs widely used in cancer treatment that promote hyperglycemia and identify dietary interventions that reverse these effects.


Assuntos
Antineoplásicos/farmacologia , Dexametasona/farmacologia , Proteína 1 de Resposta de Crescimento Precoce/metabolismo , Jejum/metabolismo , Glucose/farmacologia , Proteínas Quinases Ativadas por AMP/metabolismo , Animais , Fator Natriurético Atrial/metabolismo , Cardiotoxinas/toxicidade , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Citoproteção/efeitos dos fármacos , Dieta , Feminino , Hiperglicemia/patologia , Metformina/farmacologia , Camundongos , Camundongos Endogâmicos C57BL , Peptídeo Natriurético Encefálico/metabolismo , Estresse Fisiológico/efeitos dos fármacos , Fatores de Tempo
12.
Int J Mol Sci ; 21(22)2020 Nov 10.
Artigo em Inglês | MEDLINE | ID: mdl-33182805

RESUMO

Lipid catabolism and anabolism changes play a role in stemness acquisition by cancer cells, and cancer stem cells (CSCs) are particularly dependent on the activity of the enzymes involved in these processes. Lipidomic changes could play a role in CSCs' ability to cause disease relapse and chemoresistance. The exploration of lipid composition and metabolism changes in CSCs in the context of hepatocellular cancer (HCC) is still incomplete and their lipidomic scenario continues to be elusive. We aimed to evaluate through high-throughput mass spectrometry (MS)-based lipidomics the levels of the members of the six major classes of sphingolipids and phospholipids in two HCC cell lines (HepG2 and Huh-7) silenced for the expression of histone variant macroH2A1 (favoring stemness acquisition), or silenced for the expression of focal adhesion tyrosine kinase (FAK) (hindering aggressiveness and stemness). Transcriptomic changes were evaluated by RNA sequencing as well. We found definite lipidomic and transcriptomic changes in the HCC lines upon knockdown (KD) of macroH2A1 or FAK, in line with the acquisition or loss of stemness features. In particular, macroH2A1 KD increased total sphingomyelin (SM) levels and decreased total lysophosphatidylcholine (LPC) levels, while FAK KD decreased total phosphatidylcholine (PC) levels. In conclusion, in HCC cell lines knocked down for specific signaling/epigenetic processes driving opposite stemness potential, we defined a lipidomic signature that hallmarks hepatic CSCs to be exploited for therapeutic strategies.


Assuntos
Carcinoma Hepatocelular/metabolismo , Carcinoma Hepatocelular/patologia , Metabolismo dos Lipídeos , Neoplasias Hepáticas/metabolismo , Neoplasias Hepáticas/patologia , Células-Tronco Neoplásicas/metabolismo , Células-Tronco Neoplásicas/patologia , Biomarcadores Tumorais/genética , Biomarcadores Tumorais/metabolismo , Carcinoma Hepatocelular/genética , Linhagem Celular Tumoral , Quinase 1 de Adesão Focal/antagonistas & inibidores , Quinase 1 de Adesão Focal/deficiência , Quinase 1 de Adesão Focal/genética , Perfilação da Expressão Gênica , Regulação Neoplásica da Expressão Gênica , Técnicas de Silenciamento de Genes , Células Hep G2 , Histonas/antagonistas & inibidores , Histonas/deficiência , Histonas/genética , Humanos , Metabolismo dos Lipídeos/genética , Lipidômica , Neoplasias Hepáticas/genética , Lisofosfatidilcolinas/metabolismo , Fosfatidilcolinas/metabolismo , RNA-Seq , Esfingomielinas/metabolismo
13.
Hepatology ; 67(2): 636-650, 2018 02.
Artigo em Inglês | MEDLINE | ID: mdl-28913935

