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1.
Biomed Environ Sci ; 32(4): 272-280, 2019 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-31217063

RESUMO

OBJECTIVE: To explore the protective effect of NANOG against hydrogen peroxide (H2O2) -induced cell damage in the human hair follicle mesenchymal stem cells (hHF-MSCs). METHODS: NANOG was expressed from a lentiviral vector, pLVX-IRES-ZsGreen. NANOG hHF-MSCs and vector hHF-MSCs were treated with 400 µmol/L hydrogen peroxide (H2O2) for 2 h, the cell survival rate, cell morphology, ROS production, apoptosis and expression of AKT, ERK, and p21 were determined and compared. RESULTS: Our results showed that NANOG could activate AKT and upregulate the expression of p-AKT, but not p-ERK. When treated with 400 µmol/L H2O2, NANOG hHF-MSCs showed higher cell survival rate, lower ROS production and apoptosis, higher expression of p-AKT, higher ratio of p-AKT/AKT. CONCLUSION: Our results suggest that NANOG could protect hHF-MSCs against cell damage caused by H2O2 through activating AKT signaling pathway.


Assuntos
Folículo Piloso/citologia , Células-Tronco Mesenquimais/metabolismo , Proteína Homeobox Nanog/metabolismo , Sobrevivência Celular , Avaliação Pré-Clínica de Medicamentos , Humanos , Peróxido de Hidrogênio , Lentivirus , Células-Tronco Mesenquimais/efeitos dos fármacos , Proteína Homeobox Nanog/farmacologia , Estresse Oxidativo/efeitos dos fármacos , Fosfatidilinositol 3-Quinases/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Transdução de Sinais
2.
Int J Mol Sci ; 20(5)2019 Mar 06.
Artigo em Inglês | MEDLINE | ID: mdl-30845764

RESUMO

Recently, cancer stem cells (CSCs) have been identified as the major cause of both chemotherapy and radiotherapy resistance. Evidence from experimental studies applying both in vitro and in vivo preclinical models suggests that CSCs survive after conventional therapy protocols. Several mechanisms are proposed to be involved in CSC resistance to radiotherapy. Among them, stimulated DNA double-strand break (DSB) repair capacity in association with aldehyde dehydrogenase (ALDH) activity seems to be the most prominent mechanism. However, thus far, the pathway through which ALDH activity stimulates DSB repair is not known. Therefore, in the present study, we investigated the underlying signaling pathway by which ALDH activity stimulates DSB repair and can lead to radioresistance of breast cancer cell lines in vitro. When compared with ALDH-negative cells, ALDH-positive cells presented significantly enhanced cell survival after radiation exposure. This enhanced cell survival was associated with stimulated Nanog, BMI1 and Notch1 protein expression, as well as stimulated Akt activity. By applying overexpression and knockdown approaches, we clearly demonstrated that Nanog expression is associated with enhanced ALDH activity and cellular radioresistance, as well as stimulated DSB repair. Akt and Notch1 targeting abrogated the Nanog-mediated radioresistance and stimulated ALDH activity. Overall, we demonstrate that Nanog signaling induces tumor cell radioresistance and stimulates ALDH activity, most likely through activation of the Notch1 and Akt pathways.


Assuntos
Aldeído Desidrogenase/metabolismo , Neoplasias da Mama/metabolismo , Tolerância a Radiação , Transdução de Sinais , Neoplasias da Mama/genética , Linhagem Celular Tumoral , Quebras de DNA de Cadeia Dupla , Reparo do DNA , Feminino , Humanos , Células MCF-7 , Proteína Homeobox Nanog/metabolismo , Células-Tronco Neoplásicas/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Receptor Notch1/metabolismo , Transdução de Sinais/efeitos da radiação
3.
Eur J Pharmacol ; 852: 51-57, 2019 Jun 05.
Artigo em Inglês | MEDLINE | ID: mdl-30831081

RESUMO

Cancer incidence, metastasis, drug resistance and recurrence are still the critical issues of oncological diseases especially Ovarian cancer (OC). It has been suggested that drug resistance and disease relapse are the main causes for the aggressive nature of OC. There is an immediate need to develop novel strategies to understand the mechanism to overcome chemoresistance. Nanog has been found to regulate stemness like cells inside the cancer cells that are termed as Cancer Stem Cells (CSCs). These cells show high self-renewal capacity with a peculiar potential in tumour initiation, heterogeneity, progression, metastasis, recurrence, radiotherapy and multi drug resistance. Recent studies have demonstrated that Nanog, a key transcription factor for pluripotency, has been playing a major role in chemoresistance. In this review, we address the functions of Nanog in both normal and cancer cells, how Nanog is involved in OC tumorigenesis and chemoresistance. This review also highlights the methods that are used for targeting Nanog as a remedy for treating OC. Thus, through this review, we predict that these concepts will open new avenues of research in ovarian cancer stem cells, and would propose Nanog as a target to improve the outcome of chemotherapy.


