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1.
Gut ; 69(2): 231-242, 2020 02.
Artigo em Inglês | MEDLINE | ID: mdl-31068366

RESUMO

OBJECTIVE: Gastric cancer (GC) is a leading cause of cancer mortality. Previous studies have shown that hepatocyte nuclear factor-4α (HNF4α) is specifically overexpressed in GC and functionally required for GC development. In this study, we investigated, on a genome-wide scale, target genes of HNF4α and oncogenic pathways driven by HNF4α and HNF4α target genes. DESIGN: We performed HNF4α chromatin immunoprecipitation followed by sequencing across multiple GC cell lines, integrating HNF4α occupancy data with (epi)genomic and transcriptome data of primary GCs to define HNF4α target genes of in vitro and in vivo relevance. To investigate mechanistic roles of HNF4α and HNF4α targets, we performed cancer metabolic measurements, drug treatments and functional assays including murine xenograft experiments. RESULTS: Gene expression analysis across 19 tumour types revealed HNF4α to be specifically upregulated in GCs. Unbiased pathway analysis revealed organic acid metabolism as the top HNF4α-regulated pathway, orthogonally supported by metabolomic analysis. Isocitrate dehydrogenase 1 (IDH1) emerged as a convergent HNF4α direct target gene regulating GC metabolism. We show that wild-type IDH1 is essential for GC cell survival, and that certain GC cells can be targeted by IDH1 inhibitors. CONCLUSIONS: Our results highlight a role for HNF4α in sustaining GC oncogenic metabolism, through the regulation of IDH1. Drugs targeting wild-type IDH1 may thus have clinical utility in GCs exhibiting HNF4α overexpression, expanding the role of IDH1 in cancer beyond IDH1/2 mutated malignancies.


Assuntos
Fator 4 Nuclear de Hepatócito/genética , Isocitrato Desidrogenase/metabolismo , Neoplasias Gástricas/genética , Animais , Antineoplásicos/farmacologia , Antineoplásicos/uso terapêutico , Linhagem Celular Tumoral , Inibidores Enzimáticos/farmacologia , Inibidores Enzimáticos/uso terapêutico , Feminino , Perfilação da Expressão Gênica/métodos , Regulação Neoplásica da Expressão Gênica , Marcação de Genes/métodos , Fator 4 Nuclear de Hepatócito/metabolismo , Humanos , Isocitrato Desidrogenase/antagonistas & inibidores , Camundongos Endogâmicos NOD , Terapia de Alvo Molecular/métodos , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/metabolismo , Regiões Promotoras Genéticas/genética , Neoplasias Gástricas/tratamento farmacológico , Neoplasias Gástricas/metabolismo , Neoplasias Gástricas/patologia , Regulação para Cima/genética , Ensaios Antitumorais Modelo de Xenoenxerto
2.
Artigo em Inglês | MEDLINE | ID: mdl-31525459

RESUMO

As the first marine teleost demonstrated to biosynthesize long-chain polyunsaturated fatty acids (LC-PUFAs) from C18 precursors such as linoleic acid (LOA, 18:2n-6) and α-linolenic acid (ALA, 18:3n-3), the rabbitfish (Siganus canaliculatus) contains the complete enzymatic system for LC-PUFA biosynthesis, including Δ6/Δ5 fatty acid desaturase (Fad), Δ4 Fad, and elongase 5 (Elovl5). Previously, our group demonstrated that hepatocyte nuclear factor 4α (Hnf4α) is a transcription factor (TF) for rabbitfish Δ4 fad and elovl5, and interacts with the core promoter of Δ6/Δ5 fad. To fully clarify the role of Hnf4α in the regulation of LC-PUFA biosynthesis, the present study aimed to explore the regulatory role of Hnf4α on Δ6/Δ5 fad gene expression. First, Hnf4α overexpression and agonist assays identified the Hnf4α response region in the Δ6/Δ5 fad core promoter as -456 bp to +51 bp. Bioinformatic analysis predicted four potential Hnf4α binding elements in the core promoter, which were confirmed by site-directed mutation and functional assays in a dual luciferase assay system. Moreover, the mRNA expression levels of hnf4α, Δ6/Δ5 fad, and Δ4 fad were significantly increased in the S. canaliculatus hepatocyte line (SCHL) cells after treatment with Hnf4α agonists (Alverine and Benfluorex) or its mRNA overexpression. By contrast, the expression levels of these three genes were markedly decreased after hnf4a small interfering RNA (siRNA) transfection. The results indicated that Hnf4α has a regulatory effect on rabbitfish Δ6/Δ5 fad gene transcription, identifying Hnf4α as a TF of Δ6/Δ5 fad in vertebrates for the first time.


Assuntos
Ácidos Graxos Dessaturases/biossíntese , Proteínas de Peixes/metabolismo , Peixes/metabolismo , Regulação Enzimológica da Expressão Gênica/fisiologia , Fator 4 Nuclear de Hepatócito/metabolismo , Linoleoil-CoA Desaturase/biossíntese , Animais , Ácidos Graxos Dessaturases/genética , Proteínas de Peixes/genética , Peixes/genética , Fator 4 Nuclear de Hepatócito/genética , Linoleoil-CoA Desaturase/genética
3.
Exp Suppl ; 111: 385-416, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31588541

RESUMO

In addition to the common types of diabetes mellitus, two major monogenic diabetes forms exist. Maturity-onset diabetes of the young (MODY) represents a heterogenous group of monogenic, autosomal dominant diseases. MODY accounts for 1-2% of all diabetes cases, and it is not just underdiagnosed but often misdiagnosed to type 1 or type 2 diabetes. More than a dozen MODY genes have been identified to date, and their molecular classification is of great importance in the correct treatment decision and in the judgment of the prognosis. The most prevalent subtypes are HNF1A, GCK, and HNF4A. Genetic testing for MODY has changed recently due to the technological advancements, as contrary to the sequential testing performed in the past, nowadays all MODY genes can be tested simultaneously by next-generation sequencing. The other major group of monogenic diabetes is neonatal diabetes mellitus which can be transient or permanent, and often the diabetes is a part of a syndrome. It is a severe monogenic disease appearing in the first 6 months of life. The hyperglycemia usually requires insulin. There are two forms, permanent neonatal diabetes mellitus (PNDM) and transient neonatal diabetes mellitus (TNDM). In TNDM, the diabetes usually reverts within several months but might relapse later in life. The incidence of NDM is 1:100,000-1:400,000 live births, and PNDM accounts for half of the cases. Most commonly, neonatal diabetes is caused by mutations in KCNJ11 and ABCC8 genes encoding the ATP-dependent potassium channel of the ß cell. Neonatal diabetes has experienced a quick and successful transition into the clinical practice since the discovery of the molecular background. In case of both genetic diabetes groups, recent guidelines recommend genetic testing.