RESUMO

Hepatocellular carcinomas (HCC) contain a subpopulation of cancer stem cells (CSCs), which exhibit stem cell-like features and are responsible for tumor relapse, metastasis, and chemoresistance. The development of effective treatments for HCC will depend on a molecular-level understanding of the specific pathways driving CSC emergence and stemness. MacroH2A1 is a variant of the histone H2A and an epigenetic regulator of stem-cell function, where it promotes differentiation and, conversely, acts as a barrier to somatic-cell reprogramming. Here, we focused on the role played by the histone variant macroH2A1 as a potential epigenetic factor promoting CSC differentiation. In human HCC sections we uncovered a significant correlation between low frequencies of macroH2A1 staining and advanced, aggressive HCC subtypes with poorly differentiated tumor phenotypes. Using HCC cell lines, we found that short hairpin RNA-mediated macroH2A1 knockdown induces acquisition of CSC-like features, including the growth of significantly larger and less differentiated tumors when injected into nude mice. MacroH2A1-depleted HCC cells also exhibited reduced proliferation, resistance to chemotherapeutic agents, and stem-like metabolic changes consistent with enhanced hypoxic responses and increased glycolysis. The loss of macroH2A1 increased expression of a panel of stemness-associated genes and drove hyperactivation of the nuclear factor kappa B p65 pathway. Blocking phosphorylation of nuclear factor kappa B p65 on Ser536 inhibited the emergence of CSC-like features in HCC cells knocked down for macroH2A1. Conclusion: The absence of histone variant macroH2A1 confers a CSC-like phenotype to HCC cells in vitro and in vivo that depends on Ser536 phosphorylation of nuclear factor kappa B p65; this pathway may hold valuable targets for the development of CSC-focused treatments for HCC. (Hepatology 2018;67:636-650).


Assuntos
Carcinoma Hepatocelular/patologia , Histonas/fisiologia , Neoplasias Hepáticas/patologia , Células-Tronco Neoplásicas/patologia , Proliferação de Células , Perfilação da Expressão Gênica , Células Hep G2 , Humanos , Fosforilação , Fator de Transcrição RelA/metabolismo
14.
J Cell Physiol ; 233(2): 1202-1212, 2018 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-28471474

RESUMO

Hepatocellular carcinoma (HCC) has a poor outcome. Most HCCs develop in the context of liver fibrosis and cirrhosis caused by chronic inflammation. Short-term fasting approaches enhance the activity of chemotherapy in preclinical cancer models, other than HCC. Multi-tyrosine kinase inhibitor Sorafenib is the mainstay of treatment in HCC. However, its benefit is frequently short-lived. Whether fasting can alleviate liver fibrosis and whether combining fasting with Sorafenib is beneficial remains unknown. A 24 hr fasting (2% serum, 0.1% glucose)-induced changes on human hepatic stellate cells (HSC) LX-2 proliferation/viability/cell cycle were assessed by MTT and flow cytometry. Expression of lypolysaccharide (LPS)-induced activation markers (vimentin, αSMA) was evaluated by qPCR and immunoblotting. Liver fibrosis and inflammation were evaluated in a mouse model of steatohepatitis exposed to cycles of fasting, by histological and biochemical analyses. A 24 hr fasting-induced changes were also analyzed on the proliferation/viability/glucose uptake of human HCC cells exposed to Sorafenib. An expression panel of genes involved in survival, inflammation, and metabolism was examined by qPCR in HCC cells exposed to fasting and/or Sorafenib. Fasting decreased the proliferation and the activation of HSC. Repeated cycles of short term starvation were safe in mice but did not improve fibrosis. Fasting synergized with Sorafenib in hampering HCC cell growth and glucose uptake. Finally, fasting normalized the expression levels of genes which are commonly altered by Sorafenib in HCC cells. Fasting or fasting-mimicking diet diets should be evaluated in preclinical studies as a mean to potentiate the activity of Sorafenib in clinical use.