Assuntos
Terapia de Alvo Molecular/métodos , Proteína Homeobox Nanog/metabolismo , Neoplasias Ovarianas/tratamento farmacológico , Animais , Carcinogênese/efeitos dos fármacos , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Feminino , Humanos , Neoplasias Ovarianas/metabolismo , Neoplasias Ovarianas/patologia
4.
Gynecol Oncol ; 153(3): 651-660, 2019 06.
Artigo em Inglês | MEDLINE | ID: mdl-30904337

RESUMO

OBJECTIVE: The aim of this study was to analyze the expression, biological role and clinical relevance of cancer stem cell markers in high-grade serous carcinoma (HGSC). METHODS: mRNA expression by qRT-PCR of NANOG, OCT4, SOX2, SOX4, SOX9, LIN28A and LIN28B was analyzed in 134 HGSC specimens (84 effusions, 50 surgical specimens). Nanog, OCT3/4, SOX2 and SOX9 protein expression by immunohistochemistry was analyzed in 52 HGSC effusions. Nanog protein expression in exosomes from 80 HGSC effusions was studied by Western Blotting. OVCAR3 cells underwent CRISPR/Cas9 Nanog knockout (KO), and the effect of Nanog KO on migration, invasion, proliferation and proteolytic activity was analyzed in OVCAR3 and OVCAR8 cells. RESULTS: OCT4 mRNA was overexpressed in effusions compared to solid specimens (p = 0.046), whereas SOX9 was overexpressed in the ovarian tumors compared to effusions and solid metastases (p = 0.003). Higher SOX2 and SOX9 expression was associated with primary (intrinsic) chemoresistance (p = 0.009 and p = 0.02, respectively). Higher SOX9 levels were associated with shorter overall survival in univariate (p = 0.04) and multivariate (p = 0.049) analysis. OCT3/4, SOX2 and SOX9 proteins were found in HGSC cells, whereas Nanog was detected only in exosomes. Higher SOX2 protein expression was associated with shorter overall survival in univariate analysis (p = 0.049). OVCAR cells exposed to OVCAR3 NANOG KO exosomes had reduced migration, invasion and MMP9 activity. CONCLUSIONS: SOX2 and SOX9 mRNA levels in HGSC effusions may be markers of clinically aggressive disease. Nanog is secreted in HGSC exosomes in effusions and modulates tumor-promoting cellular processes in vitro.


Assuntos
Neoplasias Císticas, Mucinosas e Serosas/metabolismo , Neoplasias Ovarianas/metabolismo , Fatores de Transcrição SOX9/genética , Fatores de Transcrição SOX9/metabolismo , Fatores de Transcrição SOXB1/genética , Fatores de Transcrição SOXB1/metabolismo , Adulto , Idoso , Idoso de 80 Anos ou mais , Biomarcadores Tumorais , Linhagem Celular Tumoral , Movimento Celular/genética , Proliferação de Células/genética , Resistencia a Medicamentos Antineoplásicos , Exossomos , Feminino , Expressão Gênica , Técnicas de Inativação de Genes , Humanos , Pessoa de Meia-Idade , Proteína Homeobox Nanog/genética , Proteína Homeobox Nanog/metabolismo , Gradação de Tumores , Metástase Neoplásica , Neoplasias Císticas, Mucinosas e Serosas/genética , Neoplasias Císticas, Mucinosas e Serosas/secundário , Fator 3 de Transcrição de Octâmero/genética , Fator 3 de Transcrição de Octâmero/metabolismo , Neoplasias Ovarianas/genética , Neoplasias Ovarianas/patologia , Proteólise , RNA Mensageiro/metabolismo , Taxa de Sobrevida
5.
Nat Commun ; 10(1): 1109, 2019 03 07.
Artigo em Inglês | MEDLINE | ID: mdl-30846691

RESUMO

Transcription factor networks, together with histone modifications and signalling pathways, underlie the establishment and maintenance of gene regulatory architectures associated with the molecular identity of each cell type. However, how master transcription factors individually impact the epigenomic landscape and orchestrate the behaviour of regulatory networks under different environmental constraints is only partially understood. Here, we show that the transcription factor Nanog deploys multiple distinct mechanisms to enhance embryonic stem cell self-renewal. In the presence of LIF, which fosters self-renewal, Nanog rewires the pluripotency network by promoting chromatin accessibility and binding of other pluripotency factors to thousands of enhancers. In the absence of LIF, Nanog blocks differentiation by sustaining H3K27me3, a repressive histone mark, at developmental regulators. Among those, we show that the repression of Otx2 plays a preponderant role. Our results underscore the versatility of master transcription factors, such as Nanog, to globally influence gene regulation during developmental processes.


Assuntos
Autorrenovação Celular/fisiologia , Células-Tronco Embrionárias Murinas/citologia , Células-Tronco Embrionárias Murinas/metabolismo , Proteína Homeobox Nanog/metabolismo , Animais , Linhagem Celular , Autorrenovação Celular/genética , Elementos Facilitadores Genéticos , Regulação da Expressão Gênica , Redes Reguladoras de Genes , Código das Histonas/genética , Fator Inibidor de Leucemia/genética , Fator Inibidor de Leucemia/metabolismo , Camundongos , Proteína Homeobox Nanog/genética , Fatores de Transcrição Otx/genética , Fatores de Transcrição Otx/metabolismo
6.
Oxid Med Cell Longev ; 2019: 9078209, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30906504

