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1.
Gene ; 734: 144381, 2020 Apr 15.
Artigo em Inglês | MEDLINE | ID: mdl-31978510

RESUMO

Down-regulation of stemness genes expression is important in differentiation therapy against cancer stem cells (CSCs). The aim of this study was to evaluate the Oct4 , Sox2, Nanog, and C-myc expression in rat breast cancer stem cells (LA7) which treated with human ovarian follicular fluid (FF), replicative senescent fibroblast culture supernatant (P14), and 16 h serum starved fibroblast supernatant (16 h-SFS). The cells were exposed to these biological fluids for 24 h, 72 h, and 7 days. Stem-loop RT-qPCR assay was used to quantify the expression of above mentioned genes. Results showed that FF had the least cytotoxic effect on the LA7 cells. Except for Nanog gene, exposure of LA7 cell line to 16 h-SFS and P14 decreased significantly expression of the three other genes after 24 h (P < 0.05). Nanog and Sox2 genes expression was also decreased in LA7 cells which have been already treated with FF for 24 h. Moreover, compared to the control solution, the expression of Oct4 increased significantly after 7 days exposure to FF (P < 0.05). Annexin V-PE /7-AAD-, acridine orange/ethidium bromide staining and doubling time assays revealed apoptosis and necrosis induction by these biological fluids in LA7 cells. Moreover, in an in vitro model of metastasis assay, i.e., scratch test, these fluids exhibited anti-LA7 migration activity which culminated in 16 h-SFS treated cells. Generally, this study showed that FF, 16 h-SFS, and P14 have positive effects on down-regulation of Nanog, Oct4, Sox2 and C-myc expression, and consequently can increase the differentiation of breast cancer stem cells. For the first time, this study provided some evidence indicating that some biological fluids have potential to differentiate the CSCs, show anti- survival, growth-, and cell migration activity.


Assuntos
Líquidos Corporais/fisiologia , Regulação Neoplásica da Expressão Gênica , Neoplasias Mamárias Animais/genética , Células-Tronco Neoplásicas , Fatores de Transcrição/genética , Animais , Diferenciação Celular/genética , Linhagem Celular Tumoral , Proliferação de Células , Sobrevivência Celular , Meios de Cultura/farmacologia , Regulação para Baixo , Feminino , Líquido Folicular/fisiologia , Genes myc , Humanos , Proteína Homeobox Nanog/genética , Células-Tronco Neoplásicas/patologia , Fator 3 de Transcrição de Octâmero/genética , Ratos , Reação em Cadeia da Polimerase em Tempo Real , Fatores de Transcrição SOXB1/genética
2.
Cell Prolif ; 53(1): e12729, 2020 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-31746095

RESUMO

OBJECTIVES: The successional dental lamina is the distinctive structure on the lingual side of the vertebrate tooth germ. The aim of this study was to investigate the relationship among Sox2, Claudin10 and laminin5 and the role of Sox2 in successional dental lamina proliferation during vertebrate tooth development. MATERIALS AND METHODS: To understand the successional dental lamina, two types of successional tooth formation, that in geckos (with multiple rounds of tooth generation) and that in mice (with only one round of tooth generation), were analysed. RESULTS: Unique coexpression patterns of Sox2 and Claudin10 expression were compared in the successional dental lamina from the cap stage to the late bell stage in the mouse tooth germ and in juvenile gecko teeth to support continuous tooth replacement. Furthermore, Laminin5 expression was shown in the cap stage and decreased after the bell stage. Upon comparing the epithelial cell cycles and cell proliferation in successional dental lamina regions between mouse and gecko molars using BrdU and IdU staining and pulse-chase methods, distinctive patterns of continuous expression were revealed. Moreover, Sox2 overexpression with a lentiviral system resulted in hyperplastic dental epithelium in mouse molars. CONCLUSIONS: Our findings indicate that the regulation of Sox2 in dental lamina proliferation is fundamental to the successional dental lamina in both species.


Assuntos
Proliferação de Células , Células Epiteliais/metabolismo , Dente Molar/embriologia , Fatores de Transcrição SOXB1/metabolismo , Germe de Dente/embriologia , Animais , Moléculas de Adesão Celular/biossíntese , Moléculas de Adesão Celular/genética , Claudinas/biossíntese , Claudinas/genética , Células Epiteliais/citologia , Lagartos/embriologia , Camundongos , Camundongos Endogâmicos ICR , Dente Molar/citologia , Proteínas de Répteis/genética , Proteínas de Répteis/metabolismo , Fatores de Transcrição SOXB1/genética , Germe de Dente/citologia
3.
Adv Exp Med Biol ; 1185: 221-226, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31884615

RESUMO

Anophthalmia and microphthalmia (A/M) are rare distinct phenotypes that represent a continuum of structural developmental eye defects. Here, we describe three probands from an Egyptian population with various forms of A/M: two patients with bilateral anophthalmia and one with bilateral microphthalmia that were investigated using whole exome sequencing (WES). We identified three causative mutations in three different genes. A new homozygous frameshift mutation c.[422delA];[422delA], p.[N141Ifs∗19];[N141Ifs∗19] in VSX2 was identified in a patient showing bilateral anophthalmia. A previously reported SOX2 deletion c.[70_89del20] p.[N24Rfs∗65];[=] was found in one subject with bilateral anophthalmia. A novel homozygous in-frame mutation c.[431_433delACT];[431_433delACT], p.[Y144del]; [Y144del]) in FOXE3 was identified in a patient with severe bilateral microphthalmia and anterior segment dysgenesis. This study shows that whole exome sequencing (WES) is a reliable and effective strategy for the molecular diagnosis of A/M. Our results expand its allelic heterogeneity and highlight the need for the testing of patient with this developmental anomaly.