Assuntos
Antineoplásicos/farmacologia , Carcinoma Hepatocelular/tratamento farmacológico , Proliferação de Células/efeitos dos fármacos , Jejum/metabolismo , Células Estreladas do Fígado/efeitos dos fármacos , Neoplasias Hepáticas/tratamento farmacológico , Niacinamida/análogos & derivados , Compostos de Fenilureia/farmacologia , Animais , Carcinoma Hepatocelular/genética , Carcinoma Hepatocelular/metabolismo , Carcinoma Hepatocelular/patologia , Relação Dose-Resposta a Droga , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Glucose/metabolismo , Células Hep G2 , Células Estreladas do Fígado/metabolismo , Células Estreladas do Fígado/patologia , Humanos , Lipopolissacarídeos/farmacologia , Cirrose Hepática Experimental/metabolismo , Cirrose Hepática Experimental/patologia , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/metabolismo , Neoplasias Hepáticas/patologia , Camundongos Endogâmicos C57BL , Niacinamida/farmacologia , Hepatopatia Gordurosa não Alcoólica/metabolismo , Hepatopatia Gordurosa não Alcoólica/patologia , Sorafenibe , Fatores de Tempo
15.
Int J Food Sci Nutr ; 68(4): 455-466, 2017 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-27899042

RESUMO

We investigated the regulation of hepatic ER stress in healthy liver and adult or perinatally programmed diet-induced non-alcoholic fatty liver disease (NAFLD). Female mice were fed either obesogenic or control diet before mating, during pregnancy and lactation. Post-weaning, offspring from each maternal group were divided into either obesogenic or control diet. At six months, offspring were sacrificed at 4-h intervals over 24 h. Offspring fed obesogenic diets developed NAFLD phenotype, and the combination of maternal and offspring obesogenic diets exacerbated this phenotype. UPR signalling pathways (IREα, PERK, ATF6) and their downstream regulators showed different basal rhythmicity, which was modified in offspring exposed to obesogenic diet and maternal programming. The double obesogenic hit increased liver apoptosis measured by TUNEL staining, active caspase-3 and phospho-JNK and GRP78 promoter methylation levels. This study demonstrates that hepatic UPR is rhythmically activated. The combination of maternal obesity (MO) and obesogenic diets in offspring triggered altered UPR rhythmicity, DNA methylation and cellular apoptosis.


Assuntos
Dieta Hiperlipídica/efeitos adversos , Retículo Endoplasmático/fisiologia , Fígado/efeitos dos fármacos , Obesidade/induzido quimicamente , Estresse Fisiológico/efeitos dos fármacos , Ração Animal/análise , Animais , Chaperona BiP do Retículo Endoplasmático , Feminino , Homeostase , Camundongos , Camundongos Endogâmicos C57BL , Obesidade/metabolismo , Gravidez , Efeitos Tardios da Exposição Pré-Natal
16.
Mol Cancer ; 15: 6, 2016 Jan 15.
Artigo em Inglês | MEDLINE | ID: mdl-26768731

RESUMO

BACKGROUND: Circadian disruption and deranged molecular clockworks are involved in carcinogenesis. The cryptochrome genes (CRY1 and CRY2) encode circadian proteins important for the functioning of biological oscillators. Their expression in human colorectal cancer (CRC) and in colon cancer cell lines has not been evaluated so far. METHODS: We investigated CRY1 and CRY2 expression in fifty CRCs and in the CaCo2, HCT116, HT29, SW480 cell lines. RESULTS: CRY1 (p = 0.01) and CRY2 (p < 0.0001) expression was significantly changed in tumour tissue, as confirmed in a large independent CRC dataset. In addition, lower CRY1 mRNA levels were observed in patients in the age range of 62-74 years (p = 0.018), in female patients (p = 0.003) and in cancers located at the transverse colon (p = 0.008). Lower CRY2 levels were also associated with cancer location at the transverse colon (p = 0.007). CRC patients displaying CRY1 (p = 0.042) and CRY2 (p = 0.043) expression levels over the median were hallmarked by a poorer survival rate. Survey of selected colon cancer cell lines evidenced variable levels of cryptochrome genes expression and time-dependent changes in their mRNA levels. Moreover, they showed reduced apoptosis, increased proliferation and different response to 5-fluorouracil and oxaliplatin upon CRY1 and CRY2 ectopic expression. The relationship with p53 status came out as an additional layer of regulation: higher CRY1 and CRY2 protein levels coincided with a wild type p53 as in HCT116 cells and this condition only marginally affected the apoptotic and cell proliferation characteristics of the cells upon CRY ectopic expression. Conversely, lower CRY and CRY2 levels as in HT29 and SW480 cells coincided with a mutated p53 and a more robust apoptosis and proliferation upon CRY transfection. Besides, an heterogeneous pattern of ARNTL, WEE and c-MYC expression hallmarked the chosen colon cancer cell lines and likely influenced their phenotypic changes. CONCLUSION: Cryptochrome gene expression is altered in CRC, particularly in elderly subjects, female patients and cancers located at the transverse colon, affecting overall survival. Altered CRY1 and CRY2 expression patterns and the interplay with the genetic landscape in colon cancer cells may underlie phenotypic divergence that could influence disease behavior as well as CRC patients survival and response to chemotherapy.