RESUMO

Several researches revealed that propofol, a hypnotic intravenous anesthesia agent, could inhibit the cancer cell proliferation and tumor formation, which might affect cancer recurrence or metastasis and impact patients' prognosis. Cancer stem cells (CSCs) comprised a tiny fraction of tumor bulk and played a vital role in cancer recurrence and eventual mortality. This study investigates the effect of propofol on breast cancer stem cells (BCSCs) in vitro and the underlying molecular mechanisms. Tumor formation of CSCs was measured by mammosphere culture. Cultured BCSCs were exposed to different concentrations and durations of propofol. Cell proliferation and self-renewal capacity were determined by MTT assays. Expressions of PD-L1 and Nanog were measured using western blotting and real-time PCR. We knocked down the PD-L1 expression in MDA-MB-231 cells by lentivirus-mediated RNAi technique, and the mammosphere-forming ability of shControl and shPD-L1 under propofol treatment was examined. Mammosphere culture could enrich BCSCs. Compared with control, cells exposed to propofol for 24 h induced a larger number of mammosphere cells (P = 0.0072). Levels of PD-L1 and Nanog were downregulated by propofol. Compared with shControl stem cells, there was no significant difference in the inhibitory effect of propofol on the mammosphere-forming ability of shPD-L1 stem cells which indicated that the inhibition of propofol could disappear in PD-L1 knockdown breast stem cells. Propofol could reduce the mammosphere-forming ability of BCSCs in vitro. Mechanism experiments indicated that the inhibition of propofol in mammosphere formation of BCSCs might be mediated through PD-L1, which was important to maintain Nanog.


Assuntos
Antígeno B7-H1/metabolismo , Neoplasias da Mama/patologia , Proteína Homeobox Nanog/metabolismo , Células-Tronco Neoplásicas/metabolismo , Propofol/farmacologia , Esferoides Celulares/metabolismo , Esferoides Celulares/patologia , Regulação para Baixo/efeitos dos fármacos , Feminino , Humanos , Células MCF-7 , Células-Tronco Neoplásicas/efeitos dos fármacos , RNA Interferente Pequeno/metabolismo
7.
Anticancer Res ; 39(3): 1205-1216, 2019 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-30842151

RESUMO

BACKGROUND/AIM: We recently investigated the contribution of the iPS-related genes SOX2, OCT4, and Nanog to de-differentiation by assaying for their mRNA levels. Given that mRNA expression does not always correlate with the protein levels, the aim of this study was to retrospectively determine the expression of these four iPS-related factors in human OSCC specimens by immunohistochemistry and examine their association with patient prognosis. MATERIALS AND METHODS: iPS cell-related gene expression in 89 OSCC patients by tissue microarray, and its correlation with clinicopathological factors, differentiation, metastasis, and poor prognoses were investigated. RESULTS: No evidence of statistically significant relationships was found between the expression of iPS cell-related genes and clinicopathological parameters. However, our data indicated that KLF4 expression was associated with survival, and poor tumor differentiation. In addition, high expression of KLF4 was an independent poor prognostic factor (p=0.004) for OSCC patients. CONCLUSION: In preoperative biopsies, higher KLF4 and poor differentiation may be clinically effective predictors for the prognosis of oral cancer.


Assuntos
Carcinoma de Células Escamosas/genética , Células-Tronco Pluripotentes Induzidas , Fatores de Transcrição Kruppel-Like/genética , Neoplasias Bucais/genética , Adulto , Idoso , Idoso de 80 Anos ou mais , Carcinoma de Células Escamosas/metabolismo , Feminino , Humanos , Estimativa de Kaplan-Meier , Fatores de Transcrição Kruppel-Like/metabolismo , Masculino , Pessoa de Meia-Idade , Neoplasias Bucais/metabolismo , Proteína Homeobox Nanog/genética , Proteína Homeobox Nanog/metabolismo , Fator 3 de Transcrição de Octâmero/genética , Fator 3 de Transcrição de Octâmero/metabolismo , Prognóstico , RNA Mensageiro/metabolismo , Fatores de Transcrição SOXB1/genética , Fatores de Transcrição SOXB1/metabolismo , Adulto Jovem
8.
J Exp Clin Cancer Res ; 38(1): 93, 2019 Feb 21.
Artigo em Inglês | MEDLINE | ID: mdl-30791957

RESUMO

BACKGROUND: Zoledronic acid is the most potent osteoclast inhibitor and is widely used for advanced cancer patients with bone metastasis, but its role on cancer stem cells (CSCs) remains unclear. In the present study, we aimed to identify the stemness phenotypic characteristics of CSCs derived from cervical cancer cells and explore the anti-cancer efficiency of zoledronic acid on these cells, as well as the possible molecular mechanisms. METHODS: Stemness phenotypic identification of cervical cancer cells derived CSCs was performed via sphere formation efficiency (SFE), tumorigenesis, immunofluorescence staining, Transwell assay, and western blot. Anti-cancer efficiency of zoledronic acid on these cells (including proliferation, stemness phenotype, apoptosis, and cell cycle) was carried out through MTT assay, SFE, transwell, DAPI staining, flow cytometry, immunofluorescence, TUNEL staining, and western blot, both in vitro and in vivo. RESULTS: Enhanced self-renewal ability, including SFE and tumorigenesis, was verified in cervical cancer cells derived CSCs compared to parental cervical cancer cells. Specifically, the expression of ALDH1, Sox2, CD49f, Nanog, and Oct4 was significantly up-regulated in cervical cancer cells derived CSCs. Furthermore, enhanced migratory ability was observed in these cells along with up-regulated N-cadherin and Vimentin and down-regulated E-cadherin. Zoledronic acid inhibited cervical cancer cells derived CSCs proliferation in vitro and in vivo. The stemness phenotype of these CSCs including tumor sphere formation, migration, as well as the expression of the aforementioned associated markers was also suppressed. In addition, zoledronic acid significantly induced apoptosis and cell cycle arrest of cervical cancer cells derived CSCs in a dose-dependent manner. Mechanistically, the expression of phosphorylated Erk1/2 and Akt was significantly increased in cervical cancer cells derived CSCs compared to parental cervical cancer cells. Zoledronic acid inhibited phosphorylated Erk1/2 and Akt in cervical cancer cells derived CSCs. IGF-1, a potent stimulator for Erk1/2 and PI3K/Akt, attenuated the aforementioned anti-cancer effect of zoledronic acid. CONCLUSIONS: Zoledronic acid inhibited the growth of cervical cancer cells derived CSCs through attenuating their stemness phenotype, inducing apoptosis, and arresting cell cycle. The suppression of phosphorylated Erk1/2 and Akt was involved in this process.