Assuntos
Diabetes Mellitus Tipo 2/genética , Diabetes Mellitus/genética , Doenças do Recém-Nascido/genética , Testes Genéticos , Fator 1-alfa Nuclear de Hepatócito/genética , Fator 4 Nuclear de Hepatócito/genética , Humanos , Recém-Nascido , Mutação , Canais de Potássio Corretores do Fluxo de Internalização/genética , Proteínas Serina-Treonina Quinases/genética , Receptores Sulfonilureia/genética
4.
Nat Genet ; 51(10): 1459-1474, 2019 10.
Artigo em Inglês | MEDLINE | ID: mdl-31578528

RESUMO

Elevated serum urate levels cause gout and correlate with cardiometabolic diseases via poorly understood mechanisms. We performed a trans-ancestry genome-wide association study of serum urate in 457,690 individuals, identifying 183 loci (147 previously unknown) that improve the prediction of gout in an independent cohort of 334,880 individuals. Serum urate showed significant genetic correlations with many cardiometabolic traits, with genetic causality analyses supporting a substantial role for pleiotropy. Enrichment analysis, fine-mapping of urate-associated loci and colocalization with gene expression in 47 tissues implicated the kidney and liver as the main target organs and prioritized potentially causal genes and variants, including the transcriptional master regulators in the liver and kidney, HNF1A and HNF4A. Experimental validation showed that HNF4A transactivated the promoter of ABCG2, encoding a major urate transporter, in kidney cells, and that HNF4A p.Thr139Ile is a functional variant. Transcriptional coregulation within and across organs may be a general mechanism underlying the observed pleiotropy between urate and cardiometabolic traits.


Assuntos
Doenças Cardiovasculares/sangue , Marcadores Genéticos , Gota/sangue , Doenças Metabólicas/sangue , Polimorfismo de Nucleotídeo Único , Transdução de Sinais , Ácido Úrico/sangue , Membro 2 da Subfamília G de Transportadores de Cassetes de Ligação de ATP/genética , Doenças Cardiovasculares/epidemiologia , Doenças Cardiovasculares/genética , Estudos de Coortes , Loci Gênicos , Predisposição Genética para Doença , Estudo de Associação Genômica Ampla , Gota/epidemiologia , Gota/genética , Fator 1-alfa Nuclear de Hepatócito/genética , Fator 4 Nuclear de Hepatócito/genética , Humanos , Rim/metabolismo , Rim/patologia , Fígado/metabolismo , Fígado/patologia , Doenças Metabólicas/epidemiologia , Doenças Metabólicas/genética , Proteínas de Neoplasias/genética , Especificidade de Órgãos
5.
J Agric Food Chem ; 67(40): 11119-11128, 2019 Oct 09.
Artigo em Inglês | MEDLINE | ID: mdl-31525874

RESUMO

Xanthohumol (Xan) is a prenylated chalcone mainly found in hops; it has been demonstrated to function against hypercholesterolemia, hyperlipidemia, and atherosclerosis. In this study, we focused on the hypocholesterolemic effect of Xan on cholesterol uptake and the underlying molecular mechanisms of Xan in human intestinal Caco-2 cells. The microarray data showed that Niemann-Pick C1-like 1 (NPC1L1), an essential transporter for dietary cholesterol absorption, was significantly downregulated in Xan-treated Caco-2 cells. We demonstrated that Xan (10 and 20 µM) suppressed the mRNA and protein expression of NPC1L1 by 0.65 ± 0.12-fold and 0.54 ± 0.15-fold and 0.72 ± 0.04-fold and 0.44 ± 0.12-fold, respectively, compared to that of the vehicle-treated Caco-2 cells. Moreover, Xan (10 and 20 µM) significantly inhibited cholesterol uptake by approximately 12 and 32% in Caco-2 cells. NPC1L1 promoter activity was significantly suppressed by Xan, and a DNA element within the NPC1L1 promoter involved in Xan-mediated NPC1L1 reduction located between the -120 and -20 positions was identified. Moreover, Xan markedly decreased the mRNA and protein levels of hepatocyte nuclear factor 4α (HNF-4α), a critical activator of NPC1L1 transcription, and subsequently attenuated HNF-4α/NPC1L1 promoter complex formation, resulting in the suppression of NPC1L1 gene expression. Finally, we demonstrated that Xan markedly abolished lovastatin-induced NPC1L1 overexpression in Caco-2 cells. These findings reveal that Xan suppresses NPC1L1 expression via downregulation of HNF-4α and exerts inhibitory effects on cholesterol uptake in the intestinal Caco-2 cells. Our findings suggest Xan could serve as a potential cholesterol-lowering agent and supplement for statin therapy.


