RESUMO
Diisononyl phthalate (DINP) is a high-molecular-weight phthalate, and has been recently introduced as di-(2-ethyl hexyl) phthalate (DEHP) substitute and commonly used in a large variety of plastic items. The fat tissue is an important target for DINP exposure, however, very little is understood about its toxicity and mechanism(s) in adipocyte cells. Therefore, the present work aimed to investigate the role of DINP in adipogenesis using 3T3-L1 preadipocytes. DINP exposure for 10 days extensively induced adipogenesis in 3T3-L1 preadipocytes to adipocytes as assessed by lipid accumulation and gene expression of adipogenic markers. The RT-qPCR results showed that DINP could upregulate the expression of peroxisome proliferator-activated receptor-gamma (PPARγ), CCAAT/enhancer-binding protein alpha (C/EBPα) and C/EBPß, while the expression of sterol regulatory element binding transcription factor 1 (SREBF1) and C/EBPδ was not affected. The DINP-induced adipogenesis could be inhibited by using the selective PPARγ antagonist GW9662. The RNA-seq analysis was used to study the systemic toxicities of DINP on preadipocytes. A total of 1181 differently expressed genes (DEGs) (640 genes were up-regulated, 541 genes were down-regulated) were detected in 3T3-L1 preadipocytes under 50⯵M DINP. The GO enrichment showed the GO term of "fat cell differentiation" was the most significantly affected metabolic functions, and the KEGG pathway enrichment showed the PPAR pathway was the top affected pathway. The interactive pathway (iPath) analysis showed that the changed metabolic pathways were focus on the lipid metabolism.
Assuntos
Adipócitos/citologia , Adipogenia/efeitos dos fármacos , Regulação da Expressão Gênica/efeitos dos fármacos , Metabolismo dos Lipídeos/efeitos dos fármacos , Ácidos Ftálicos/toxicidade , Células 3T3-L1 , Tecido Adiposo/metabolismo , Anilidas/farmacologia , Animais , Proteína alfa Estimuladora de Ligação a CCAAT/biossíntese , Proteína beta Intensificadora de Ligação a CCAAT/biossíntese , Proteínas Estimuladoras de Ligação a CCAAT/biossíntese , Linhagem Celular , Regulação para Baixo , Expressão Gênica/efeitos dos fármacos , Camundongos , PPAR gama/antagonistas & inibidores , PPAR gama/biossíntese , Proteína de Ligação a Elemento Regulador de Esterol 1/biossíntese , Ativação Transcricional , Regulação para CimaRESUMO
BACKGROUND: To investigate the role of microRNA (miR)-27a and miR-27b in adipogenesis in an in vitro model of Graves' orbitopathy (GO). METHODS: Orbital fat tissues were harvested from GO and non-GO participants for primary orbital fibroblast cultures. The expression levels of miR-27a and miR-27b between GO and non-GO orbital fat tissues were compared. During adipogenesis of GO orbital fibroblasts, the expression levels of miR-27a and miR-27b were determined, and the effects of mimics of miR-27a and miR-27b transfection on adipogenesis of GO orbital fibroblast were investigated. RESULTS: Real time-polymerase chain reaction showed significantly more decreases in miR-27a and miR-27b levels in orbital fat tissues in GO participants than in non-GO participants (p < 0.05). The expression of both miR-27a and miR-27b was highest in orbital fibroblasts at day 0 and declined gradually after the induction of adipogenic differentiation. The expression levels of PPARγ, CCAAT/enhancer binding protein (C/EBP)α and C/EBPß were decreased and Oil Red O-stained lipid droplets were lower in GO orbital fibroblasts transfected with miR-27a and miR-27b mimics than in negative controls. CONCLUSIONS: Our results indicated that miR-27a and miR-27b inhibited adipogenesis in orbital fibroblasts from GO patients. Further studies are required to examine the potential of miR-27a and miR-27b as targets for therapeutic strategies.
Assuntos
Adipogenia , Diferenciação Celular , Fibroblastos/metabolismo , Oftalmopatia de Graves/metabolismo , MicroRNAs/biossíntese , Órbita/metabolismo , Proteína beta Intensificadora de Ligação a CCAAT/biossíntese , Proteínas Estimuladoras de Ligação a CCAAT/biossíntese , Feminino , Fibroblastos/patologia , Regulação da Expressão Gênica , Oftalmopatia de Graves/patologia , Humanos , Masculino , Órbita/patologia , PPAR gama/biossínteseRESUMO
CCAAT/enhancer binding protein ε (CEBPE) is an essential transcription factor for granulocytic differentiation. Mutations of CEBPE occur in individuals with neutrophil-specific granule deficiency (SGD), which is characterized by defects in neutrophil maturation. Cebpe-knockout mice also exhibit defects in terminal differentiation of granulocytes, a phenotype reminiscent of SGD. Analysis of DNase I hypersensitive sites sequencing data revealed an open chromatin region 6 kb downstream of the transcriptional start site of Cebpe in murine myeloid cells. We identified an interaction between this +6-kb region and the core promoter of Cebpe using circular chromosome conformation capture sequencing (4C-seq). To understand the role of this putative enhancer in transcriptional regulation of Cebpe, we targeted it using catalytically inactive Cas9 fused to Krüppel-associated box (KRAB) domain and observed a significant downregulation of transcript and protein levels of CEBPE in cells expressing guide RNA targeting the +6-kb region. To further investigate the role of this novel enhancer further in myelopoiesis, we generated mice with deletion of this region using CRISPR/Cas9 technology. Germline deletion of the +6-kb enhancer resulted in reduced levels of CEBPE and its target genes and caused a severe block in granulocytic differentiation. We also identified binding of CEBPA and CEBPE to the +6-kb enhancer, which suggests their role in regulating the expression of Cebpe In summary, we have identified a novel enhancer crucial for regulating expression of Cebpe and required for normal granulocytic differentiation.