Assuntos
Apoptose/efeitos dos fármacos , Pontos de Checagem do Ciclo Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Células-Tronco Neoplásicas/efeitos dos fármacos , Transdução de Sinais/efeitos dos fármacos , Neoplasias do Colo do Útero/tratamento farmacológico , Ácido Zoledrônico/farmacologia , Antineoplásicos/farmacologia , Caderinas/metabolismo , Carcinogênese/efeitos dos fármacos , Carcinogênese/metabolismo , Linhagem Celular Tumoral , Movimento Celular/efeitos dos fármacos , Regulação para Baixo/efeitos dos fármacos , Feminino , Células HeLa , Humanos , Integrina alfa6/metabolismo , Isoenzimas/metabolismo , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Proteína Homeobox Nanog/metabolismo , Células-Tronco Neoplásicas/metabolismo , Fator 3 de Transcrição de Octâmero/metabolismo , Fenótipo , Fosfatidilinositol 3-Quinases/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Retinal Desidrogenase/metabolismo , Fatores de Transcrição SOXB1/metabolismo , Regulação para Cima/efeitos dos fármacos , Neoplasias do Colo do Útero/metabolismo
9.
Genome Res ; 29(3): 383-395, 2019 03.
Artigo em Inglês | MEDLINE | ID: mdl-30674556

RESUMO

The zebrafish embryo is transcriptionally mostly quiescent during the first 10 cell cycles, until the main wave of zygotic genome activation (ZGA) occurs, accompanied by fast chromatin remodeling. At ZGA, homologs of the mammalian stem cell transcription factors (TFs) Pou5f3, Nanog, and Sox19b bind to thousands of developmental enhancers to initiate transcription. So far, how these TFs influence chromatin dynamics at ZGA has remained unresolved. To address this question, we analyzed nucleosome positions in wild-type and maternal-zygotic (MZ) mutants for pou5f3 and nanog by MNase-seq. We show that Nanog, Sox19b, and Pou5f3 bind to the high nucleosome affinity regions (HNARs). HNARs are spanning over 600 bp, featuring high in vivo and predicted in vitro nucleosome occupancy and high predicted propeller twist DNA shape value. We suggest a two-step nucleosome destabilization-depletion model, in which the same intrinsic DNA properties of HNAR promote both high nucleosome occupancy and differential binding of TFs. In the first step, already before ZGA, Pou5f3 and Nanog destabilize nucleosomes at HNAR centers genome-wide. In the second step, post-ZGA, Nanog, Pou5f3, and SoxB1 maintain open chromatin state on the subset of HNARs, acting synergistically. Nanog binds to the HNAR center, whereas the Pou5f3 stabilizes the flanks. The HNAR model will provide a useful tool for genome regulatory studies in a variety of biological systems.


Assuntos
Montagem e Desmontagem da Cromatina , Proteína Homeobox Nanog/metabolismo , Nucleossomos/genética , Fator 3 de Transcrição de Octâmero/metabolismo , Fatores de Transcrição SOX/metabolismo , Proteínas de Peixe-Zebra/metabolismo , Zigoto/metabolismo , Animais , Regulação da Expressão Gênica no Desenvolvimento , Proteína Homeobox Nanog/genética , Nucleossomos/metabolismo , Fator 3 de Transcrição de Octâmero/genética , Ligação Proteica , Fatores de Transcrição SOX/genética , Peixe-Zebra , Proteínas de Peixe-Zebra/genética
10.
Dev Cell ; 48(3): 345-360.e7, 2019 02 11.
Artigo em Inglês | MEDLINE | ID: mdl-30595535

RESUMO

NANOG is an essential transcriptional factor for the maintenance of embryonic stem cells (ESCs) and cancer stem cells (CSCs) in prostate cancer (PCa). However, the regulation mechanism of NANOG protein stability in cancer progression is still elusive. Here, we report that NANOG is degraded by SPOP, a frequently mutated tumor suppressor of PCa. Cancer-associated mutations of SPOP or the mutation of NANOG at S68Y abrogates the SPOP-mediated NANOG degradation, leading to elevated PCa cancer stemness and poor prognosis. In addition, SPOP-mediated NANOG degradation is controlled by the AMPK-BRAF signal axis through the phosphorylation of NANOG at Ser68, which blocked the interaction between SPOP and NANOG. Thus, our study provides a regulation mechanism of PCa stemness controlled by phosphorylation-mediated NANOG stability, which helps to identify novel drug targets and improve therapeutic strategy for PCa.