Assuntos
Anoftalmia/genética , Fatores de Transcrição Forkhead/genética , Proteínas de Homeodomínio/genética , Microftalmia/genética , Fatores de Transcrição SOXB1/genética , Fatores de Transcrição/genética , Egito , Mutação da Fase de Leitura , Humanos , Mutação , Fenótipo , Deleção de Sequência
4.
Cytogenet Genome Res ; 159(3): 143-150, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31760386

RESUMO

Two species of spiny rats, Tokudaia osimensis and Tokudaia tokunoshimensis, show an X0/X0 sex chromosome constitution due to the lack of a Y chromosome. The Sry gene has been completely lost from the genome of these species. We hypothesized that Sox3, which is thought to be originally a homologue of Sry, could function in sex determination in these animals in the absence of Sry. Sox3 was localized in a region of the X chromosome in T. osimensis homologous to mouse. A similar testis- and ovary-specific pattern of expression was observed in mouse and T. osimensis. Although the sequence of the Sox3 gene and its promoter are highly conserved, a 13-bp deletion was specifically found in the promoter region of the 2 spiny rat species. Reporter gene assays were performed to examine the effect of the 13-bp deletion in the promoter region on Sox3 regulation. Although an approximately 60% decrease in activity was observed using the Tokudaia promoters with the 13-bp deletion, the activity was recovered using a mutated promoter in which the deletion was filled with mouse sequence. To evaluate whether SOX3 could regulate Sox9 expression, a reporter gene assay was carried out using testis-specific enhancer of Sox9 core (TESCO). Co-transfection with a combination of mouse SF1 and mouse SOX3 or T. osimensis SOX3 resulted in a greater than 2-fold increase in activity of mouse and T. osimensis TESCO. These results support the idea that the function of SOX3 as a transcription factor, as has been reported in mice and humans, is conserved in T. osimensis. Therefore, we conclude that the Sox3 gene has no function in sex determination in Sry-lacking Tokudaia species.


Assuntos
Murinae/genética , Fatores de Transcrição SOXB1/genética , Proteína da Região Y Determinante do Sexo/genética , Sequência de Aminoácidos , Animais , Espécies em Perigo de Extinção , Feminino , Deleção de Genes , Genes Reporter , Masculino , Regiões Promotoras Genéticas , Fatores de Transcrição SOXB1/química , Homologia de Sequência de Aminoácidos
5.
J Ovarian Res ; 12(1): 93, 2019 Oct 14.
Artigo em Inglês | MEDLINE | ID: mdl-31610800

RESUMO

BACKGROUND: The ST6Gal-I glycosyltransferase, which adds α2-6-linked sialic acids to N-glycosylated proteins is upregulated in a wide range of malignancies including ovarian cancer. Prior studies have shown that ST6Gal-I-mediated sialylation of select surface receptors remodels intracellular signaling to impart cancer stem cell (CSC) characteristics. However, the mechanisms that contribute to ST6Gal-I expression in stem-like cancer cells are poorly understood. RESULTS: Herein, we identify the master stem cell transcription factor, Sox2, as a novel regulator of ST6Gal-I expression. Interestingly, SOX2 and ST6GAL1 are located within the same tumor-associated amplicon, 3q26, and these two genes exhibit coordinate gains in copy number across multiple cancers including ~ 25% of ovarian serious adenocarcinomas. In conjunction with genetic co-amplification, our studies suggest that Sox2 directly binds the ST6GAL1 promoter to drive transcription. ST6Gal-I expression is directed by at least four distinct promoters, and we identified the P3 promoter as the predominant promoter utilized by ovarian cancer cells. Chromatin Immunoprecipitation (ChIP) assays revealed that Sox2 binds regions proximal to the P3 promoter. To confirm that Sox2 regulates ST6Gal-I expression, Sox2 was either overexpressed or knocked-down in various ovarian cancer cell lines. Sox2 overexpression induced an increase in ST6Gal-I mRNA and protein, as well as surface α2-6 sialylation, whereas Sox2 knock-down suppressed levels of ST6Gal-I mRNA, protein and surface α2-6 sialylation. CONCLUSIONS: These data suggest a process whereby SOX2 and ST6GAL1 are coordinately amplified in cancer cells, with the Sox2 protein then binding the ST6GAL1 promoter to further augment ST6Gal-I expression. Our collective results provide new insight into mechanisms that upregulate ST6Gal-I expression in ovarian cancer cells, and also point to the possibility that some of the CSC characteristics commonly attributed to Sox2 may, in part, be mediated through the sialyltransferase activity of ST6Gal-I.