Assuntos
Proteínas Estimuladoras de Ligação a CCAAT/biossíntese , Diferenciação Celular/genética , Regulação da Expressão Gênica/genética , Granulócitos/metabolismo , Mielopoese/genética , Animais , Proteínas Estimuladoras de Ligação a CCAAT/genética , Camundongos , Camundongos Endogâmicos C57BL , Camundongos KnockoutRESUMO
To unravel molecular mechanisms by which Runt-related transcription factor 1 (RUNX1) mutations contribute to leukemic transformation, we introduced the RUNX1-S291fs300X mutation in human CD34+ stem/progenitor cells and in human induced pluripotent stem cells (iPSCs). In both models, RUNX1mut overexpression strongly impaired myeloid commitment. Instead, self-renewal was enhanced, as shown, by increased long-term culture-initiating cell frequencies and enhanced colony-forming cell replating capacity. Long-term suspension cultures with RUNX1mut-transduced cord blood (CB) CD34+ cells continued for more than 100 days, during which the cells displayed an immature granulocyte-macrophage progenitor-like CD34+/CD123+/CD45RA+ phenotype. The CD34+/CD38- hematopoietic stem cell (HSC) population most likely acted as cell of origin, as HSCs provided the best long-term proliferative potential on overexpression of RUNX1mut. CEBPA expression was reduced in RUNX1mut cells, and reexpression of CEBPA partly restored differentiation. RNA-seq analysis on CB/iPSC systems and on primary patient samples confirmed that RUNX1 mutations induce a myeloid differentiation block, and that a common set of RUNX1mut-upregulated target genes was strongly enriched for gene ontology terms associated with nucleosome assembly and chromatin structure. Interestingly, in comparison with AML1-ETO binding in acute myeloid leukemias (AMLs), we found significantly distinct genomic distribution and differential expression for RUNX1mut of genes such as TCF4, MEIS1, and HMGA2 that may potentially contribute to the underlying difference in clinical outcomes between RUNX1mut and AML1-ETO patients. In conclusion, RUNX1mut appears to induce a specific transcriptional program that contributes to leukemic transformation.
Assuntos
Subunidade alfa 2 de Fator de Ligação ao Core/genética , Granulócitos/patologia , Leucemia Mieloide Aguda/genética , Leucemia Mieloide Aguda/patologia , Mutação , Proteínas Estimuladoras de Ligação a CCAAT/biossíntese , Proteínas Estimuladoras de Ligação a CCAAT/genética , Diferenciação Celular/genética , Transformação Celular Neoplásica/genética , Imunoprecipitação da Cromatina , Subunidade alfa 2 de Fator de Ligação ao Core/biossíntese , Subunidade alfa 2 de Fator de Ligação ao Core/sangue , Subunidade alfa 2 de Fator de Ligação ao Core/metabolismo , Sangue Fetal/metabolismo , Granulócitos/metabolismo , Humanos , Leucemia Mieloide Aguda/metabolismo , Ligação ProteicaRESUMO
BACKGROUND: The transcription factor CCAAT-enhancer-binding protein alpha (CEBPA) is a crucial regulator of cell proliferation and differentiation. Expression levels of CEBPA have been suggested to be prognostic in various tumor types. METHODS: Here, we analyzed the immunohistochemical expression of CEBPA in a tissue microarray containing more than 17 000 prostate cancer specimens with annotated clinical and molecular data including for example TMPRSS2:ERG fusion and PTEN deletion status. RESULTS: Normal prostate glands showed moderate to strong CEBPA staining, while CEBPA expression was frequently reduced (40%) or lost (30%) in prostate cancers. Absence of detectable CEBPA expression was markedly more frequent in ERG negative (45%) as compared to ERG positive cancers (20%, P < 0.0001). Reduced CEBPA expression was linked to unfavorable phenotype (P < 0.0001) and poor prognosis (P = 0.0008). Subgroup analyses revealed, that the prognostic value of CEBPA loss was entirely driven by tumors carrying both TMPRSS2:ERG fusions and PTEN deletions. In this subgroup, CEBPA loss was tightly linked to advanced tumor stage (P < 0.0001), high Gleason grade (P < 0.0001), positive nodal stage (0.0003), and early biochemical recurrence (P = 0.0007), while these associations were absent or markedly diminished in tumors with normal PTEN copy numbers and/or absence of ERG fusion. CONCLUSIONS: CEBPA is down regulated in about one third of prostate cancers, but the clinical impact of CEBPA loss is strictly limited to the subset of about 10% prostate cancers carrying both ERG fusion and deletions of the PTEN tumor suppressor. Our findings challenge the concept that prognostic molecular markers may be generally applicable to all prostate cancers.