Assuntos
Proteínas Quinases Ativadas por AMP/metabolismo , Proteína Homeobox Nanog/metabolismo , Proteínas Nucleares/metabolismo , Neoplasias da Próstata/metabolismo , Proteínas Repressoras/metabolismo , Animais , Linhagem Celular Tumoral , Proteínas Culina/metabolismo , Genes Supressores de Tumor , Humanos , Masculino , Camundongos Nus , Mutação/genética , Neoplasias da Próstata/genética , Fatores de Transcrição/metabolismo , Ubiquitinação/fisiologia
11.
Int J Biochem Cell Biol ; 108: 21-28, 2019 03.
Artigo em Inglês | MEDLINE | ID: mdl-30633985

RESUMO

The cancer cell population is heterogeneous, and cancer stem cells (CSCs) are important for tumor growth and maintenance. The CSC population is associated with different neoplastic characteristics, such as cell migration, resistance to apoptosis, radiation therapy and chemotherapy. To increase the knowledge of CSCs in canine prostate cancer (PC), we characterized CSC markers in canine PC tissues and tumorspheres. We performed immunohistochemistry of OCT3/4, Nestin, NANOG, CD44 and CD24 in 10 normal canine prostatic tissue samples, 10 prostatic hyperplastic (PH) tissue samples and 28 PC tissue samples. Then, we established two canine prostate cancer cell cultures and characterized the CSC profile of tumorspheres grown from these cultures. Normal and PH tissues were positive for Nestin, NANOG, CD44 and CD24 only in the basal cell layer. OCT3/4 was expressed in the luminal cells of normal and PH tissues. There was no significant difference in Nestin expression among the prostatic tissues. However, we found higher expression of NANOG and CD44 in canine PC tissues than that in normal and PH tissues. Tumorspheres from canine prostate cancer cells express OCT3/4, Nestin, NANOG and CD44, indicating that these markers may be potential cancer stem cell markers in canine PC. The results obtained can be useful to better characterize the stem cell population in canine prostatic cancer and to guide future studies in comparative oncology.


Assuntos
Antígeno CD24/metabolismo , Receptores de Hialuronatos/metabolismo , Proteína Homeobox Nanog/metabolismo , Células-Tronco Neoplásicas/metabolismo , Nestina/metabolismo , Fator 3 de Transcrição de Octâmero/metabolismo , Neoplasias da Próstata/metabolismo , Animais , Biomarcadores Tumorais/metabolismo , Linhagem Celular Tumoral , Autorrenovação Celular , Cães , Regulação Neoplásica da Expressão Gênica , Masculino , Fenótipo , Neoplasias da Próstata/patologia
12.
ACS Appl Mater Interfaces ; 11(4): 3679-3689, 2019 Jan 30.
Artigo em Inglês | MEDLINE | ID: mdl-30614683

RESUMO

The development of human-induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) provides significant advances to cell therapy, disease modeling, and drug screening applications. However, the current differentiation protocol is inefficient in mimicking biophysical and biochemical characteristics of cardiac niche. Hence, immature cardiomyocytes are often generated. In this study, hiPSC-CMs were generated on a new family of substrates called monolayer binary colloidal crystals (BCCs). Four BCCs were fabricated with different sizes (2 or 5 or 0.4 or 0.2 µm) and materials [Si or polystyrene (PS) or poly(methyl methacrylate)] abbreviated as 2PS, 5PS, 2PM, and 5PM. BCCs have complex surface micro-/nanotopographies and heterogeneous chemistries which are important modulators in microenvironments in vitro. The results showed that hiPSCs formed adhered spheroids with strong pluripotent markers ( Oct4, Nanog, and Sox2) on PM surfaces compared to PS and flat surfaces. After 30-day differentiation, hiPSC-CMs on PM surfaces showed markedly improved myofibril ultrastructures, Ca2+ handling, and electrophysiological properties, indicating that more mature hiPSC-CMs were generated. hiPSC-CMs generated on 5PM are more similar to adult heart tissue compared to other surfaces in terms of genes ( ACTC1, TNNT2, RYR2, SERCA2a, SCN5a, KCNJ2, CACNA1c, ITGB1, GJA1, MYH6, and MYH7) and protein (ssTnI and cTnI) expressions. We further demonstrated that 5PM surfaces facilitated cadherin switching (from E- to N-) during cardiac differentiation and mature N-cadherin expression, which were positively correlated with the cardiogensis markers ( GATA4, MEF2c, and NKX2.5). This study illuminated that a tailored surface nanotopography was beneficial in hiPSC culture and in situ cardiac differentiation. This one-step approach and BCCs can be a next-generation tool for hiPSC expansion and CM differentiation.