Assuntos
Antígenos CD/genética , Proliferação de Células/genética , Neoplasias Ovarianas/genética , Fatores de Transcrição SOXB1/genética , Sialiltransferases/genética , Apoptose/genética , Linhagem Celular Tumoral , Feminino , Regulação Neoplásica da Expressão Gênica , Glicosiltransferases/genética , Humanos , Células-Tronco Neoplásicas/metabolismo , Neoplasias Ovarianas/patologia , Ligação Proteica , Transdução de Sinais/genética
6.
Medicine (Baltimore) ; 98(38): e17225, 2019 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-31567982

RESUMO

The present study is to analyze the difference of gene methylation in early cervical adenocarcinoma and to find molecular markers for predicting the occurrence and development of cervical adenocarcinoma.A total of 15 cases of primary cervical adenocarcinoma and 10 cases of primary cervical squamous cell carcinoma at stages IB1 or IIA1 were included in the study. Infinium MethylationEPIC BeadChip (850K) was used to screen specifically expressed genes in cervical adenocarcinoma tissues. Bisulfite sequencing polymerase chain reaction (BSP) and quantitative real-time polymerase chain reaction (qRT-PCR) were used to verify the methylation levels in cervical adenocarcinoma, cervical squamous cell carcinoma, and normal cervical tissues.Sex determining region Y-box 1 (SOX1) and cyclin D1 (CCND1) genes participated in multiple signaling pathways, being the central nodes of gene regulatory networks. SOX1 gene, but not CCND1 gene, was a specifically methylated gene in cervical adenocarcinoma according to BSP. According to qRT-PCR, methylation level of SOX1 in cervical adenocarcinoma tissues is significantly different from that in cervical squamous cell carcinoma tissues or normal cervical tissues, and the methylation level of CCND1 in cervical adenocarcinoma tissues or cervical squamous cell carcinoma tissues is significantly different from that in normal cervical tissues.The present study demonstrates that tumor-suppressor gene SOX1 is a methylation-specific expression gene of cervical adenocarcinoma and is expected to become a specific molecular marker for the diagnosis of cervical adenocarcinoma. However, CCND1 gene was not proven to be a specific methylation expression gene in cervical adenocarcinoma in the present study.


Assuntos
Adenocarcinoma/genética , Metilação de DNA/genética , Fatores de Transcrição SOXB1/genética , Neoplasias do Colo do Útero/genética , Adenocarcinoma/metabolismo , Carcinoma de Células Escamosas/genética , Carcinoma de Células Escamosas/metabolismo , Colo do Útero/metabolismo , Ciclina D1/genética , Feminino , Regulação Neoplásica da Expressão Gênica , Redes Reguladoras de Genes/genética , Marcadores Genéticos/genética , Humanos , Reação em Cadeia da Polimerase em Tempo Real , Neoplasias do Colo do Útero/metabolismo
7.
Yi Chuan ; 41(10): 950-961, 2019 Oct 20.
Artigo em Chinês | MEDLINE | ID: mdl-31624057

RESUMO

SOX2 (sex determining region Y-box2) is one of the critical pluripotent factors that play a crucial role in the first lineage differentiation and maintenance of pluripotency in inner cell mass during early embryonic development. However, there are few researches about the regulation of the SOX2 promoter, especially in Sus scrofa. To analyzed the activity of SOX2 promoter in early porcine embryos, we determined the control system and established the microinjection system for assessing SOX2 promoter activity by analyzing the embryonic development and the expression of enhanced green fluorescence protein (EGFP) after micro-injected different EGFP plasmids at different times after activation of the oocytes. Then, we analyzed the structure of 5000 bp upstream of the SOX2 translation initiation site and found there were four transcription factor binding site clusters. Next, we designed and constructed promoter-containing plasmids to analyze the function of each cluster. To detect the activity of different promoters, we assessed the mCherry expression in protein levels and mRNA levels by analyzing the mCherry fluorescence intensity and qRT-PCR after injecting plasmids into embryos. These results showed that the activity of the shorted promoter, with the region from 2254 bp to 2442 bp upstream of translation initiation site deleted, decreased to 17.8% at 4-cell and 8-cell stages compared with the full-length promoter. This region included two NF-AT transcription factor binding sites, which indicated that the NF-AT binding site is a key region to regulate the activity of the SOX2 promoter. The results provide important data for determination the mechanism of porcine SOX2 regulation.


Assuntos
Desenvolvimento Embrionário , Regulação da Expressão Gênica no Desenvolvimento , Regiões Promotoras Genéticas , Fatores de Transcrição SOXB1/genética , Animais , Sítios de Ligação , Diferenciação Celular , Embrião de Mamíferos , Proteínas de Fluorescência Verde , Fatores de Transcrição NFATC , Plasmídeos , Suínos
8.
Int J Mol Sci ; 20(18)2019 Sep 10.
Artigo em Inglês | MEDLINE | ID: mdl-31509934

RESUMO

In addition to its role as an endocrine messenger, growth hormone (GH) also acts as a neurotrophic factor in the central nervous system (CNS), whose effects are involved in neuroprotection, axonal growth, and synaptogenic modulation. An increasing amount of clinical evidence shows a beneficial effect of GH treatment in patients with brain trauma, stroke, spinal cord injury, impaired cognitive function, and neurodegenerative processes. In response to injury, Müller cells transdifferentiate into neural progenitors and proliferate, which constitutes an early regenerative process in the chicken retina. In this work, we studied the long-term protective effect of GH after causing severe excitotoxic damage in the retina. Thus, an acute neural injury was induced via the intravitreal injection of kainic acid (KA, 20 µg), which was followed by chronic administration of GH (10 injections [300 ng] over 21 days). Damage provoked a severe disruption of several retinal layers. However, in KA-damaged retinas treated with GH, we observed a significant restoration of the inner plexiform layer (IPL, 2.4-fold) and inner nuclear layer (INL, 1.5-fold) thickness and a general improvement of the retinal structure. In addition, we also observed an increase in the expression of several genes involved in important regenerative pathways, including: synaptogenic markers (DLG1, NRXN1, GAP43); glutamate receptor subunits (NR1 and GRIK4); pro-survival factors (BDNF, Bcl-2 and TNF-R2); and Notch signaling proteins (Notch1 and Hes5). Interestingly, Müller cell transdifferentiation markers (Sox2 and FGF2) were upregulated by this long-term chronic GH treatment. These results are consistent with a significant increase in the number of BrdU-positive cells observed in the KA-damaged retina, which was induced by GH administration. Our data suggest that GH is able to facilitate the early proliferative response of the injured retina and enhance the regeneration of neurite interconnections.