Assuntos
Proteínas Estimuladoras de Ligação a CCAAT/deficiência , Proteínas de Fusão Oncogênica/metabolismo , PTEN Fosfo-Hidrolase/deficiência , Neoplasias da Próstata/metabolismo , Idoso , Proteínas Estimuladoras de Ligação a CCAAT/biossíntese , Proteínas Estimuladoras de Ligação a CCAAT/metabolismo , Dosagem de Genes , Humanos , Imuno-Histoquímica , Masculino , Pessoa de Meia-Idade , Proteínas de Fusão Oncogênica/genética , PTEN Fosfo-Hidrolase/genética , PTEN Fosfo-Hidrolase/metabolismo , Prognóstico , Prostatectomia , Neoplasias da Próstata/genética , Neoplasias da Próstata/patologia , Neoplasias da Próstata/cirurgia , Análise Serial de TecidosRESUMO
3'-untranslated regions (UTRs) can vary through the use of alternative polyadenylation sites during pre-mRNA processing. Multiple publically available pipelines combining high profiling technologies and bioinformatics tools have been developed to catalog changes in 3'-UTR lengths. In our recent RNA-seq experiments using cells with hyper-activated mammalian target of rapamycin (mTOR), we found that cellular mTOR activation leads to transcriptome-wide alternative polyadenylation (APA), resulting in the activation of multiple cellular pathways. Here, we developed a novel bioinformatics algorithm, IntMAP, which integrates RNA-Seq and PolyA Site (PAS)-Seq data for a comprehensive characterization of APA events. By applying IntMAP to the datasets from cells with hyper-activated mTOR, we identified novel APA events that could otherwise not be identified by either profiling method alone. Several transcription factors including Cebpg (CCAAT/enhancer binding protein gamma) were among the newly discovered APA transcripts, indicating that diverse transcriptional networks may be regulated by mTOR-coordinated APA. The prevention of APA in Cebpg using the CRISPR/cas9-mediated genome editing tool showed that mTOR-driven 3'-UTR shortening in Cebpg is critical in protecting cells from endoplasmic reticulum (ER) stress. Taken together, we present IntMAP as a new bioinformatics algorithm for APA analysis by which we expand our understanding of the physiological role of mTOR-coordinated APA events to ER stress response. IntMAP toolbox is available at http://compbio.cs.umn.edu/IntMAP/.
Assuntos
Algoritmos , Estresse do Retículo Endoplasmático/genética , Poliadenilação , Serina-Treonina Quinases TOR/metabolismo , Regiões 3' não Traduzidas , Animais , Proteínas Estimuladoras de Ligação a CCAAT/biossíntese , Proteínas Estimuladoras de Ligação a CCAAT/genética , Células Cultivadas , CamundongosRESUMO
CCAAT/enhancer binding protein α, ß, δ (C/EBP α, ß, δ) are essential transcriptional factors in regulating adipose development. However, information about their sequence characteristics and functions during adipocyte development still remains scarce in birds. In present study, we found that duck C/EBP α, ß, δ differed in their phosphorylation sites and low complexity regions (LCRs) among their orthologs and paralogs. Phylogenetic analysis showed that C/EBP α, ß, δ had different evolutionary patterns, and each of duck C/EBP α, ß, δ was strikingly diverged from orthologs of other Aves. Results of quantitative real-time PCR exhibited that C/EBP α, ß, δ were all highly expressed in duck adipose tissues. Indeed, investigations of changes in both their mRNA levels and lipid droplet content during duck adipocytes differentiation showed that their expression profiles were closely related to cellular lipid accumulation. Furthermore, hierarchical clustering analysis of the C/EBPs and lipid metabolism-related genes expression profiles showed that C/EBP α was clustered with genes related to lipolysis, lipogenesis and fatty acid desaturation, whereas C/EBP ß, δ were clustered with genes related to de novo lipogenesis and fatty acid elongation, which were different from mammals. In summary, C/EBP α, ß, δ of duck differ from other species in their structures and have different effects on lipid metabolism during adipocytes differentiation. This research serve as a foundation for further investigations about avian C/EBP α, ß, δ in adipocytes differentiation and adipose development.
Assuntos
Adipócitos/metabolismo , Tecido Adiposo/metabolismo , Proteínas Aviárias , Proteínas Estimuladoras de Ligação a CCAAT , Clonagem Molecular , Patos , Regulação da Expressão Gênica/fisiologia , Adipócitos/citologia , Tecido Adiposo/citologia , Animais , Proteínas Aviárias/biossíntese , Proteínas Aviárias/genética , Proteínas Estimuladoras de Ligação a CCAAT/biossíntese , Proteínas Estimuladoras de Ligação a CCAAT/genética , Patos/genética , Patos/metabolismoRESUMO
Obesity is characterized by excess body fat accumulation due to an increase in the size and number of differentiated mature adipocytes. Adipocyte differentiation is regulated by genetic and environmental factors, and its inhibition could represent a strategy for obesity prevention and treatment. The current study was designed with two aims: (i) to evaluate the changes in the expression of adipogenic markers (C/EBPα, PPARγ variant 1 and variant 2, and GLUT4) in 3T3-L1 murine preadipocytes at four stages of the differentiation process and (ii) to compare the effectiveness of sulforaphane, genistein, and docosahexaenoic acid in reducing lipid accumulation and modulating C/EBPα, PPARγ1, PPARγ2, and GLUT4 mRNA expression in mature adipocytes. All bioactive compounds were shown to suppress adipocyte differentiation, although with different effectiveness. These results set the stage for further studies considering natural food constituents as important agents in preventing or treating obesity.