Assuntos
Células-Tronco Pluripotentes Induzidas/citologia , Células-Tronco Pluripotentes Induzidas/metabolismo , Miócitos Cardíacos/citologia , Miócitos Cardíacos/metabolismo , Esferoides Celulares/citologia , Esferoides Celulares/metabolismo , Diferenciação Celular/fisiologia , Células Cultivadas , Humanos , Proteína Homeobox Nanog/metabolismo , Fator 3 de Transcrição de Octâmero/metabolismo , Fatores de Transcrição SOXB1/metabolismo
13.
Eur J Obstet Gynecol Reprod Biol ; 234: 143-148, 2019 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-30690190

RESUMO

OBJECTIVE: To study the molecular features of mesenchymal stem cells from Wharton Jelly (WJ-MSCs) of umbilical cord to predict their differentiation capacity. DESIGN: Comparison of gene expression from mesenchymal stem cells of male and female umbilical cord SETTING: University hospital PATIENT (S): umbilical cords (n = 12, 6 males and 6 females) retrieved from spontaneous full-term vaginal delivery of healthy women INTERVENTION: we analyzed the expression of the stemness related genes C-MYC, OCT4, SOX2 and NANOG and of the epigenetic modulating gene DNA-methyltransferase 1 (DNMT1). MEAN OUTCOME MEASURE: WJ-MSCs were isolated by standard procedures and immunophenotypically characterized. Gene expression analysis of stemness related genes and the epigenetic modulating gene DNMT1 were performed by real-time PCR RESULTS: expression of the OCT4 and DNMT1 genes was significantly higher in WJ- MSCs isolated from male subjects, as compared to MSCs isolated from female-derived WJ. The resulting higher expression of OCT4 and DNMT1 in WJ-MSCs from males as compared with female WJ-MSCs for the first time identifies a specific relationship between stemness genes, an epigenetic modulator, and gender differences. CONCLUSION: our findings disclose novel biomedical implications in WJ-MSCs related to the sex of the donor, thus providing additional cues to exploit their regenerative potential in allogenic transplantation.


Assuntos
Células-Tronco Mesenquimais/metabolismo , Caracteres Sexuais , Cordão Umbilical/citologia , Geleia de Wharton/citologia , Diferenciação Celular , DNA (Citosina-5-)-Metiltransferase 1/metabolismo , Feminino , Expressão Gênica/fisiologia , Genes myc/fisiologia , Voluntários Saudáveis , Humanos , Masculino , Proteína Homeobox Nanog/metabolismo , Fator 3 de Transcrição de Octâmero/metabolismo , Gravidez , Fatores de Transcrição SOXB1/metabolismo , Vagina
14.
Dev Biol ; 446(1): 43-55, 2019 02 01.
Artigo em Inglês | MEDLINE | ID: mdl-30529251

RESUMO

Teratomas are tumors consisting of components of the three germ layers that differentiate from pluripotent stem cells derived from germ cells. In the normal mouse testis, teratomas rarely form, but a deficiency in Dead-end1 (Dnd1) in mice with a 129/Sv genetic background greatly enhances teratoma formation. Thus, DND1 is crucial for suppression of teratoma development from germ cells. In the Dnd1 mutant testis, nascent teratoma cells emerge at E15.5. To understand the nature of early teratoma cells, we established cell lines in the presence of serum and leukemia inhibitory factor (LIF) from teratoma-forming cells in neonatal Dnd1 mutant testis. These cells, which we designated cultured Dnd1 mutant germ cells (CDGCs), were morphologically similar to embryonic stem cells (ESCs) and could be maintained in the naïve pluripotent condition. In addition, the cells expressed pluripotency genes including Oct4, Nanog, and Sox2; differentiated into cells of the three germ layers in culture; and contributed to chimeric mice. The expression levels of pluripotency genes and global transcriptomes in CDGCs as well as these cells' adaption to culture conditions for primed pluripotency suggested that their pluripotent status is intermediate between naïve and primed pluripotency. In addition, the teratoma-forming cells in the neonatal testis from which CDGCs were derived also showed gene expression profiles intermediate between naïve and primed pluripotency. The results suggested that germ cells in embryonic testes of Dnd1 mutants acquire the intermediate pluripotent status during the course of conversion into teratoma cells.


Assuntos
Diferenciação Celular/genética , Células-Tronco Embrionárias Murinas/metabolismo , Proteínas de Neoplasias/genética , Células-Tronco Pluripotentes/metabolismo , Animais , Animais Recém-Nascidos , Células Cultivadas , Perfilação da Expressão Gênica , Regulação da Expressão Gênica no Desenvolvimento , Masculino , Camundongos , Camundongos da Linhagem 129 , Camundongos Knockout , Camundongos Transgênicos , Células-Tronco Embrionárias Murinas/citologia , Proteína Homeobox Nanog/genética , Proteína Homeobox Nanog/metabolismo , Proteínas de Neoplasias/deficiência , Fator 3 de Transcrição de Octâmero/genética , Fator 3 de Transcrição de Octâmero/metabolismo , Células-Tronco Pluripotentes/citologia , Fatores de Transcrição SOXB1/genética , Fatores de Transcrição SOXB1/metabolismo , Teratoma/genética , Teratoma/metabolismo , Teratoma/patologia , Testículo/citologia , Testículo/embriologia , Testículo/metabolismo
15.
Cell Physiol Biochem ; 48(5): 2205-2218, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-30110679