Assuntos
Hormônio do Crescimento/farmacologia , Ácido Caínico/toxicidade , Regeneração/efeitos dos fármacos , Retina/efeitos dos fármacos , Animais , Animais Recém-Nascidos , Fator Neurotrófico Derivado do Encéfalo/genética , Embrião de Galinha , Galinhas , Regulação da Expressão Gênica no Desenvolvimento/efeitos dos fármacos , Neurogênese/efeitos dos fármacos , Neurogênese/genética , Neurogênese/fisiologia , Fármacos Neuroprotetores/farmacologia , Neurotoxinas/toxicidade , Receptor Notch1/genética , Regeneração/genética , Regeneração/fisiologia , Retina/metabolismo , Retina/fisiopatologia , Fatores de Transcrição SOXB1/genética
9.
Nat Commun ; 10(1): 4269, 2019 09 19.
Artigo em Inglês | MEDLINE | ID: mdl-31537794

RESUMO

Embryonic development yields many different cell types in response to just a few families of inductive signals. The property of signal-receiving cells that determines how they respond to inductive signals is known as competence, and it differs in different cell types. Here, we explore the ways in which maternal factors modify chromatin to specify initial competence in the frog Xenopus tropicalis. We identify early-engaged regulatory DNA sequences, and infer from them critical activators of the zygotic genome. Of these, we show that the pioneering activity of the maternal pluripotency factors Pou5f3 and Sox3 determines competence for germ layer formation by extensively remodelling compacted chromatin before the onset of inductive signalling. This remodelling includes the opening and marking of thousands of regulatory elements, extensive chromatin looping, and the co-recruitment of signal-mediating transcription factors. Our work identifies significant developmental principles that inform our understanding of how pluripotent stem cells interpret inductive signals.


Assuntos
Montagem e Desmontagem da Cromatina/genética , Proteínas de Homeodomínio/genética , Células-Tronco Pluripotentes/citologia , Fatores de Transcrição SOXB1/genética , Fatores de Transcrição/genética , Proteínas de Xenopus/genética , Xenopus/embriologia , Animais , Diferenciação Celular/genética , Cromatina/metabolismo , Desenvolvimento Embrionário/genética , Células-Tronco Embrionárias/citologia , Regulação da Expressão Gênica no Desenvolvimento/genética , Camadas Germinativas/crescimento & desenvolvimento , Sequências Reguladoras de Ácido Nucleico/genética , Xenopus/genética
10.
Int J Mol Sci ; 20(18)2019 Sep 13.
Artigo em Inglês | MEDLINE | ID: mdl-31540269

RESUMO

The Sox2 transcription factor, encoded by a gene conserved in animal evolution, has become widely known because of its functional relevance for stem cells. In the developing nervous system, Sox2 is active in neural stem cells, and important for their self-renewal; differentiation to neurons and glia normally involves Sox2 downregulation. Recent evidence, however, identified specific types of fully differentiated neurons and glia that retain high Sox2 expression, and critically require Sox2 function, as revealed by functional studies in mouse and in other animals. Sox2 was found to control fundamental aspects of the biology of these cells, such as the development of correct neuronal connectivity. Sox2 downstream target genes identified within these cell types provide molecular mechanisms for cell-type-specific Sox2 neuronal and glial functions. SOX2 mutations in humans lead to a spectrum of nervous system defects, involving vision, movement control, and cognition; the identification of neurons and glia requiring Sox2 function, and the investigation of Sox2 roles and molecular targets within them, represents a novel perspective for the understanding of the pathogenesis of these defects.


Assuntos
Células-Tronco Neurais/citologia , Neuroglia/citologia , Fatores de Transcrição SOXB1/genética , Fatores de Transcrição SOXB1/metabolismo , Animais , Diferenciação Celular , Autorrenovação Celular , Regulação para Baixo , Humanos , Camundongos , Mutação , Células-Tronco Neurais/metabolismo , Neurogênese , Neuroglia/metabolismo , Transdução de Sinais
11.
Nucleic Acids Res ; 47(19): 10115-10133, 2019 11 04.
Artigo em Inglês | MEDLINE | ID: mdl-31555818

RESUMO

Pluripotency and cell fates can be modulated through the regulation of super-enhancers; however, the underlying mechanisms are unclear. Here, we showed a novel mechanism in which Ash2l directly binds to super-enhancers of several stemness genes to regulate pluripotency and self-renewal in pluripotent stem cells. Ash2l recruits Oct4/Sox2/Nanog (OSN) to form Ash2l/OSN complex at the super-enhancers of Jarid2, Nanog, Sox2 and Oct4, and further drives enhancer activation, upregulation of stemness genes, and maintains the pluripotent circuitry. Ash2l knockdown abrogates the OSN recruitment to all super-enhancers and further hinders the enhancer activation. In addition, CRISPRi/dCas9-mediated blocking of Ash2l-binding motifs at these super-enhancers also prevents OSN recruitment and enhancer activation, validating that Ash2l directly binds to super-enhancers and initiates the pluripotency network. Transfection of Ash2l with W118A mutation to disrupt Ash2l-Oct4 interaction fails to rescue Ash2l-driven enhancer activation and pluripotent gene upregulation in Ash2l-depleted pluripotent stem cells. Together, our data demonstrated Ash2l formed an enhancer-bound Ash2l/OSN complex that can drive enhancer activation, govern pluripotency network and stemness circuitry.