Assuntos
Adipócitos/efeitos dos fármacos , Adipogenia/efeitos dos fármacos , Ácidos Docosa-Hexaenoicos/farmacologia , Genisteína/farmacologia , Isotiocianatos/farmacologia , Obesidade/tratamento farmacológico , Células 3T3-L1 , Adipócitos/citologia , Adipócitos/metabolismo , Animais , Proteínas Estimuladoras de Ligação a CCAAT/biossíntese , Proteínas Estimuladoras de Ligação a CCAAT/genética , Diferenciação Celular/efeitos dos fármacos , Expressão Gênica/efeitos dos fármacos , Transportador de Glucose Tipo 4/biossíntese , Transportador de Glucose Tipo 4/genética , Metabolismo dos Lipídeos/efeitos dos fármacos , Camundongos , Obesidade/genética , Obesidade/metabolismo , Obesidade/patologia , PPAR gama/biossíntese , PPAR gama/genética , SulfóxidosRESUMO
Stratum corneum forms the UV barrier. The effect of ultraviolet B (UVB) on normal skin was extensively studied; however, its effect on barrier perturbed skin remains undefined. Both barrier perturbation and UVB irradiation induce endoplasmic reticulum (ER) stress and unfolded protein response (UPR) in keratinocytes. Mild ER stress activates homeostatic UPR, while severe ER stress leads to abnormal UPR, promoting apoptosis and inflammation. Here, we investigated UV sensitivity and UVB-induced UPR in barrier-disrupted human skin and the effects of pseudoceramide-dominant emollient on UVB-induced skin responses. Tape-stripped skin of healthy volunteers showed enhanced susceptibility to erythema and augmented proinflammatory cytokines induction following suberythemal UVB irradiation. Suberythemal UVB activated XBP1 in normal skin, while increased CHOP transcription in barrier perturbed skin. After tape stripping, pseudoceramide-dominant emollient was applied for 3 days, and then, the areas were irradiated with suberythemal UVB. Pretreatment with topical pseudoceramide protected against UVB-induced upregulation of IL-1ß, IL-6, and TNF-α transcription and reduced susceptibility to erythema following UVB. Topical pseudoceramide also suppressed suberythemal UVB-induced CHOP transcription in barrier-disrupted skin. Taken together, these data indicate that permeability barrier disruption increases UV sensitivity in human skin, partly via switch the UVB-induced UPR, from homeostatic signals to pro-apoptotic and proinflammatory signals. In addition, we conclude that pseudoceramide-dominant emollient suppresses excessive ER stress induction and CHOP activation following UVB in barrier damaged skin, providing evidence that pseudoceramide-dominant emollients can be promising strategies for photoprotection of the barrier damaged skin.
Assuntos
Estresse do Retículo Endoplasmático/efeitos da radiação , Eritema/tratamento farmacológico , Queratinócitos/efeitos da radiação , Espermina/análogos & derivados , Tirosina/análogos & derivados , Raios Ultravioleta/efeitos adversos , Resposta a Proteínas não Dobradas/efeitos da radiação , Adulto , Proteínas Estimuladoras de Ligação a CCAAT/biossíntese , Eritema/prevenção & controle , Feminino , Humanos , Interleucina-1beta/biossíntese , Interleucina-1beta/genética , Interleucina-6/biossíntese , Interleucina-6/genética , Queratinócitos/metabolismo , RNA Mensageiro/biossíntese , Pele/patologia , Pele/efeitos da radiação , Espermina/farmacologia , Junções Íntimas/fisiologia , Fator de Necrose Tumoral alfa/biossíntese , Fator de Necrose Tumoral alfa/genética , Tirosina/farmacologiaRESUMO
DNMT1 is recruited to substrate sites by PCNA and UHRF1 to maintain DNA methylation after replication. The cell cycle dependent recruitment of DNMT1 is mediated by the PCNA-binding domain (PBD) and the targeting sequence (TS) within the N-terminal regulatory domain. The TS domain was found to be mutated in patients suffering from hereditary sensory and autonomic neuropathies with dementia and hearing loss (HSANIE) and autosomal dominant cerebellar ataxia deafness and narcolepsy (ADCA-DN) and is associated with global hypomethylation and site specific hypermethylation. With functional complementation assays in mouse embryonic stem cells, we showed that DNMT1 mutations P496Y and Y500C identified in HSANIE patients not only impair DNMT1 heterochromatin association, but also UHRF1 interaction resulting in hypomethylation. Similar DNA methylation defects were observed when DNMT1 interacting domains in UHRF1, the UBL and the SRA domain, were deleted. With cell-based assays, we could show that HSANIE associated mutations perturb DNMT1 heterochromatin association and catalytic complex formation at methylation sites and decrease protein stability in late S and G2 phase. To investigate the neuronal phenotype of HSANIE mutations, we performed DNMT1 rescue assays and could show that cells expressing mutated DNMT1 were prone to apoptosis and failed to differentiate into neuronal lineage. Our results provide insights into the molecular basis of DNMT1 dysfunction in HSANIE patients and emphasize the importance of the TS domain in the regulation of DNA methylation in pluripotent and differentiating cells.