RESUMO

Background /Aims: Recent studies of microRNA (miRNA) involvement in tumorigenesis have indicated the critical role of these non-coding small RNAs in malignant transformation, but the prognostic role, if any, of miRNAs in breast cancer remains undetermined. Therefore, we assessed the prognostic significance of microRNA-9 (miR-9) and miR-221 in breast cancer toward the goal of understanding the contribution(s) of these miRNAs to cancer cell stemness. METHODS: The level of each of miR-9 and miR-221 in 206 paired laser capture microdissected tumor cells and non-tumor cells was determined by quantitative reverse transcription-PCR (qRT-PCR). The relationship between the miRNA signature and clinicopathological data and prognosis of breast cancer was assessed. Identification of a stem cell-enriched side population was achieved with fluorescence-activated cell sorting and a sphere-forming assay. Wound healing, Boyden chamber assays, and western blotting were used to study the contribution of each miRNA to tumor cell migration and invasion. RESULTS: We found that induction of miR-9 and miR-221 mimics conferred side-population cells to form spheroidal tumor colonies in suspension culture that maintained the mesenchymal stem-cell potential in non-invasive MCF-7 breast cancer cells. In contrast, knockdown of both miR-9 and miR-221 in invasive MDA-MB-231 breast cancer cells dramatically decreased the number of side-population colonies with stem cell-like potency, which reduced the capacity for tumor-cell renewal, invasion, and migration. Clinically, the mean proportion of miR-9- or miR-221-overexpressing cells was significantly greater in tumor cells compared with non-tumor cells (P < 0.05). Increased levels of miR-9 and miR-221 in breast tissue portended a significantly elevated risk of progression to malignancy with respect to larger tumor size, poor differentiation, late-stage evolution, lymph-node metastasis (P < 0.05), and lower overall survival (Ptrend = 0.017; eight-year follow-up). CONCLUSION: Our findings provide strong evidence that miR-9 and miR-221 can enhance the generation of cancer stem cells to yield an invasive phenotype and that overexpression of these miRNAs predicts a poor outcome for breast cancer patients.


Assuntos
Neoplasias da Mama/diagnóstico , MicroRNAs/metabolismo , Antígeno AC133/metabolismo , Adulto , Antagomirs/metabolismo , Área Sob a Curva , Neoplasias da Mama/genética , Neoplasias da Mama/mortalidade , Neoplasias da Mama/patologia , Linhagem Celular Tumoral , Movimento Celular , Feminino , Humanos , Metástase Linfática , Células MCF-7 , MicroRNAs/antagonistas & inibidores , MicroRNAs/genética , Pessoa de Meia-Idade , Proteína Homeobox Nanog/metabolismo , Células-Tronco Neoplásicas/citologia , Células-Tronco Neoplásicas/metabolismo , Prognóstico , Modelos de Riscos Proporcionais , Curva ROC , Taxa de Sobrevida
16.
Theriogenology ; 120: 33-39, 2018 Oct 15.
Artigo em Inglês | MEDLINE | ID: mdl-30092372

RESUMO

Nanog as a novel pluripotent cell-specific gene plays important roles in regulation of signaling pathways for maintenance and induction of pluripotency in inner cell mass (ICM) and embryonic stem cells (ESC) in mouse. The molecular features and transcription regulation of NANOG gene in domestic animals are not well defined. In this study, we performed knockdown of NANOG mRNA in goat embryos and examined its effect on early embryonic development. Presumptive zygotes were injected with a volume of 8-10 pl NANOG or scrambled (SCR) siRNA, and subsequently cleavage and blastocyst formation rate were assessed. Furthermore, gene expression analysis was carried out in 6-8 cell and blastocyst derived embryos from non-injected controls, SCR - and siRNA-injected presumptive zygotes. Cleavage and blastocyst rates in siRNA groups were insignificantly lower than the control and SCR groups. Embryos with reduced expression of NANOG showed decrease in number of trophectoderm (TE) and total cells in blastocysts. Analysis of expression of developmentally important genes (SOX2, OCT4 and NANOG), which work as a network, showed that NANOG knockdown results in significant increase in expression of SOX2 and OCT4 and among the possible target genes (CDX2, REX1 and GATA4) of this network, only GATA4 showed increased expression. Our results suggest that NANOG is likely to be required for proliferation of trophoblastic cells.


Assuntos
Blastocisto/metabolismo , Desenvolvimento Embrionário , Cabras/embriologia , Proteína Homeobox Nanog/fisiologia , Análise de Variância , Animais , Blastocisto/citologia , Proliferação de Células/genética , Regulação da Expressão Gênica no Desenvolvimento , Proteína Homeobox Nanog/genética , Proteína Homeobox Nanog/metabolismo , RNA Mensageiro/metabolismo , Transdução de Sinais/genética
17.
Vet Pathol ; 55(6): 849-852, 2018 11.
Artigo em Inglês | MEDLINE | ID: mdl-30025495

RESUMO

Mast cell tumors are one of the most frequent skin tumors in dogs. Treatment decisions often depend on a wide range of clinical information and the main criteria for prognostic formulation are histological grade, mitotic count, Ki67 index, and KIT immunostaining pattern. NANOG is a pluripotency factor expressed by normal and cancer stem cells, which is a prognostic marker and a potential therapeutic target for several human tumors. In the present study, mast cell tumor samples from 41 dogs were evaluated for NANOG and Ki67 by immunohistochemistry. All samples were positive for NANOG but its expression was not correlated with Ki67 index and no significant differences were found with respect to histopathological grades, disease-related mortality, or survival. Our results suggest that, although related to pluripotency, NANOG expression does not correlate with proliferative activity, and is not a reliable prognostic factor for canine cutaneous mast cell tumors.