Assuntos
Colesterol/metabolismo , Flavonoides/farmacologia , Fator 4 Nuclear de Hepatócito/metabolismo , Proteínas de Membrana/genética , Propiofenonas/farmacologia , Anticolesterolemiantes/farmacologia , Transporte Biológico/efeitos dos fármacos , Células CACO-2 , Regulação para Baixo/efeitos dos fármacos , Expressão Gênica/efeitos dos fármacos , Fator 4 Nuclear de Hepatócito/genética , Humanos , Proteínas de Membrana/metabolismo , Regiões Promotoras Genéticas
6.
Artif Cells Nanomed Biotechnol ; 47(1): 3072-3078, 2019 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-31343368

RESUMO

Objective: To investigate the potential mechanism of microRNA-34a (miR-34a) on proliferation, migration and invasion of paediatric neuroblastoma cells. Methods: The expression of miR-34a and hepatocyte nuclear factor 4α (HNF4α) in paediatric neuroblastoma tissues were detected by RT-q PCR and Western blot, respectively. Cell proliferation, migration, invasion and the expression of matrix metalloproteinase 2 (MMP-2) and matrix metalloproteinase 14 (MMP-14) after transfection of miR-34a mimics or HNF4α siRNA into SH-SY5Y cells were detected by MTT assay, Transwell assay and Western blot assay, respectively. The target relationship between miR-34a and HNF4α was verified by TargetScan online prediction and dual-luciferase assay. Cell proliferation, migration and invasion of SH-SY5Y cells after overexpression of miR-34a and HNF4α were detected. Results: The expression level of miR-34a was decreased (p < .05) while the expression level of HNF4α was increased (p < .05) in paediatric neuroblastoma tissues. Over- expression of Mi-34a or knockdown of HNF4α in SH-SY5Y cells could lead to a decreased of cell proliferation, migration, invasion and the expression of MMP-2 and MMP-14 (p < .05). The results of TargetScan online prediction and dual-luciferase assay indicted that HNF4α was a potential target gene for miR-34a. Overexpression of HNF4α could reverse the inhibition of miR-34a on proliferation, migration and invasion of SH-SY5Y cells. Conclusion: The expression of miR-34a was down-regulated in paediatric neuroblastoma tissues, and overexpression of miR-34a could inhibit proliferation, migration and invasion of SH-SY5Y cells by targeting HNF4α.


Assuntos
Movimento Celular/genética , Fator 4 Nuclear de Hepatócito/genética , MicroRNAs/genética , Neuroblastoma/genética , Neuroblastoma/patologia , Sequência de Bases , Linhagem Celular Tumoral , Proliferação de Células/genética , Criança , Pré-Escolar , Feminino , Regulação Neoplásica da Expressão Gênica , Técnicas de Silenciamento de Genes , Fator 4 Nuclear de Hepatócito/deficiência , Humanos , Lactente , Masculino , Metaloproteinase 14 da Matriz/genética , Metaloproteinase 2 da Matriz/genética , Invasividade Neoplásica/genética
7.
Medicine (Baltimore) ; 98(28): e16424, 2019 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-31305461

RESUMO

Although the changes in DNA methylation are assumed to be due to the association between adverse intrauterine conditions and adult metabolic health, evidence from human studies is rare. Little is known about the changes in DNA methylation present at birth that affect metabolic profiles in childhood. Previous studies have shown that the melanocortin 4 receptor (MC4R) and hepatocyte nuclear factor 4 alpha (HNF4α) genes are associated with obesity and metabolic disorders. Thus, we investigated the associations of the DNA methylation statuses of MC4R and HNF4α in cord blood with metabolic profiles in childhood.We collected data from 90 children 7 to 9 years of age included in the Ewha Birth & Growth Cohort Study in Korea. DNA methylation was analyzed by pyrosequencing. The children were split into 2 groups according to the cutoff triglyceride (TG) levels (<110 and ≥110 mg/dL).The methylation statuses of MC4R and HNF4α at birth were significantly associated with the TG level in childhood (P < .05). It was interesting to note that the methylation statuses of MC4R and HNF4α in cord blood were significantly decreased, whereas childhood body mass index was significantly increased, in children with high TG levels compared with children with low TG levels (P < .05).Our findings show that the methylation statuses of MC4R and HNF4α at birth are associated with metabolic profiles in childhood. These epigenetic modifications occurring in early life may contribute to subsequent metabolic-related disorders. Thus, we suggest that DNA methylation status in cord blood may be predictive of the risk of developing metabolic syndrome.


Assuntos
Metilação de DNA , Fator 4 Nuclear de Hepatócito/genética , Regiões Promotoras Genéticas , Receptor Tipo 4 de Melanocortina/genética , Triglicerídeos/sangue , Índice de Massa Corporal , Criança , Desenvolvimento Infantil , Ilhas de CpG , Epigênese Genética , Feminino , Sangue Fetal/metabolismo , Seguimentos , Fator 4 Nuclear de Hepatócito/metabolismo , Humanos , Estudos Longitudinais , Masculino , Estudos Prospectivos , Receptor Tipo 4 de Melanocortina/metabolismo
8.
Hematol Oncol ; 37(4): 474-482, 2019 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-31325181

RESUMO

LncRNAs play critical roles in various pathophysiological and biological processes, such as protein translation, RNA splicing, and epigenetic modification. Indeed, abundant evidences demonstrated that lncRNA act as competing endogenous RNAs (ceRNAs) to participate in tumorigenesis. However, little is known about the underlying function of lncRNA in nonhomologous end joining (NHEJ) pathway 1 (LINP1) in pediatric and adolescent acute myeloid leukemia (AML). The expression of LINP1 was examined in AML patient samples by qRT-PCR. Cell proliferation was examined by CCK-8 and Edu assays. ß-Galactosidase senescence assay, mGlucose uptake assay, lactate production assay, and Gene Ontology (GO) analysis were performed for functional analysis. We found that LINP1 was significantly overexpressed in AML patients at diagnosis, whereas downregulated after complete remission (CR). Furthermore, knockdown of LINP1 expression remarkably suppressed glucose uptake and AML cell maintenance. Mechanistically, LINP1 was found to inhibit the glucose metabolism by suppressing the expression of HNF4a. Both LINP1 and HNF4a knockdown reduced the expression levels of AMPK phosphorylation and WNT5A, indicating for the first time that LINP1 strengthened the HNF4a-AMPK/WNT5A signaling pathway involved in cell glucose metabolism modulation and AML cell survival. Taken together, our results indicated that LINP1 promotes the malignant phenotype of AML cells and stimulates glucose metabolism, which can be regarded as a potential prognostic marker and therapeutic target for AML.