Assuntos
Neoplasias Colorretais/genética , Criptocromos/genética , Regulação Neoplásica da Expressão Gênica , Idoso , Antineoplásicos/farmacologia , Apoptose/efeitos dos fármacos , Apoptose/genética , Ciclo Celular/efeitos dos fármacos , Ciclo Celular/genética , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Neoplasias Colorretais/patologia , Criptocromos/metabolismo , Feminino , Dosagem de Genes , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , Hibridização in Situ Fluorescente , Masculino , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Fatores de Tempo , Transfecção
17.
FASEB J ; 29(5): 1676-87, 2015 May.
Artigo em Inglês | MEDLINE | ID: mdl-25526730

RESUMO

The importance of epigenetic changes in the development of hepatic steatosis is largely unknown. The histone variant macroH2A1 under alternative splicing gives rise to macroH2A1.1 and macroH2A1.2. In this study, we show that the macroH2A1 isoforms play an important role in the regulation of lipid accumulation in hepatocytes. Hepatoma cell line and immortalized human hepatocytes transiently transfected or knocked down with macroH2A1 isoforms were used as in vitro model of fat-induced steatosis. Gene expressions were analyzed by quantitative PCR array and Western blot. Chromatin immunoprecipitation analysis was performed to check the association of histone H3 lysine 27 trimethylation (H3K27me3) and histone H3 lysine 4 trimethylation (H3K4me3) with the promoter of lipogenic genes. Livers from knockout mice that are resistant to lipid deposition despite a high-fat diet were used for histopathology. We found that macroH2A1.2 is regulated by fat uptake and that its overexpression caused an increase in lipid uptake, triglycerides, and lipogenic genes compared with macroH2A1.1. This suggests that macroH2A1.2 is important for lipid uptake, whereas macroH2A1.1 was found to be protective. The result was supported by a high positivity for macroH2A1.1 in knockout mice for genes targeted by macroH2A1 (Atp5a1 and Fam73b), that under a high-fat diet presented minimal lipidosis. Moreover, macroH2A1 isoforms differentially regulate the expression of lipogenic genes by modulating the association of the active (H3K4me3) and repressive (H3K27me3) histone marks on their promoters. This study underlines the importance of the replacement of noncanonical histones in the regulation of genes involved in lipid metabolism in the progression of steatosis.


Assuntos
Biomarcadores/metabolismo , Carcinoma Hepatocelular/patologia , Dieta Hiperlipídica/efeitos adversos , Epigenômica , Fígado Gorduroso/metabolismo , Fígado Gorduroso/patologia , Hepatócitos/patologia , Histonas/metabolismo , Animais , Western Blotting , Carcinoma Hepatocelular/metabolismo , Células Cultivadas , Imunoprecipitação da Cromatina , Fígado Gorduroso/etiologia , Feminino , Imunofluorescência , Perfilação da Expressão Gênica , Hepatócitos/metabolismo , Histonas/genética , Humanos , Técnicas Imunoenzimáticas , Peroxidação de Lipídeos , Fígado/metabolismo , Fígado/patologia , Neoplasias Hepáticas/metabolismo , Neoplasias Hepáticas/patologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , ATPases Mitocondriais Próton-Translocadoras/fisiologia , Análise de Sequência com Séries de Oligonucleotídeos , Regiões Promotoras Genéticas/genética , Isoformas de Proteínas , RNA Mensageiro/genética , Reação em Cadeia da Polimerase em Tempo Real , Reação em Cadeia da Polimerase Via Transcriptase Reversa
18.
J Gastroenterol Hepatol ; 31(2): 475-83, 2016 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-26270240