Assuntos
Proteínas de Ligação a DNA/genética , Elementos Facilitadores Genéticos , Células-Tronco Embrionárias Murinas/metabolismo , Fator 3 de Transcrição de Octâmero/genética , Fatores de Transcrição/genética , Animais , Sistemas CRISPR-Cas/genética , Diferenciação Celular/genética , Linhagem da Célula/genética , Autorrenovação Celular/genética , Reprogramação Celular/genética , Elementos Facilitadores Genéticos/genética , Regulação da Expressão Gênica no Desenvolvimento/genética , Humanos , Camundongos , Mutação/genética , Proteína Homeobox Nanog/genética , Células-Tronco Pluripotentes/metabolismo , Fatores de Transcrição SOXB1/genética , Transfecção
12.
Mol Cell ; 76(3): 473-484.e7, 2019 11 07.
Artigo em Inglês | MEDLINE | ID: mdl-31494034

RESUMO

Enhancers can regulate the promoters of their target genes over very large genomic distances. It is widely assumed that mechanisms of enhancer action involve the reorganization of three-dimensional chromatin architecture, but this is poorly understood. The predominant model involves physical enhancer-promoter interaction by looping out the intervening chromatin. However, studying the enhancer-driven activation of the Sonic hedgehog gene (Shh), we have identified a change in chromosome conformation that is incompatible with this simple looping model. Using super-resolution 3D-FISH and chromosome conformation capture, we observe a decreased spatial proximity between Shh and its enhancers during the differentiation of embryonic stem cells to neural progenitors. We show that this can be recapitulated by synthetic enhancer activation, is impeded by chromatin-bound proteins located between the enhancer and the promoter, and appears to involve the catalytic activity of poly (ADP-ribose) polymerase. Our data suggest that models of enhancer-promoter communication need to encompass chromatin conformations other than looping.


Assuntos
Montagem e Desmontagem da Cromatina , Elementos Facilitadores Genéticos , Proteínas Hedgehog/metabolismo , Células-Tronco Embrionárias Murinas/metabolismo , Neurogênese , Neurônios/metabolismo , Regiões Promotoras Genéticas , Ativação Transcricional , Animais , Linhagem Celular , Regulação da Expressão Gênica no Desenvolvimento , Proteínas Hedgehog/genética , Camundongos , Modelos Genéticos , Neurogênese/genética , Conformação de Ácido Nucleico , Poli(ADP-Ribose) Polimerase-1/genética , Poli(ADP-Ribose) Polimerase-1/metabolismo , Fatores de Transcrição SOXB1/genética , Fatores de Transcrição SOXB1/metabolismo
13.
BMC Cancer ; 19(1): 864, 2019 Aug 30.
Artigo em Inglês | MEDLINE | ID: mdl-31470802

RESUMO

BACKGROUND: Bronchial carcinoids are neuroendocrine tumors that present as typical (TC) and atypical (AC) variants, the latter being more aggressive, invasive and metastatic. Studies of tumor initiating cell (TIC) biology in bronchial carcinoids has been hindered by the lack of appropriate in-vitro and xenograft models representing the bronchial carcinoid phenotype and behavior. METHODS: Bronchial carcinoid cell lines (H727, TC and H720, AC) were cultured in serum-free growth factor supplemented medium to form 3D spheroids and serially passaged up to the 3rd generation permitting expansion of the TIC population as verified by expression of stemness markers, clonogenicity in-vitro and tumorigenicity in both subcutaneous and orthotopic (lung) models. Acetazolamide (AZ), sulforaphane (SFN) and the AZ + SFN combination were evaluated for targeting TIC in bronchial carcinoids. RESULTS: Data demonstrate that bronchial carcinoid cell line 3rd generation spheroid cells show increased drug resistance, clonogenicity, and tumorigenic potential compared with the parental cells, suggesting selection and expansion of a TIC fraction. Gene expression and immunolabeling studies demonstrated that the TIC expressed stemness factors Oct-4, Sox-2 and Nanog. In a lung orthotopic model bronchial carcinoid, cell line derived spheroids, and patient tumor derived 3rd generation spheroids when supported by a stroma, showed robust tumor formation. SFN and especially the AZ + SFN combination were effective in inhibiting tumor cell growth, spheroid formation and in reducing tumor formation in immunocompromised mice. CONCLUSIONS: Human bronchial carcinoid tumor cells serially passaged as spheroids contain a higher fraction of TIC exhibiting a stemness phenotype. This TIC population can be effectively targeted by the combination of AZ + SFN. Our work portends clinical relevance and supports the therapeutic use of the novel AZ+ SFN combination that may target the TIC population of bronchial carcinoids.