Assuntos
Adenilato Quinase/fisiologia , Fator 4 Nuclear de Hepatócito/fisiologia , Leucemia Mieloide Aguda/genética , RNA Longo não Codificante/fisiologia , RNA Neoplásico/fisiologia , Transdução de Sinais/fisiologia , Proteína Wnt-5a/fisiologia , Adolescente , Animais , Medula Óssea/patologia , Divisão Celular , Criança , Regulação Leucêmica da Expressão Gênica , Técnicas de Silenciamento de Genes , Ontologia Genética , Glucose/metabolismo , Fator 4 Nuclear de Hepatócito/biossíntese , Fator 4 Nuclear de Hepatócito/genética , Humanos , Leucemia Mieloide Aguda/metabolismo , Leucemia Mieloide Aguda/patologia , Masculino , Camundongos , Camundongos Endogâmicos NOD , Camundongos SCID , Transplante de Neoplasias , Púrpura Trombocitopênica Idiopática/metabolismo , Interferência de RNA , RNA Longo não Codificante/biossíntese , RNA Longo não Codificante/genética , RNA Neoplásico/biossíntese , RNA Neoplásico/genética , RNA Interferente Pequeno/genética , Distribuição Aleatória , Indução de Remissão , Transdução de Sinais/genética , Células THP-1
9.
Nat Commun ; 10(1): 3126, 2019 07 16.
Artigo em Inglês | MEDLINE | ID: mdl-31311938

RESUMO

Alcoholic hepatitis (AH) is a life-threatening condition characterized by profound hepatocellular dysfunction for which targeted treatments are urgently needed. Identification of molecular drivers is hampered by the lack of suitable animal models. By performing RNA sequencing in livers from patients with different phenotypes of alcohol-related liver disease (ALD), we show that development of AH is characterized by defective activity of liver-enriched transcription factors (LETFs). TGFß1 is a key upstream transcriptome regulator in AH and induces the use of HNF4α P2 promoter in hepatocytes, which results in defective metabolic and synthetic functions. Gene polymorphisms in LETFs including HNF4α are not associated with the development of AH. In contrast, epigenetic studies show that AH livers have profound changes in DNA methylation state and chromatin remodeling, affecting HNF4α-dependent gene expression. We conclude that targeting TGFß1 and epigenetic drivers that modulate HNF4α-dependent gene expression could be beneficial to improve hepatocellular function in patients with AH.


Assuntos
Hepatite Alcoólica/genética , Fator 4 Nuclear de Hepatócito/metabolismo , Hepatócitos/patologia , Fígado/patologia , Fator de Crescimento Transformador beta1/metabolismo , Adulto , Idoso , Animais , Biópsia , Montagem e Desmontagem da Cromatina , Metilação de DNA , Progressão da Doença , Epigênese Genética , Feminino , Perfilação da Expressão Gênica , Regulação da Expressão Gênica , Hepatite Alcoólica/patologia , Fator 4 Nuclear de Hepatócito/genética , Humanos , Fígado/citologia , Masculino , Pessoa de Meia-Idade , Polimorfismo Genético , Análise de Sequência de RNA , Fator de Crescimento Transformador beta1/genética
10.
Oncol Rep ; 42(3): 1066-1074, 2019 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-31322246

RESUMO

Renal cell carcinoma (RCC) is the most common malignant disease of the kidneys in adults. Patients with metastatic RCC have an unusually poor prognosis and exhibit resistance to all current therapies. Therefore, it is necessary to explore novel molecules involved in the progression of RCC and to identify effective therapeutic targets. Hepatocyte nuclear factor­4α (HNF­4α) serves an important role in hepatocyte differentiation and is involved in the progression of liver cancer; however, the functional role of HNF­4α has not been well established in RCC. The present study reported that HNF­4α expression was markedly downregulated in RCC tissue samples compared with in normal controls by immunohistochemistry and RNA­sequencing analysis. Statistical analysis demonstrated that HNF­4α downregulation was significantly associated with tumor stage, recurrence, metastasis and poor prognosis in patients with RCC. Furthermore, wound­healing and Transwell assays revealed that downregulation of HNF­4α promoted cell migration and invasion by transcriptionally regulating E­cadherin in RCC. Finally, a positive correlation was revealed between HNF­4α expression and E­cadherin expression, and patients with low E­cadherin expression also had a poor prognosis. These findings may provide novel insights into the biological effects of HNF­4α and lay the foundation for the discovery of molecular therapeutic targets in RCC.


Assuntos
Antígenos CD/metabolismo , Biomarcadores Tumorais/metabolismo , Caderinas/metabolismo , Carcinoma de Células Renais/patologia , Movimento Celular , Fator 4 Nuclear de Hepatócito/metabolismo , Neoplasias Renais/secundário , Recidiva Local de Neoplasia/patologia , Idoso , Antígenos CD/genética , Apoptose , Biomarcadores Tumorais/genética , Caderinas/genética , Carcinoma de Células Renais/genética , Carcinoma de Células Renais/metabolismo , Estudos de Casos e Controles , Proliferação de Células , Feminino , Seguimentos , Regulação Neoplásica da Expressão Gênica , Fator 4 Nuclear de Hepatócito/genética , Humanos , Neoplasias Renais/genética , Neoplasias Renais/metabolismo , Masculino , Pessoa de Meia-Idade , Invasividade Neoplásica , Recidiva Local de Neoplasia/genética , Recidiva Local de Neoplasia/metabolismo , Prognóstico , Taxa de Sobrevida , Células Tumorais Cultivadas
11.
J Agric Food Chem ; 67(28): 8007-8019, 2019 Jul 17.
Artigo em Inglês | MEDLINE | ID: mdl-31268702