Assuntos
Proteínas Estimuladoras de Ligação a CCAAT/genética , Diferenciação Celular/genética , DNA (Citosina-5-)-Metiltransferases/genética , Metilação de DNA/genética , Neuropatias Hereditárias Sensoriais e Autônomas/genética , Animais , Apoptose/genética , Proteínas Estimuladoras de Ligação a CCAAT/biossíntese , DNA (Citosina-5-)-Metiltransferase 1 , DNA (Citosina-5-)-Metiltransferases/biossíntese , Regulação da Expressão Gênica , Neuropatias Hereditárias Sensoriais e Autônomas/patologia , Heterocromatina/genética , Humanos , Camundongos , Células-Tronco Embrionárias Murinas/metabolismo , Mutação , Neurônios/metabolismo , Neurônios/patologia , Domínios Proteicos/genética , Domínios e Motivos de Interação entre Proteínas/genética , Estabilidade Proteica , Ubiquitina-Proteína LigasesRESUMO
Esophageal adenocarcinoma (EAC) is one of the most common malignancies in the world which is associated the increased prevalence of obesity. In the context of obesity, leptin can directly contribute to progression of EAC. Adiponectin inhibits leptin-induced oncogenic signaling in EAC cells. However, the exact molecular mechanisms linking obesity, adipokines, and EAC remain far from completely understood. In the present study, we tested the role of ubiquitin-like with PHD and ring finger domains 1 (UHRF1) in adiponectin-induced protective effects against leptin-induced EAC cell proliferation. We found that globular adiponectin (gAD) significantly inhibited leptin-induced increase of cell proliferation and decrease of apoptosis in OE 19 cells. Moreover, leptin-induced increase of UHRF1 expression was suppressed by gAD. Compared with normal controls, UHRF1 expression was markedly increased in EAC tissues and cell lines. Silence of UHRF1 increased the expression of cleaved caspase 3 and 9 and Bax, reduced the expression of Bcl-2, promoted apoptosis, and inhibited cell proliferation in OE 19 cells. Overexpression of UHRF1 significantly blocked gAD-induced decrease of cell proliferation and increase of apoptosis in leptin-treated cells. Silence of adiponectin receptor 1/2 (AdipoR1/2) could inhibit gAD-induced decrease of cell proliferation and increase of apoptosis in leptin-treated cells. Silence of AdipoR2, but not AdipoR1, suppressed gAD-induced decrease of UHRF1 expression in leptin-treated cells. The results indicated that gAD inhibited the prooncogenic effects of leptin via AdipoR2-mediated suppression of UHRF1. Our study provides novel insights into the role of UHRF1 in the development of EAC and the mechanism of antitumor effect of gAD.
Assuntos
Adenocarcinoma/metabolismo , Adiponectina/metabolismo , Proteínas Estimuladoras de Ligação a CCAAT/biossíntese , Proliferação de Células , Neoplasias Esofágicas/metabolismo , Leptina/metabolismo , Proteínas de Neoplasias/metabolismo , Receptores de Adiponectina/metabolismo , Adenocarcinoma/patologia , Linhagem Celular Tumoral , Neoplasias Esofágicas/patologia , Humanos , Ubiquitina-Proteína LigasesRESUMO
As the second most common cancer in men around the world, prostate cancer is increasingly gaining more attention. Dihydroartemisinin (DHA) has been proven to be a promising anticancer agent in vitro as well as in vivo in accumulating data. However, the detailed mechanisms of how DHA action in human prostate cancer PC-3 cells remain elusive. This study aimed to investigate the effects of DHA, a novel anticancer agent, by inhibiting the expression of ubiquitin like containing PHD and ring finger 1 (UHRF1) in PC-3 cells. The apoptosis and cell-cycle distribution were detected by flow cytometry. Quantitative real-time PCR was performed to examine both UHRF1 and DNA methyltransferase 1 (DNMT1) expressions at mRNA levels, whereas the expressions of UHRF1, DNMT1, and p16 proteins at protein levels were detected by Western blotting. Methylation levels of p16 CpG islands were determined by bisulfite genomic sequencing. We showed that DHA induced the downregulation of UHRF1 and DNMT1, accompanied by an upregulation of p16 in PC-3 cells. Decreased p16 promoter methylation levels in DHA-treated groups were also observed in PC-3 cells. Furthermore, DHA significantly induced apoptosis and G1/S cell-cycle arrest in PC-3 cells. Our results suggested that downregulation of UHRF1/DNMT1 is upstream to many cellular events, including G1 cell arrest, demethylation of p16, and apoptosis. Together, our study provides new evidence that DHA may serve as a potential therapeutic agent in the treatment of prostate cancer.