Assuntos
Acetazolamida/administração & dosagem , Anticarcinógenos/administração & dosagem , Neoplasias Brônquicas/tratamento farmacológico , Tumor Carcinoide/tratamento farmacológico , Isotiocianatos/administração & dosagem , Células-Tronco Neoplásicas/efeitos dos fármacos , Acetazolamida/farmacologia , Animais , Anticarcinógenos/farmacologia , Protocolos de Quimioterapia Combinada Antineoplásica/administração & dosagem , Protocolos de Quimioterapia Combinada Antineoplásica/farmacologia , Neoplasias Brônquicas/genética , Neoplasias Brônquicas/metabolismo , Tumor Carcinoide/genética , Tumor Carcinoide/metabolismo , Técnicas de Cultura de Células , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Feminino , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , Isotiocianatos/farmacologia , Camundongos , Proteína Homeobox Nanog/genética , Proteína Homeobox Nanog/metabolismo , Células-Tronco Neoplásicas/citologia , Células-Tronco Neoplásicas/metabolismo , Fator 3 de Transcrição de Octâmero/genética , Fator 3 de Transcrição de Octâmero/metabolismo , Fatores de Transcrição SOXB1/genética , Fatores de Transcrição SOXB1/metabolismo , Esferoides Celulares/citologia , Esferoides Celulares/efeitos dos fármacos , Esferoides Celulares/metabolismo , Ensaios Antitumorais Modelo de Xenoenxerto
14.
Phytomedicine ; 63: 153058, 2019 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-31394414

RESUMO

BACKGROUND: Sulforaphane (SFN), a natural compound present in cruciferous vegetable, has been shown to possess anti-cancer activities. Cancer stem cell (CSC) in bulk tumor is generally considered as treatment resistant cell and involved in cancer recurrence. The effects of SFN on nasopharyngeal carcinoma (NPC) CSCs have not yet been explored. PURPOSE: The present study aims to examine the anti-tumor activities of SFN on NPC cells with CSC-like properties and the underlying mechanisms. METHODS: NPC cells growing in monolayer culture, CSCs-enriched NPC tumor spheres, and also the NPC nude mice xenograft were used to study the anti-tumor activities of SFN on NPC. The population of cells expressing CSC-associated markers was evaluated using flow cytometry and aldehyde dehydrogenase (ALDH) activity assay. The effect of DNA methyltransferase 1 (DNMT1) on the growth of NPC cells was analyzed by using small interfering RNA (siRNA)-mediated silencing method. RESULTS: SFN was found to inhibit the formation of CSC-enriched NPC tumor spheres and reduce the population of cells with CSC-associated properties (SRY (Sex determining Region Y)-box 2 (SOX2) and ALDH). In the functional study, SFN was found to restore the expression of Wnt inhibitory factor 1 (WIF1) and the effect was accompanied with the downregulation of DNMT1. The functional activities of WIF1 and DNMT1 were confirmed using exogenously added recombinant WIF1 and siRNA knockdown of DNMT1. Moreover, SFN was found to inhibit the in vivo growth of C666-1 cells and enhance the anti-tumor effects of cisplatin. CONCLUSION: Taken together, we demonstrated that SFN could suppress the growth of NPC cells via the DNMT1/WIF1 axis.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Antineoplásicos Fitogênicos/farmacologia , DNA (Citosina-5-)-Metiltransferase 1/metabolismo , Isotiocianatos/farmacologia , Carcinoma Nasofaríngeo/tratamento farmacológico , Neoplasias Nasofaríngeas/tratamento farmacológico , Proteínas Adaptadoras de Transdução de Sinal/genética , Animais , Antineoplásicos Fitogênicos/administração & dosagem , Protocolos de Quimioterapia Combinada Antineoplásica/farmacologia , Brassicaceae/química , Linhagem Celular Tumoral , Cisplatino/administração & dosagem , DNA (Citosina-5-)-Metiltransferase 1/genética , Humanos , Isotiocianatos/administração & dosagem , Masculino , Camundongos Endogâmicos BALB C , Camundongos Nus , Carcinoma Nasofaríngeo/metabolismo , Carcinoma Nasofaríngeo/patologia , Neoplasias Nasofaríngeas/metabolismo , Neoplasias Nasofaríngeas/patologia , Células-Tronco Neoplásicas/efeitos dos fármacos , Células-Tronco Neoplásicas/patologia , Fatores de Transcrição SOXB1/genética , Fatores de Transcrição SOXB1/metabolismo , Ensaios Antitumorais Modelo de Xenoenxerto
15.
Genes Cells ; 24(10): 667-673, 2019 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-31386786

RESUMO

Analysis of gene expression in single cells is required to understand somatic cell reprogramming into human induced pluripotent stem cells (iPSCs). To facilitate this, we established intermediately reprogrammed stem cells (iRSCs), pre-iPSC lines. The iRSC-iPSC conversion system enables the reproducible monitoring of reprogramming events and the analysis of progressive gene expression profiles using single-cell microarray analysis and genome editing. Here, single-cell microarray analysis showed the stage-specific sequential gene activation during the conversion of iRSCs into iPSCs, using OCT4, TDGF1 and E-CADHERIN as marker genes. Out of 75 OCT4-related genes, which were significantly up-regulated after the activation of OCT4, and entry into the mesenchymal-to-epithelial transition (MET), LIN28 (LIN28A) and FOXO1 were selected for applying to gene expression visualization. Multicolored visualization was achieved by the genome editing of LIN28 or FOXO1 with mCherry into OCT4-GFP iRSCs. Fluorescent analysis of gene activity in individual cells showed that OCT4 was dispensable for maintenance, but required for activation, of the LIN28 and FOXO1 expression in reprogramming.