RESUMO

Cow and human milk have been reported to contain dioxins ranging from 0.023 to 26.46 and 0.88 to 19 pg/g of fat, respectively. However, the toxic effects of the dioxins in the milk in this range of concentrations were not explored. Therefore, considering the outbred livestock tissues as better models than inbred laboratory animals, the present study targeted to study the effect of dioxins present in the milk on three-dimensionally (3D) cultured buffalo primary hepatocyte spheroids. The spheroids were treated with a model dioxin, 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), directly and also through milk fat at different concentrations (i.e, 0.02-20 pg/mL) for 24 h. Among the liver-cell-specific (ALB, HNF4α, and AFP) genes, a similar ALB and upregulated HNF4α expression at all treatments indicated the functional and transcriptionally active hepatocyte spheroids. Supportingly, no significant difference in the antiapoptotic gene expression between the treatments of milk fat and milk fat containing dioxins indicated the survivability of the spheroids during dioxin treatments. Among the selected TCDD responsive (CYP1A1, CYP1A2, AHR, CYP1B1, and TIPARP) genes, a nonsignificant increasing trend of the CYP1A1 expression was observed from 0.2 to 10 pg/mL of TCDD concentration through milk fat. This pattern was similar to the reported insensitive response of human primary hepatocytes toward dioxins than that of rat primary hepatocytes. This may indicate that the buffalo hepatocyte spheroids could be better models than rats for TCDD hepatotoxic studies. Further, TCDD in the milk in the range of 0.02-20 pg/mL concentration may not be very hepatotoxic.


Assuntos
Dioxinas/farmacologia , Hepatócitos/efeitos dos fármacos , Leite/química , Animais , Búfalos , Células Cultivadas , Citocromo P-450 CYP1A1/genética , Citocromo P-450 CYP1A1/metabolismo , Citocromo P-450 CYP1A2/genética , Citocromo P-450 CYP1A2/metabolismo , Dioxinas/análise , Contaminação de Alimentos/análise , Fator 4 Nuclear de Hepatócito/genética , Fator 4 Nuclear de Hepatócito/metabolismo , Hepatócitos/metabolismo , Fígado/citologia , Fígado/efeitos dos fármacos , Fígado/metabolismo , Modelos Animais , Ratos , Esferoides Celulares/efeitos dos fármacos , Esferoides Celulares/metabolismo , alfa-Fetoproteínas/genética , alfa-Fetoproteínas/metabolismo
12.
J Biotechnol ; 301: 11-17, 2019 Aug 10.
Artigo em Inglês | MEDLINE | ID: mdl-31158411

RESUMO

Chicken ovalbumin upstream promoter transcription factor II (COUP-TFII) is a member of the steroid/thyroid hormone receptor superfamily, but its ligand has not yet been identified. Little is known about the role of the COUP-TFII nuclear receptor in cancer cells. In this study, we mapped the cistrome of COUP-TFII in three different cancer cells, namely breast cancer cells (MCF-7), myelogenous leukaemia cells (K562) and liver cancer cells (HepG2) using publicly available ChIP-seq data. Our results show that COUP-TFII co-localises with master transcription factors (TFs) in a cell-specific manner such as estrogen receptor alpha in MCF-7, hepatocyte nuclear factor alpha in HepG2, and GATA-binding factor in K562, while the shared, non-specific COUP-TFII binding sites are co-occupied by CTCF. We identified chromatin environments for these COUP-TFII and master TF co-bound sites together with COUP-TFII and CTCF co-bound sites. Our results show that COUP-TFII and master TF co-bound sites are marked with active enhancer specific histone modifications (H3K27ac and H3K4me1), while COUP-TFII and CTCF co-bound sites reveal active promoter specific histone marks (H3K27ac and H3K4me3). These results describe the genomic context and role of COUP-TFII in the cell-type specific transcriptional programs. Furthermore, we report that the VEGFA gene regulated by shared COUP-TFII and CTCF co-bound regulatory elements is involved in long-range looping in a cell-type-independent manner. These findings provide a genomic insight into the regulation and angiogenic role of COUP-TFII.


Assuntos
Fator II de Transcrição COUP/genética , Regulação Neoplásica da Expressão Gênica/genética , Genômica/métodos , Regiões Promotoras Genéticas/genética , Bases de Dados Genéticas , Receptor alfa de Estrogênio/genética , Células Hep G2 , Fator 4 Nuclear de Hepatócito/genética , Humanos , Células K562 , Células MCF-7 , Especificidade de Órgãos
13.
ACS Appl Mater Interfaces ; 11(22): 19808-19818, 2019 Jun 05.
Artigo em Inglês | MEDLINE | ID: mdl-31066542

RESUMO

Hepatocellular carcinoma (HCC) is one of the most prevalent and deadly malignancies characterized by high rate of recurrence. Tumor recurrence is often attributed to the presence of a subpopulation of cells with stem cell properties, referred to as cancer stem cells (CSCs). Traditionally, cancer therapies target the entire bulk of tumor cells; however, they are poorly effective against CSCs, characterized by higher drug resistance. Therefore, approaches targeting CSCs may be required in addition to conventional chemotherapy to prevent tumor recurrence. In this study, we investigated an approach to target HCC by combining the conventional chemotherapeutic drug, cisplatin, to target the bulk of tumor cells, and differentiation therapy by delivering the gene encoding HNF4α, an important regulator of hepatocyte differentiation, to target CSCs. We used the Huh7 cell line as an in vitro model of HCC, which is characterized by a high proportion of CD133-expressing CSCs. By using flow cytometry, we separated CD133+ and CD133- Huh7 cell subpopulations and have shown that the former has highly pronounced in vivo tumorigenic capacity in contrast to the latter, which could not generate tumors in vivo. For the dual delivery of HNF4α-encoding plasmid and cisplatin, we used polyethyleneimine-modified mesoporous silica nanoparticles (PMSNs) as the nanocarriers. Here, we show that the treatment of CD133-expressing Huh7 cells with HNF4α-loaded PMSNs can suppress their proliferation rate, decrease the proportion of CSCs, downregulate stemness-associated genes, and increase the expression of mature hepatocyte-associated genes. At the same time, the treatment of Huh7 with PMSNs loaded with both HNF4α-encoding plasmid and cisplatin could block them in the S-phase of the cell cycle and cause apoptosis. In addition, dually loaded PMSNs were the most efficient formulation in suppressing tumor growth in vivo. To summarize, in this study, we tested the nanoparticle-based delivery system as both chemotherapy and gene-based therapy agents, which has great potential for development of effective treatment of HCC.