RESUMO

BACKGROUND: The parasympathetic nervous system (PNS), via neurotransmitter acetylcholine (ACh), modulates fibrogenesis in animal models. However, the role of ACh in human hepatic fibrogenesis is unclear. AIMS: We aimed to determine the fibrogenic responses of human hepatic stellate cells (hHSC) to ACh and the relevance of the PNS in hepatic fibrosis in patients with non-alcoholic steatohepatitis (NASH). METHODS: Primary hHSC were analyzed for synthesis of endogenous ACh and acetylcholinesterase and gene expression of choline acetyltransferase and muscarinic ACh receptors (mAChR). Cell proliferation and fibrogenic markers were analyzed in hHSC exposed to ACh, atropine, mecamylamine, methoctramine, and 4-diphenylacetoxy-N-methylpiperidine methiodide. mAChR expression was analyzed in human NASH scored for fibrosis. RESULTS: We observed that hHSC synthesize ACh and acetylcholinesterase and express choline acetyltransferase and M1-M5 mAChR. We also show that M2 was increased during NASH progression, while both M2 and M3 were found upregulated in activated hHSC. Furthermore, endogenous ACh is required for hHSC basal growth. Exogenous ACh resulted in hHSC hyperproliferation via mAChR and phosphoinositide 3-kinase and Mitogen-activated protein kinase kinase (MEK) signaling pathways, as well as increased fibrogenic markers. CONCLUSION: We show that ACh regulates hHSC activation via M2 and M3 mAChR involving the phosphoinositide 3-kinase and MEK pathways in vitro. Finally, we provide evidence that the PNS may be involved in human NASH fibrosis.


Assuntos
Acetilcolina/efeitos adversos , Acetilcolina/fisiologia , Células Estreladas do Fígado/patologia , Cirrose Hepática/etiologia , Cirrose Hepática/patologia , Hepatopatia Gordurosa não Alcoólica/etiologia , Receptores Muscarínicos/fisiologia , 1-Fosfatidilinositol 4-Quinase/fisiologia , Acetilcolina/biossíntese , Acetilcolinesterase/biossíntese , Células Cultivadas , Colina O-Acetiltransferase/genética , Colina O-Acetiltransferase/metabolismo , Progressão da Doença , Fibrose , Expressão Gênica , Células Estreladas do Fígado/metabolismo , Humanos , Quinases de Proteína Quinase Ativadas por Mitógeno/fisiologia , Hepatopatia Gordurosa não Alcoólica/patologia , Sistema Nervoso Parassimpático/fisiologia , Receptores Muscarínicos/genética , Receptores Muscarínicos/metabolismo , Transdução de Sinais/fisiologia , Regulação para Cima
19.
J Mol Cell Cardiol ; 88: 111-9, 2015 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-26456066

RESUMO

MicroRNAs are endogenously expressed small noncoding RNAs that regulate gene expression. Laminar blood flow induces atheroprotective gene expression in endothelial cells (ECs) in part by upregulating the transcription factor KLF2. Here, we identified KLF2- and flow-responsive miRs that affect gene expression in ECs. Bioinformatic assessment of mRNA expression patterns identified the miR-30-5p seed sequence to be highly enriched in mRNAs that are downregulated by KLF2. Indeed, KLF2 overexpression and shear stress stimulation in vitro and in vivo increased the expression of miR-30-5p family members. Furthermore, we identified angiopoietin 2 (Ang2) as a target of miR-30. MiR-30 overexpression reduces Ang2 levels, whereas miR-30 inhibition by LNA-antimiRs induces Ang2 expression. Consistently, miR-30 reduced basal and TNF-α-induced expression of the inflammatory cell­cell adhesion molecules E-selectin, ICAM1 and VCAM1, which was rescued by stimulation with exogenous Ang2. In summary, KLF2 and shear stress increase the expression of the miR-30-5p family which acts in an anti-inflammatory manner in ECs by impairing the expression of Ang2 and inflammatory cell­cell adhesion molecules. The upregulation of miR-30-5p family members may contribute to the atheroprotective effects of shear stress.