Assuntos
Técnicas de Reprogramação Celular/métodos , Reprogramação Celular/genética , Células-Tronco Pluripotentes Induzidas/citologia , Animais , Caderinas/metabolismo , Diferenciação Celular/genética , Células Cultivadas , Transição Epitelial-Mesenquimal , Proteínas Ligadas por GPI/metabolismo , Humanos , Células-Tronco Pluripotentes Induzidas/metabolismo , Peptídeos e Proteínas de Sinalização Intercelular/metabolismo , Camundongos , Proteínas de Neoplasias/metabolismo , Fator 3 de Transcrição de Octâmero/genética , Fator 3 de Transcrição de Octâmero/metabolismo , Fatores de Transcrição SOXB1/genética , Análise de Célula Única/métodos , Ativação Transcricional
16.
Mol Cell ; 75(5): 891-904.e7, 2019 09 05.
Artigo em Inglês | MEDLINE | ID: mdl-31375262

RESUMO

Mammalian SWI/SNF complexes are multi-subunit chromatin remodeling complexes associated with an ATPase (either SMARCA4 or SMARCA2). Heterozygous mutations in the SMARCA2 ATPase cause Nicolaides-Baraitser syndrome (NCBRS), an intellectual disability syndrome associated with delayed speech onset. We engineered human embryonic stem cells (hESCs) to carry NCBRS-associated heterozygous SMARCA2 K755R or R1159Q mutations. While SMARCA2 mutant hESCs were phenotypically normal, differentiation to neural progenitors cells (NPCs) was severely impaired. We find that SMARCA2 mutations cause enhancer reorganization with loss of SOX3-dependent neural enhancers and prominent emergence of astrocyte-specific de novo enhancers. Changes in chromatin accessibility at enhancers were associated with an increase in SMARCA2 binding and retargeting of SMARCA4. We show that the AP-1 family member FRA2 is aberrantly overexpressed in SMARCA2 mutant NPCs, where it functions as a pioneer factor at de novo enhancers. Together, our results demonstrate that SMARCA2 mutations cause impaired differentiation through enhancer reprogramming via inappropriate targeting of SMARCA4.


Assuntos
DNA Helicases/metabolismo , Elementos Facilitadores Genéticos , Heterozigoto , Células-Tronco Embrionárias Humanas/metabolismo , Mutação de Sentido Incorreto , Proteínas Nucleares/metabolismo , Fatores de Transcrição/metabolismo , Substituição de Aminoácidos , Diferenciação Celular/genética , Cromatina/genética , Cromatina/metabolismo , DNA Helicases/genética , Facies , Deformidades Congênitas do Pé/genética , Deformidades Congênitas do Pé/metabolismo , Deformidades Congênitas do Pé/patologia , Antígeno 2 Relacionado a Fos/biossíntese , Antígeno 2 Relacionado a Fos/genética , Células HEK293 , Células-Tronco Embrionárias Humanas/patologia , Humanos , Hipotricose/genética , Hipotricose/metabolismo , Hipotricose/patologia , Deficiência Intelectual/genética , Deficiência Intelectual/metabolismo , Deficiência Intelectual/patologia , Células-Tronco Neurais/metabolismo , Células-Tronco Neurais/patologia , Proteínas Nucleares/genética , Fatores de Transcrição SOXB1/genética , Fatores de Transcrição SOXB1/metabolismo , Fatores de Transcrição/genética
17.
DNA Cell Biol ; 38(10): 1100-1111, 2019 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-31418589

RESUMO

Members of the Sox gene family play crucial roles during reproduction and development, but their genome-wide identification has not yet been performed in large yellow croaker, Larimichthys crocea. In this study, a total of 26 members of the Sox gene family were identified from the genome of large yellow croaker and classified into seven subgroups based on the conserved HMG-box domain they contain. Among the identified Sox gene family members, eight belonged to the SoxB subgroup (five in B1 and three in B2), four belonged to the SoxC subgroup, four belonged to the SoxD subgroup, six belonged to the SoxE subgroup, three belonged to the SoxF subgroup, and one belonged to the SoxK subgroup. During evolution, members of the SoxE subgroup (Sox8, Sox9, Sox10), Sox1, Sox4, Sox6, and Sox11 evolved into two copies, which may be a result of teleost-specific whole-genome duplication. Sox genes were distributed unevenly across 15 chromosomes. The number of introns in large yellow croaker Sox genes varied from 0 to 14. Results of the expression profile during embryogenesis revealed that most of the members of the Sox gene family had lower expression, except several Sox genes, and expression patterns also differed among each Sox gene group and duplicated gene. This study systematically characterized and analyzed the Sox gene family in large yellow croaker and provided new insights into its function during embryogenesis.