Assuntos
Antígeno AC133/metabolismo , Carcinoma Hepatocelular/metabolismo , Cisplatino/administração & dosagem , Cisplatino/farmacologia , Fator 4 Nuclear de Hepatócito/metabolismo , Nanopartículas/química , Dióxido de Silício/química , Células-Tronco/efeitos dos fármacos , Células-Tronco/metabolismo , Animais , Western Blotting , Ciclo Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Citometria de Fluxo , Fator 4 Nuclear de Hepatócito/genética , Humanos , Marcação In Situ das Extremidades Cortadas , Camundongos , Camundongos SCID , Reação em Cadeia da Polimerase Via Transcriptase Reversa
14.
Biochim Biophys Acta Gene Regul Mech ; 1862(6): 619-624, 2019 06.
Artigo em Inglês | MEDLINE | ID: mdl-31005673

RESUMO

Engineered transcription factors (TF) have expanded our ability to modulate gene expression and hold great promise as bio-therapeutics. The first-generation TF, based on Zinc Fingers or Transcription-Activator-like Effectors (TALE), required complex and time-consuming assembly protocols, and were indeed replaced in recent years by the CRISPR activation (CRISPRa) technology. Here, with coagulation F7/F8 gene promoters as models, we exploited a CRISPRa system based on deactivated (d)Cas9, fused with a transcriptional activator (VPR), which is driven to its target by a single guide (sg)RNA. Reporter gene assays in hepatoma cells identified a sgRNA (sgRNAF7.5) triggering a ~35-fold increase in the activity of F7 promoter, either wild-type, or defective due to the c.-61T>G mutation. The effect was higher (~15-fold) than that of an engineered TALE-TF (TF4) targeting the same promoter region. Noticeably, when challenged on the endogenous F7 gene, the dCas9-VPR/sgRNAF7.5 combination was more efficient (~6.5-fold) in promoting factor VII (FVII) protein secretion/activity than TF4 (~3.8-fold). The approach was translated to the promoter of F8, whose reduced expression causes hemophilia A. Reporter gene assays in hepatic and endothelial cells identified sgRNAs that, respectively, appreciably increased F8 promoter activity (sgRNAF8.1, ~8-fold and 3-fold; sgRNAF8.2, ~19-fold and 2-fold) with synergistic effects (~38-fold and 2.7-fold). Since modest increases in F7/F8 expression would ameliorate patients' phenotype, the CRISPRa-mediated transactivation extent might approach the low therapeutic threshold. Through this pioneer study we demonstrated that the CRISPRa system is easily tailorable to increase expression, or rescue disease-causing mutations, of different promoters, with potential intriguing implications for human disease models.


Assuntos
Sistemas CRISPR-Cas , Regiões Promotoras Genéticas/genética , Fatores de Transcrição/genética , Ativação Transcricional/genética , Sítios de Ligação , Biomarcadores Tumorais/genética , Carcinoma Hepatocelular , Células Endoteliais , Fator VII , Fator VIII/genética , Expressão Gênica , Genes Reporter , Células Hep G2 , Fator 4 Nuclear de Hepatócito/genética , Humanos , Mutação , RNA Guia , Receptores Imunológicos/genética
15.
Nat Genet ; 51(5): 777-785, 2019 05.
Artigo em Inglês | MEDLINE | ID: mdl-30988513

RESUMO

BMP/SMAD signaling is a crucial regulator of intestinal differentiation1-4. However, the molecular underpinnings of the BMP pathway in this context are unknown. Here, we characterize the mechanism by which BMP/SMAD signaling drives enterocyte differentiation. We establish that the transcription factor HNF4A acts redundantly with an intestine-restricted HNF4 paralog, HNF4G, to activate enhancer chromatin and upregulate the majority of transcripts enriched in the differentiated epithelium; cells fail to differentiate on double knockout of both HNF4 paralogs. Furthermore, we show that SMAD4 and HNF4 function via a reinforcing feed-forward loop, activating each other's expression and co-binding to regulatory elements of differentiation genes. This feed-forward regulatory module promotes and stabilizes enterocyte cell identity; disruption of the HNF4-SMAD4 module results in loss of enterocyte fate in favor of progenitor and secretory cell lineages. This intersection of signaling and transcriptional control provides a framework to understand regenerative tissue homeostasis, particularly in tissues with inherent cellular plasticity5.


Assuntos
Enterócitos/citologia , Enterócitos/metabolismo , Fator 4 Nuclear de Hepatócito/metabolismo , Proteína Smad4/metabolismo , Animais , Sítios de Ligação/genética , Células CACO-2 , Diferenciação Celular/genética , Diferenciação Celular/fisiologia , Elementos Facilitadores Genéticos , Fator 4 Nuclear de Hepatócito/deficiência , Fator 4 Nuclear de Hepatócito/genética , Humanos , Camundongos , Camundongos Knockout , Transdução de Sinais , Proteína Smad4/deficiência , Proteína Smad4/genética
16.
J Pediatr Endocrinol Metab ; 32(3): 301-304, 2019 Mar 26.
Artigo em Inglês | MEDLINE | ID: mdl-30730840