Assuntos
Artemisininas/farmacologia , Proteínas Estimuladoras de Ligação a CCAAT/biossíntese , Neoplasias de Próstata Resistentes à Castração/tratamento farmacológico , Apoptose/efeitos dos fármacos , Proteínas Estimuladoras de Ligação a CCAAT/genética , Proteínas Estimuladoras de Ligação a CCAAT/metabolismo , Linhagem Celular Tumoral , Ilhas de CpG , Inibidor p16 de Quinase Dependente de Ciclina/biossíntese , Inibidor p16 de Quinase Dependente de Ciclina/genética , Inibidor p16 de Quinase Dependente de Ciclina/metabolismo , DNA (Citosina-5-)-Metiltransferase 1/biossíntese , DNA (Citosina-5-)-Metiltransferase 1/genética , DNA (Citosina-5-)-Metiltransferase 1/metabolismo , Metilação de DNA , Regulação para Baixo/efeitos dos fármacos , Pontos de Checagem da Fase G1 do Ciclo Celular/efeitos dos fármacos , Expressão Gênica/efeitos dos fármacos , Humanos , Masculino , Neoplasias de Próstata Resistentes à Castração/genética , Neoplasias de Próstata Resistentes à Castração/metabolismo , Neoplasias de Próstata Resistentes à Castração/patologia , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Fase S/efeitos dos fármacos , Ubiquitina-Proteína Ligases , Regulação para Cima/efeitos dos fármacosRESUMO
Increasing energy expenditure through brown adipocyte recruitment is a promising approach to combat obesity. We report here the comprehensive profiling of the epigenome and transcriptome throughout the lineage commitment and differentiation of C3H10T1/2 mesenchymal stem cell line into brown adipocytes. Through direct comparison to datasets from differentiating white adipocytes, we systematically identify stage- and lineage-specific coding genes, lncRNAs and microRNAs. Utilizing chromatin state maps, we also define stage- and lineage-specific enhancers, including super-enhancers, and their associated transcription factor binding motifs and genes. Through these analyses, we found that in brown adipocytes, brown lineage-specific genes are pre-marked by both H3K4me1 and H3K27me3, and the removal of H3K27me3 at the late stage is necessary but not sufficient to promote brown gene expression, while the pre-deposition of H3K4me1 plays an essential role in poising the brown genes for expression in mature brown cells. Moreover, we identify SOX13 as part of a p38 MAPK dependent transcriptional response mediating early brown cell lineage commitment. We also identify and subsequently validate PIM1, SIX1 and RREB1 as novel regulators promoting brown adipogenesis. Finally, we show that SIX1 binds to adipogenic and brown marker genes and interacts with C/EBPα, C/EBPß and EBF2, suggesting their functional cooperation during adipogenesis.
Assuntos
Adipogenia/genética , Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Proteína beta Intensificadora de Ligação a CCAAT/genética , Proteínas Estimuladoras de Ligação a CCAAT/genética , Proteínas de Homeodomínio/genética , Obesidade/genética , Tecido Adiposo Marrom/crescimento & desenvolvimento , Tecido Adiposo Marrom/metabolismo , Animais , Autoantígenos/genética , Fatores de Transcrição Hélice-Alça-Hélice Básicos/biossíntese , Proteína beta Intensificadora de Ligação a CCAAT/biossíntese , Proteína beta Intensificadora de Ligação a CCAAT/metabolismo , Proteínas Estimuladoras de Ligação a CCAAT/biossíntese , Proteínas Estimuladoras de Ligação a CCAAT/metabolismo , Diferenciação Celular/genética , Linhagem da Célula/genética , Proteínas de Ligação a DNA/biossíntese , Proteínas de Ligação a DNA/genética , Metabolismo Energético/genética , Regulação da Expressão Gênica no Desenvolvimento , Proteínas de Homeodomínio/metabolismo , Células-Tronco Mesenquimais , Camundongos , Obesidade/metabolismo , Obesidade/patologia , RNA Longo não Codificante/biossíntese , Fatores de Transcrição/biossíntese , Fatores de Transcrição/genética , Transcriptoma/genéticaRESUMO
Studies have showed the involvement of ubiquitin-like with PHD and RING finger domains 1 (UHRF1) in tumorigenesis and progression. This study focused on the relationships between UHRF1 and medulloblastoma (MB). Immunostaining and western blotting demonstrated differential expression of UHRF1 in MB tissues and no UHRF1 expression in normal cerebellum tissues. Univariate survival analysis revealed MB patients with high UHRF1 expression had significantly shorter OS and PFS than patients with low UHRF1 (OS p = 0.009, PFS p = 0.003). Multivariate analysis illustrated that UHRF1 expression level is an independent prognostic factor influencing the OS and PFS (OS p = 0.038, PFS p = 0.014). UHRF1 expression levels were significantly different among molecular subgroups of MB (p = 0.003). Down-regulation of UHRF1 by RNAi inhibited proliferation and clonogenic ability of MB cell lines with cell cycle arrest in G1/G2-phase. Meanwhile, cells transfected with lenti-shUHRF1 showed increased p16 expression and location shift of CDK4 in MB cells. These findings indicate UHRF1 may promote cell proliferation and be a potential biomarker that can be used as a prognostic parameter and a therapeutic target for MB.