Assuntos
Proteínas de Peixes/genética , Regulação da Expressão Gênica no Desenvolvimento , Genoma , Perciformes/genética , Filogenia , Fatores de Transcrição SOXB1/genética , Sequência de Aminoácidos , Animais , Evolução Biológica , Mapeamento Cromossômico , Biologia Computacional , Embrião não Mamífero , Desenvolvimento Embrionário , Éxons , Proteínas de Peixes/classificação , Duplicação Gênica , Íntrons , Família Multigênica , Perciformes/classificação , Isoformas de Proteínas/classificação , Isoformas de Proteínas/genética , Fatores de Transcrição SOXB1/classificação , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos
18.
PLoS One ; 14(7): e0215494, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31365524

RESUMO

Roles for SOX2 have been extensively studied in several types of cancer, including colorectal cancer, glioblastoma and breast cancer, with particular emphasis placed on the roles of SOX2 in cancer stem cell. Our previous study identified SOX2 as a marker in cervical cancer stem cells driven by a full promoter element of SOX2 EGFP reporter. Here, dual-luciferase reporter and mutagenesis analyses were employed, identifying key cis-elements in the SOX2 promoter, including binding sites for SOX2, OCT4 and NF-YA factors in SOX2 promoter. Mutagenesis analysis provided additional evidence to show that one high affinity-binding domain CCAAT box was precisely recognized and bound by the transcription factor NF-YA. Furthermore, overexpression of NF-YA in primitive cervical cancer cells SiHa and C33A significantly activated the transcription and the protein expression of SOX2. Collectively, our data identified NF-YA box CCAAT as a key cis-element in the SOX2 promoter, suggesting that NF-YA is a potent cellular regulator in the maintenance of SOX2-positive cervical cancer stem cell by specific transcriptional activation of SOX2.


Assuntos
Fator de Ligação a CCAAT/metabolismo , Células-Tronco Neoplásicas/metabolismo , Fatores de Transcrição SOXB1/genética , Ativação Transcricional , Neoplasias do Colo do Útero/genética , Linhagem Celular Tumoral , Feminino , Regulação Neoplásica da Expressão Gênica , Humanos , Regiões Promotoras Genéticas
19.
Eur J Pharmacol ; 861: 172599, 2019 Oct 15.
Artigo em Inglês | MEDLINE | ID: mdl-31404537

RESUMO

The promoting roles of transcriptional factor six1 have been shown in various tumors, such as breast cancer and colorectal Cancer. However, its roles in hepatocellular carcinoma (HCC) cell stemness and chemotherapeutic sensitivity are never been revealed. In the present study, we showed that six1 expression was negatively correlated the overall survival of HCC patients and significantly increased in HCC tissues. Analysis on normal hepatic cells and HCC cells obtained the consistent result. Functional experiments revealed that six1 knockdown enhanced 5-fluorouracil (5-FU) sensitivity and reduced the stemness of HCC cells. Additionally, six1 knockdown partially reversed 5-FU resistance and attenuated the stemness in 5-FU-resistant HCC cells. Furthermore, we demonstrated that six1 directly bound to sox2 (a stemness master regulator) promoter, enhanced its transcription and expression. Overexpression of sox2 rescued the inhibitory effects of six1 knockdown on the stemness and 5-FU sensitivity of HCC cells. Thus, our work identified a novel six1/sox2 axis in regulating the stemness of HCC cells.


Assuntos
Carcinoma Hepatocelular/patologia , Fluoruracila/farmacologia , Proteínas de Homeodomínio/metabolismo , Neoplasias Hepáticas/patologia , Células-Tronco Neoplásicas/efeitos dos fármacos , Carcinoma Hepatocelular/diagnóstico , Resistencia a Medicamentos Antineoplásicos/genética , Feminino , Técnicas de Silenciamento de Genes , Proteínas de Homeodomínio/genética , Humanos , Neoplasias Hepáticas/diagnóstico , Masculino , Pessoa de Meia-Idade , Prognóstico , Fatores de Transcrição SOXB1/genética , Análise de Sobrevida
20.
Mol Cell ; 75(5): 905-920.e6, 2019 09 05.
Artigo em Inglês | MEDLINE | ID: mdl-31422875

RESUMO

Variable levels of DNA methylation have been reported at tissue-specific differential methylation regions (DMRs) overlapping enhancers, including super-enhancers (SEs) associated with key cell identity genes, but the mechanisms responsible for this intriguing behavior are not well understood. We used allele-specific reporters at the endogenous Sox2 and Mir290 SEs in embryonic stem cells and found that the allelic DNA methylation state is dynamically switching, resulting in cell-to-cell heterogeneity. Dynamic DNA methylation is driven by the balance between DNA methyltransferases and transcription factor binding on one side and co-regulated with the Mediator complex recruitment and H3K27ac level changes at regulatory elements on the other side. DNA methylation at the Sox2 and the Mir290 SEs is independently regulated and has distinct consequences on the cellular differentiation state. Dynamic allele-specific DNA methylation at the two SEs was also seen at different stages in preimplantation embryos, revealing that methylation heterogeneity occurs in vivo.


Assuntos
Diferenciação Celular/fisiologia , Metilação de DNA/fisiologia , Elementos Facilitadores Genéticos/fisiologia , Células-Tronco Embrionárias Murinas/metabolismo , Transcrição Genética/fisiologia , Animais , Linhagem Celular , Histona Desmetilases com o Domínio Jumonji/genética , Histona Desmetilases com o Domínio Jumonji/metabolismo , Camundongos , MicroRNAs/genética , MicroRNAs/metabolismo , Células-Tronco Embrionárias Murinas/citologia , Fatores de Transcrição SOXB1/genética , Fatores de Transcrição SOXB1/metabolismo
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