RESUMO

Background Congenital hyperinsulinism (CHI) occurs due to an unregulated insulin secretion from the pancreatic ß-cells resulting in hypoglycaemia. Causative mutations in multiple genes have been reported. Phenotypic variability exists both within and between different genetic subgroups. Case presentation A male infant born at 35+6 weeks' gestation with a birth weight of 4.3 kg [+3.6 standard deviation score (SDS)] had recurrent hypoglycaemic episodes from birth. Biochemical investigations confirmed a diagnosis of CHI. Diazoxide was started and the dose was progressively increased to maintain euglycaemia. His father was slim and had been diagnosed with type 2 diabetes in his 30s. Sequence analysis identified a heterozygous hepatocyte nuclear factor 4 alpha (HNF4A) mutation (p.Arg245Pro, c.734G>C) and compound heterozygous ABCC8 mutations (p.Gly92Ser, c.274G>A and p.Ala1185Val, c.3554C>T) in the patient. The p.Ala1185Val ABCC8 mutation was inherited from his unaffected mother and the p.Arg245Pro HNF4A and p.Gly92Ser ABCC8 mutations from his father. All three mutations were predicted to be pathogenic. Identification of the HNF4A mutation in the father established a diagnosis of maturity-onset diabetes of the young (MODY), which enabled medication change resulting in improved glycaemic control. Conclusions We report a rare patient with CHI due to dual genetic aetiology. Although he is currently responsive to the maximum dose of diazoxide, the long-term prognosis remains unclear.


Assuntos
Hiperinsulinismo Congênito/genética , Fator 4 Nuclear de Hepatócito/genética , Receptores Sulfonilureia/genética , Peso ao Nascer , Glicemia/análise , Humanos , Recém-Nascido , Masculino , Mutação
17.
Biochim Biophys Acta Gene Regul Mech ; 1862(5): 567-581, 2019 05.
Artigo em Inglês | MEDLINE | ID: mdl-30753902

RESUMO

MiR-15/16 play an important role in liver development and hepatocyte differentiation, but the mechanisms by which these miRNAs regulate their targets and downstream genes to influence cell fate are poorly understood. In this study, we showed up-regulation of miR-15/16 during HGF- and FGF4-induced hepatocyte differentiation from amniotic epithelial cells (AECs). To elucidate the role of miR-15/16 and their targets in hepatocyte differentiation, we investigated the roles of miR-15/16 in both the MAPK and Wnt/ß-catenin pathways, which were predicted to be involved in miR-15/16 signaling. Our results demonstrated that the transcription of miR-15/16 was enhanced by c-Fos, c-Jun, and CREB, important elements of the MAPK pathway, and miR-15/16 in turn directly targeted adenomatous polyposis coli (APC) protein, a major member of the ß-catenin degradation complex. MiR-15/16 destroyed these degradation complexes to activate ß-catenin, and the activated ß-catenin combined with LEF/TCF7L1 to form a transcriptional complex that enhanced transcription of hepatocyte nuclear factor 4 alpha (HNF4α). HNF4α also bound the promoter region of miR-15/16 and promoted its transcription, thereby forming a regulatory circuit to promote the differentiation of AECs into hepatocytes. Endogenous miRNAs are, therefore, involved in hepatocyte differentiation from AECs and should be considered during the development of an effective hepatocyte transplant therapy for liver damage.


Assuntos
Âmnio/citologia , Regulação da Expressão Gênica , Hepatócitos/metabolismo , Sistema de Sinalização das MAP Quinases , MicroRNAs/fisiologia , Via de Sinalização Wnt , Diferenciação Celular , Células Cultivadas , Células Epiteliais/efeitos dos fármacos , Células HEK293 , Fator 4 Nuclear de Hepatócito/biossíntese , Fator 4 Nuclear de Hepatócito/genética , Hepatócitos/citologia , Hepatócitos/enzimologia , Humanos , Peptídeos e Proteínas de Sinalização Intercelular/farmacologia , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , MicroRNAs/biossíntese , MicroRNAs/genética , Transcrição Genética , Via de Sinalização Wnt/efeitos dos fármacos
18.
Mutat Res ; 814: 1-6, 2019 03.
Artigo em Inglês | MEDLINE | ID: mdl-30648609

RESUMO

HNF4α is a culprit gene product for a monogenic and dominantly-inherited form of diabetes, referred to as MODY1 (Maturity Onset Diabetes of the Young type 1). Reduced HNF4α activities have been linked to impaired insulin secretion and ß-cell function. Numerous mutations have been identified from the patients and they have been instructive as to the individual residue's role in protein structure-function and dysfunction. As a member of the nuclear receptor (NR) superfamily, HNF4α is made of characteristic modular domains and it functions exclusively as a homodimer despite its sequence homology to RXR, a common heterodimer partner of non-steroidal NRs. Transcription factors commonly dimerize to enhance their molecular functions mainly by facilitating the recognition of double helix target DNAs that display an intrinsic pseudo-2-fold symmetry and the recruitment of the remainder of the main transcriptional machinery. HNF4α is no exception and its dimerization is maintained by the ligand binding domain (LBD) mainly through the leucine-zipper-like interactions at the stalk of two interacting helices. Although many MODY1 mutations have been previously characterized, including DNA binding disruptors, ligand binding disruptors, coactivator binding disruptors, and protein stability disruptors, protein dimerization disruptors have not been formally reported. In this report, we present a set of data for the two MODY1 mutations found right at the dimerization interface (L332 P and L328del mutations) which clearly exhibit the disruptive effects of directly affecting dimerization, protein stability, and transcriptional activities. These data reinforced the fact that MODY mutations are loss-of-function mutations and HNF4α dimerization is essential for its optimal function and normal physiology.