Assuntos
Proteínas Estimuladoras de Ligação a CCAAT/biossíntese , Neoplasias Cerebelares/metabolismo , Neoplasias Cerebelares/patologia , Meduloblastoma/metabolismo , Meduloblastoma/patologia , Proteínas Estimuladoras de Ligação a CCAAT/genética , Linhagem Celular Tumoral , Proliferação de Células/fisiologia , Neoplasias Cerebelares/genética , Inibidor p16 de Quinase Dependente de Ciclina/biossíntese , Inibidor p16 de Quinase Dependente de Ciclina/genética , Regulação para Baixo , Humanos , Imuno-Histoquímica , Meduloblastoma/genética , Inclusão em Parafina , Prognóstico , RNA Interferente Pequeno/administração & dosagem , RNA Interferente Pequeno/genética , Fixação de Tecidos , Transfecção , Ubiquitina-Proteína LigasesRESUMO
MEK/ERK signal pathway was required for the differentiation of granulocytes, megakaryocytes and erythrocytes. Recently, MEK/ERK cascade was reported to be involved in all-trans retinoic acid (ATRA) induced differentiation in acute promyelocytic leukemia (APL) cells. However, the upstream and downstream molecules of MEK/ERK signal pathway in this cell model remains to be elucidated. In this work, we showed that RAF-1 was activated and the blockade of RAF-1 activation attenuated MEK/ERK activation as well as ATRA-induced differentiation. ATRA-enhanced protein levels of C/EBPß, C/EBPε and PU.1, which were required for differentiation in APL cells, were suppressed by the specific inhibitor of MEK. However, MEK inhibition had no effect on the degradation of PML-RARα fusion protein or the restoration of PML nuclear bodies by ATRA treatment. Taken together, our study suggested that RAF-1/MEK/ERK cascade was involved in ATRA-induced differentiation in APL cells through enhancing the protein level of C/EBPß, C/EBPε and PU.1.
Assuntos
Diferenciação Celular/efeitos dos fármacos , Leucemia Promielocítica Aguda/patologia , Sistema de Sinalização das MAP Quinases/fisiologia , Proteínas Proto-Oncogênicas c-raf/fisiologia , Tretinoína/farmacologia , Antineoplásicos/farmacologia , Proteína beta Intensificadora de Ligação a CCAAT/biossíntese , Proteína beta Intensificadora de Ligação a CCAAT/efeitos dos fármacos , Proteínas Estimuladoras de Ligação a CCAAT/biossíntese , Proteínas Estimuladoras de Ligação a CCAAT/efeitos dos fármacos , Linhagem Celular Tumoral , Granulócitos/efeitos dos fármacos , Granulócitos/patologia , Humanos , Leucemia Promielocítica Aguda/tratamento farmacológico , Leucemia Promielocítica Aguda/metabolismo , Proteínas Proto-Oncogênicas/biossíntese , Proteínas Proto-Oncogênicas/efeitos dos fármacos , Transativadores/biossíntese , Transativadores/efeitos dos fármacosRESUMO
OBJECTIVE: To investigate the association between the polymorphism of miR-146a and The ubiquitin-like with PHD and ring-finger domains 1 (UHRF1) expression in patients with gastric cancer. METHODS: MiR-146a rs2910164 was genotyped in 130 patients with gastric cancer and 130 cancer-free individuals using polymerase chain reaction (PCR)-restriction fragment length polymorphism. UHRF1 expression was analyzed in 22 gastric cancer tissues and their adjacent normal tissues using quantitative real-time PCR. RESULTS: No significant differences in genotype distributions of miR-146a rs2910164 were found between cases and controls, but we observed that grade II tumors were more frequently detected in patients with CG/CC genotype compared to those with CC genotype. UHRF1 expressions in cancerous tissues were significantly higher than in noncancerous tissues (1.89-fold). Patients with CC genotype showed a significant increase in UHRF1 expression in comparison to the carriers of GG/CG genotype. A higher UHRF1 expression was associated with cancer stage IV and grade III (P<0.05). CONCLUSION: The overexpression of UHRF1 was correlated with the stage and grade of gastric cancer and is associated with the genotype distribution of rs2910164.
Assuntos
Proteínas Estimuladoras de Ligação a CCAAT/biossíntese , MicroRNAs/genética , Polimorfismo de Nucleotídeo Único , Neoplasias Gástricas/genética , Idoso , Idoso de 80 Anos ou mais , Proteínas Estimuladoras de Ligação a CCAAT/genética , Estudos de Casos e Controles , Feminino , Regulação Neoplásica da Expressão Gênica , Predisposição Genética para Doença , Genótipo , Humanos , Masculino , Pessoa de Meia-Idade , Gradação de Tumores , Proteínas de Neoplasias/biossíntese , Proteínas de Neoplasias/genética , Estadiamento de Neoplasias , RNA Neoplásico/genética , Neoplasias Gástricas/metabolismo , Neoplasias Gástricas/patologia , Ubiquitina-Proteína Ligases , Regulação para CimaRESUMO
BACKGROUND/AIMS: This study aimed to examine whether UHRF-1 and p53 overexpression is a prognostic marker for gastric cancer. PATIENTS AND METHODS: Sixty-four patients with gastric cancer (study group) and 23 patients with gastritis (control group) were evaluated. Immunohistochemistry was used to examine expression of UHRF-1 and p53 in gastric cancers and a control group diagnosed with gastritis. RESULTS: The median age was 63 years (18-83 years) in the study group. UHRF-1 was positive in 15 (23%) patients with gastric cancer and fi ve (21.7%) patients with gastritis (P = 0.559). UHRF1 expression level in gastric cancer is more powerful than in gastritis (P = 0.046). Thirty-seven (61%) patients with gastric cancer and only one patient with gastritis were p53 positive (P < 0.001). After a median follow-up of 12 months (1-110), the 2-year overall survival rates were 55% and 30% in negative and positive p53, respectively (P = 0.084). Also, the 2-year overall survival rates were 45% and 53% in negative and positive UHRF-1, respectively (P = 0.132). CONCLUSION: According to this study, UHRF-1 and p53 were not prognostic factors for gastric cancer, whereas they may have a diagnostic value for differentiating between gastric cancer and gastritis.