Assuntos
Células Endoteliais da Veia Umbilical Humana/metabolismo , Fatores de Transcrição Kruppel-Like/genética , MicroRNAs/genética , RNA Mensageiro/genética , Estresse Mecânico , Proteínas de Transporte Vesicular/genética , Adenoviridae/genética , Sequência de Bases , Biologia Computacional , Selectina E/genética , Selectina E/metabolismo , Regulação da Expressão Gênica , Hemorreologia , Células Endoteliais da Veia Umbilical Humana/citologia , Células Endoteliais da Veia Umbilical Humana/efeitos dos fármacos , Humanos , Molécula 1 de Adesão Intercelular/genética , Molécula 1 de Adesão Intercelular/metabolismo , Fatores de Transcrição Kruppel-Like/metabolismo , Lentivirus/genética , MicroRNAs/metabolismo , Dados de Sequência Molecular , RNA Mensageiro/metabolismo , Transdução de Sinais , Transdução Genética , Fator de Necrose Tumoral alfa/farmacologia , Molécula 1 de Adesão de Célula Vascular/genética , Molécula 1 de Adesão de Célula Vascular/metabolismo , Proteínas de Transporte Vesicular/metabolismo
20.
Biochim Biophys Acta ; 1843(6): 1225-36, 2014 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-24631504

RESUMO

UNLABELLED: MicroRNAs (miRNAs) regulate diverse biological processes by inhibiting translation or inducing degradation of target mRNAs. miR-145 is a candidate tumor suppressor in colorectal carcinoma (CRC). Colorectal carcinogenesis involves deregulation of cellular processes controlled by a number of intertwined chief transcription factors, such as PPARγ and SOX9. Since PPAR family members are able to modulate complex miRNAs networks, we hypothesized a role of miRNA-145 in the interaction between PPARγ and SOX9 in colorectal carcinogenesis. To address this issue, we evaluated gene expression in tissue specimens of CRC patients and we took advantage of invitro models represented by CRC derived cell lines (CaCo2, SW480, HCT116, and HT-29), employing PPARγ activation and/or miRNA-145 ectopic overexpression to analyze how their interplay impact the expression of SOX9 and the development of a malignant phenotype. RESULTS: PPARγ regulates the expression of miR-145 by directly binding to a PPAR response element (PPRE) in its promoter at -1207/-1194bp from the transcription start site. The binding is essential for miR-145 upregulation by PPARγ upon rosiglitazone treatment. Ectopic expression of miR-145, in turn, regulates SOX9 expression through the binding to specific seed motifs. The PPARγ-miR-145-SOX9 axis overarches cell cycle progression, invasiveness and differentiation of CRC derived cell lines. Together, these results suggest that miR-145 is a novel target of PPARγ, acts as a tumor suppressor in CRC cell lines and is a key regulator of intestinal cell differentiation by directly targeting SOX9, a marker of undifferentiated progenitors in the colonic crypts.


Assuntos
Neoplasias Colorretais/genética , Regulação Neoplásica da Expressão Gênica , MicroRNAs/genética , PPAR gama/metabolismo , Fatores de Transcrição SOX9/metabolismo , Idoso , Western Blotting , Ciclo Celular , Movimento Celular , Proliferação de Células , Estudos de Coortes , Colo/metabolismo , Colo/patologia , Neoplasias Colorretais/metabolismo , Neoplasias Colorretais/patologia , Primers do DNA/química , Primers do DNA/genética , Feminino , Humanos , Luciferases/metabolismo , Masculino , Mutagênese , PPAR gama/genética , Fenótipo , Regiões Promotoras Genéticas/genética , RNA Mensageiro/genética , Reação em Cadeia da Polimerase em Tempo Real , Reto/metabolismo , Reto/patologia , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Fatores de Transcrição SOX9/genética , Ativação Transcricional , Células Tumorais Cultivadas
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