Assuntos
Diabetes Mellitus Tipo 2/genética , Fator 4 Nuclear de Hepatócito/genética , Fator 4 Nuclear de Hepatócito/metabolismo , Mutação , Domínios e Motivos de Interação entre Proteínas/genética , Multimerização Proteica , Dimerização , Células HeLa , Fator 4 Nuclear de Hepatócito/química , Humanos , Mutação com Perda de Função/genética , Modelos Moleculares , Mutagênese Sítio-Dirigida , Ligação Proteica , Multimerização Proteica/genética , Estabilidade Proteica , Estrutura Quaternária de Proteína/genética , Ativação Transcricional/genética
19.
Int J Mol Sci ; 20(2)2019 Jan 16.
Artigo em Inglês | MEDLINE | ID: mdl-30654452

RESUMO

Although human liver tumor cells have reduced metabolic functions as compared to primary human hepatocytes (PHH) they are widely used for pre-screening tests of drug metabolism and toxicity. The aim of the present study was to modify liver cancer cell lines in order to improve their drug-metabolizing activities towards PHH. It is well-known that epigenetics is strongly modified in tumor cells and that epigenetic regulators influence the expression and function of Cytochrome P450 (CYP) enzymes through altering crucial transcription factors responsible for drug-metabolizing enzymes. Therefore, we screened the epigenetic status of four different liver cancer cell lines (Huh7, HLE, HepG2 and AKN-1) which were reported to have metabolizing drug activities. Our results showed that HepG2 cells demonstrated the highest similarity compared to PHH. Thus, we modified the epigenetic status of HepG2 cells towards 'normal' liver cells by 5-Azacytidine (5-AZA) and Vitamin C exposure. Then, mRNA expression of Epithelial-mesenchymal transition (EMT) marker SNAIL and CYP enzymes were measured by PCR and determinate specific drug metabolites, associated with CYP enzymes by LC/MS. Our results demonstrated an epigenetic shift in HepG2 cells towards PHH after exposure to 5-AZA and Vitamin C which resulted in a higher expression and activity of specific drug metabolizing CYP enzymes. Finally, we observed that 5-AZA and Vitamin C led to an increased expression of Hepatocyte nuclear factor 4α (HNF4α) and E-Cadherin and a significant down regulation of Snail1 (SNAIL), the key transcriptional repressor of E-Cadherin. Our study shows, that certain phase I genes and their enzyme activities are increased by epigenetic modification in HepG2 cells with a concomitant reduction of EMT marker gene SNAIL. The enhancing of liver specific functions in hepatoma cells using epigenetic modifiers opens new opportunities for the usage of cell lines as a potential liver in vitro model for drug testing and development.


Assuntos
Epigênese Genética , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/metabolismo , Preparações Farmacêuticas/metabolismo , Ácido Ascórbico/farmacologia , Azacitidina/farmacologia , Biomarcadores Tumorais/genética , Biomarcadores Tumorais/metabolismo , Cromatina/metabolismo , Sistema Enzimático do Citocromo P-450/genética , Sistema Enzimático do Citocromo P-450/metabolismo , Regulação para Baixo/efeitos dos fármacos , Epigênese Genética/efeitos dos fármacos , Transição Epitelial-Mesenquimal/efeitos dos fármacos , Transição Epitelial-Mesenquimal/genética , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Células Hep G2 , Fator 4 Nuclear de Hepatócito/genética , Fator 4 Nuclear de Hepatócito/metabolismo , Hepatócitos/efeitos dos fármacos , Hepatócitos/metabolismo , Humanos , Hidrocortisona/farmacologia , Insulina/farmacologia , Neoplasias Hepáticas/enzimologia , Fatores de Transcrição da Família Snail/metabolismo , Xenobióticos/metabolismo
20.
J Clin Endocrinol Metab ; 104(3): 845-855, 2019 03 01.
Artigo em Inglês | MEDLINE | ID: mdl-30535056

RESUMO

Objective: To characterize the initial presentation and clinical course of patients with hepatocyte nuclear factor (HNF) 4A‒ and HNF1B‒MODY in a multinational registry. Design, Setting, and Participants: Within the Diabetes Patienten Verlaufsdokumentation (DPV) registry, 44 patients with HNF4A- and 35 patients with HNF1B-MODY were characterized and compared with patients <20 years old with type 1 diabetes (T1D)/type 2 diabetes (T2D). Main Outcome Measure: Clinical and laboratory parameters, therapy, metabolic control, and extrapancreatic symptoms in patients with HNF1B-MODY. Results: Patients with both MODY types were significantly older than patients with T1D at diagnosis (HNF4A, 13.8 years, and HNF1B, 13.5 years, vs T1D, 8.8 years; P < 0.0001). Mean C-peptide at diagnosis was higher for HNF4A-MODY than for T1D (1.8 vs 0.9 ng/mL; P < 0.01); 36.4% of patients with HNF4A-MODY and 65.7% of patients with HNF1B-MODY were treated with insulin, whereas 20.5% and 8.6% received oral antidiabetics only (P < 0.05 and P < 0.01 vs T2D). At the most recent visit, glycated hemoglobin levels were lower in HNF4A- and HNF1B-MODY (mean, 6.5% and 6.1%) than in T1D (7.9%; P < 0.0001). In 40% of patients with HNF1B-MODY, extrapancreatic symptoms were reported. Several clinical predictors previously described to differentiate between MODY and T1D or T2D were revalidated by logistic regression analyses in this cohort. Conclusion: The DPV registry enabled us to precisely characterize phenotype and treatment in these two rare MODY types. Although phenotype of HNF4A- and HNF1B-MODY showed distinct differences from those of T1D and T2D, 38% of patients were initially misclassified as having T1D or T2D.


Assuntos
Diabetes Mellitus Tipo 1/diagnóstico , Diabetes Mellitus Tipo 2/diagnóstico , Fator 1-beta Nuclear de Hepatócito/genética , Fator 4 Nuclear de Hepatócito/genética , Doenças Raras/diagnóstico , Adolescente , Fatores Etários , Criança , Diabetes Mellitus Tipo 1/tratamento farmacológico , Diabetes Mellitus Tipo 2/tratamento farmacológico , Diabetes Mellitus Tipo 2/genética , Diagnóstico Diferencial , Feminino , Humanos , Hipoglicemiantes/uso terapêutico , Insulina/uso terapêutico , Masculino , Estudos Prospectivos , Doenças Raras/tratamento farmacológico , Doenças Raras/genética , Sistema de Registros/estatística & dados numéricos
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