Assuntos
Biomarcadores Tumorais/biossíntese , Proteínas Estimuladoras de Ligação a CCAAT/biossíntese , Neoplasias Gástricas/metabolismo , Neoplasias Gástricas/patologia , Proteína Supressora de Tumor p53/biossíntese , Adulto , Idoso , Idoso de 80 Anos ou mais , Feminino , Gastrite/metabolismo , Gastrite/patologia , Humanos , Masculino , Pessoa de Meia-Idade , Prognóstico , Neoplasias Gástricas/diagnóstico , Análise de Sobrevida , Ubiquitina-Proteína Ligases , Adulto JovemRESUMO
Over the last few decades, research focusing on canola (Brassica napus L.) seed oil content and composition has expanded. Oil production and accumulation are influenced by genes participating in embryo and seed development. The Arabidopsis LEAFY COTYLEDON1 (LEC1) is a well characterized regulator of embryo development that also enhances the expression of genes involved in fatty acid (FA) synthesis. B. napus lines over-expressing or down-regulating BnLEC1 were successfully generated by Agrobacterium-mediated transformation. The constitutive expression of BnLEC1 in B. napus var. Polo, increased seed oil content by 7-16%, while the down-regulation of BnLEC1 in B. napus var. Topas reduced oil content by 9-12%. Experimental manipulation of BnLEC1 caused transcriptional changes in enzymes participating in sucrose metabolism, glycolysis, and FA biosynthesis, suggesting an enhanced carbon flux towards FA biosynthesis in tissues over-expressing BnLEC1. The increase in oil content induced by BnLEC1 was not accompanied by alterations in FA composition, oil nutritional value or glucosinolate (GLS) levels. Suppression of BnLEC1 reduced seed oil accumulation and elevated the level of GLS possibly through the transcriptional regulation of BnST5a (Sulphotransferase5a), the last GLS biosynthetic enzyme. Collectively, these findings demonstrate that experimental alterations of BnLEC1 expression can be used to influence oil production and quality in B. napus.
Assuntos
Brassica napus/metabolismo , Proteínas Estimuladoras de Ligação a CCAAT/biossíntese , Ácidos Graxos Monoinsaturados/metabolismo , Regulação da Expressão Gênica de Plantas , Glucosinolatos/biossíntese , Proteínas de Plantas/biossíntese , Sementes/metabolismo , Brassica napus/genética , Proteínas Estimuladoras de Ligação a CCAAT/genética , Glucosinolatos/genética , Proteínas de Plantas/genética , Óleo de Brassica napus , Sementes/genéticaRESUMO
The tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is a potent anticancer agent possessing the ability to induce apoptosis in various cancer cells but not in nonmalignant cells. However, certain type of cancer cells are resistant to TRAILinduced apoptosis and some acquire resistance after the first treatment. So development of an agent that can reduce or avoid resistance in TRAILinduced apoptosis has garnered significant attention. The present study evaluated the anticancer potential of hispolon in TRAILinduced apoptosis and indicated hispolon can sensitize cancer cells to TRAIL. As the mechanism of action was examined, hispolon was found to activate caspase3, caspase8 and caspase9, while downregulating the expression of cell survival proteins such as cFLIP, Bcl2 and BclxL and upregulating the expression of Bax and truncated Bid. We also found hispolon induced death receptors in a noncell typespecific manner. Upregulation of death receptors by hispolon was found to be p53-independent but linked to the induction of CAAT enhancer binding protein homologous protein (CHOP). Overall, hispolon was demonstrated to potentiate the apoptotic effects of TRAIL through downregulation of antiapoptotic proteins and upregulation of death receptors linked with CHOP and pERK elevation.
Assuntos
Antineoplásicos Fitogênicos/farmacologia , Proteínas Reguladoras de Apoptose/biossíntese , Apoptose/efeitos dos fármacos , Caspases/biossíntese , Catecóis/farmacologia , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Proteínas de Neoplasias/biossíntese , Polissacarídeos/química , Receptores do Ligante Indutor de Apoptose Relacionado a TNF/biossíntese , Transdução de Sinais/fisiologia , Ligante Indutor de Apoptose Relacionado a TNF/farmacologia , Antineoplásicos Fitogênicos/isolamento & purificação , Proteínas Reguladoras de Apoptose/genética , Proteínas Estimuladoras de Ligação a CCAAT/biossíntese , Proteínas Estimuladoras de Ligação a CCAAT/genética , Caspases/genética , Catecóis/isolamento & purificação , Linhagem Celular Tumoral , Regulação para Baixo , Humanos , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Sistema de Sinalização das MAP Quinases/fisiologia , Proteínas de Neoplasias/genética , Phellinus , Extratos Vegetais , Receptores do Ligante Indutor de Apoptose Relacionado a TNF/genética , Transdução de Sinais/efeitos dos fármacos , Regulação para Cima/efeitos dos fármacosRESUMO
One of the major obstacles in generating induced pluripotent stem (iPS) cells suitable for therapeutic application is the low efficiency of the process and the long time required, with many iPS lines acquiring genomic aberrations. In this chapter we describe a highly efficient iPS reprogramming system based on the transient expression in pre-B cells of the transcription factor C/EBPα, followed by the induction of the four Yamanaka factors (OSKM). In addition, the process is very rapid, yielding Oct4 positive cells within 2 days and Nanog-positive iPS cell colonies within a week.