Assuntos
Doenças do Cão/patologia , Mastocitoma/veterinária , Proteína Homeobox Nanog/metabolismo , Neoplasias Cutâneas/veterinária , Animais , Biomarcadores Tumorais/metabolismo , Proliferação de Células , Doenças do Cão/diagnóstico , Doenças do Cão/metabolismo , Cães , Antígeno Ki-67/metabolismo , Masculino , Mastocitoma/diagnóstico , Mastocitoma/metabolismo , Mastocitoma/patologia , Prognóstico , Neoplasias Cutâneas/diagnóstico , Neoplasias Cutâneas/metabolismo , Neoplasias Cutâneas/patologia
18.
Mol Med Rep ; 18(3): 2705-2714, 2018 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-30015965

RESUMO

One approach to cell differentiation is to use the natural capacity of pluripotent stem cells to form three germ layers via embryoid bodies (EB). However, unification of this process during in vitro culture remains challenging and many microenvironmental factors including the number of cells in the culture can influence differentiation patterns. The number of cells serves a crucial role as it determines access to nutrients, the distribution of oxygen concentration and cellular interactions, all of which influence the fate of the differentiated cells. The influence of EBs derived from human pluripotent cells on the chondrogenic potential of such cells is not well understood. For this reason, the present study sought to determine the effect of varying amounts of cells on the properties of EBs derived from human embryonic stem cells (BG01V cell line). In the present study, 500­2,000 cells per well were cultivated from 5 to 15 days in suspension cell culture. Expression of pluripotency genes and germ layer markers were evaluated in order to determine the EBs with the greatest and least mesodermal properties. Genes associated with pluripotency and chondrogenesis were also evaluated to assess the influence of suspension culture duration and EB size on chondrogenic differentiation. Immunofluorescence staining for pluripotent and chondrocyte­associated proteins confirmed successful differentiation into chondrocyte­like cells. Alcian blue staining confirmed deposition of proteoglycans. These results suggested that EBs formed in 500­cell wells possess the highest mesodermal and prochondrogenic properties. Differentiation of EBs into chondrocytes on day 5 in 500­cell wells was more efficient than in that observed in larger and older EBs.


Assuntos
Diferenciação Celular , Condrogênese , Corpos Embrioides/metabolismo , Linhagem Celular , Condrócitos/citologia , Condrócitos/metabolismo , Colágeno Tipo II/metabolismo , Técnica Indireta de Fluorescência para Anticorpo , Humanos , Células-Tronco Pluripotentes Induzidas/citologia , Células-Tronco Pluripotentes Induzidas/metabolismo , Proteína Homeobox Nanog/metabolismo , Fatores de Transcrição SOX9/metabolismo
19.
Acta Biochim Biophys Sin (Shanghai) ; 50(8): 793-799, 2018 Aug 01.
Artigo em Inglês | MEDLINE | ID: mdl-29945210

RESUMO

The ability to self-renew is one of the most important properties of embryonic stem (ES) cells. Pluripotin (SC1), a small molecule with high activity and low toxicity, promotes self-renewal in mouse ES cells. SC1 can noticeably change the morphology of retinoic acid (RA)-induced F9 embryonic carcinoma cells (F9 cells). However, in the long term, RA and SC1 together cause cell apoptosis. When being added after 18-24 h of RA-induced F9 cell differentiation, SC1 transitorily activated Nanog and Oct4. Both Nanog and Oct4 were downregulated when SC1 and RA were added simultaneously. On the other hand, Klf4 was continually activated when SC1 was added between 6 and 24 h. Phosphorylated Erk1/2 protein levels were reduced from 6 to 24 h, whereas unphosphorylated Erk1 protein levels remained unchanged. A higher concentration of SC1 promoted cell self-renewal by strengthening the inhibition of Erk1/2 protein phosphorylation in F9 cells. Furthermore, SC1 and RA affect global DNA methylation by influencing the expressions of methylation-associated proteins, including Dnmt3b, Dnmt3l, Tet1, Tet2, and Tet3. In conclusion, SC1 inhibits the differentiation of RA-induced F9 cells mainly by reducing the levels of phosphorylated Erk1/2 and enhancing the expression of Klf4, although it also reduces DNA methylation, which may have an additional effect on ES cell differentiation.


Assuntos
Apoptose/efeitos dos fármacos , Diferenciação Celular/efeitos dos fármacos , Pirazóis/farmacologia , Pirimidinas/farmacologia , Tretinoína/farmacologia , Animais , Antineoplásicos/farmacologia , Apoptose/genética , Carcinoma Embrionário/genética , Carcinoma Embrionário/metabolismo , Carcinoma Embrionário/patologia , Diferenciação Celular/genética , Linhagem Celular Tumoral , DNA (Citosina-5-)-Metiltransferases/genética , DNA (Citosina-5-)-Metiltransferases/metabolismo , Metilação de DNA/efeitos dos fármacos , Metilação de DNA/genética , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Camundongos , Proteína Homeobox Nanog/genética , Proteína Homeobox Nanog/metabolismo , Fator 3 de Transcrição de Octâmero/genética , Fator 3 de Transcrição de Octâmero/metabolismo , Proteínas Proto-Oncogênicas/genética , Proteínas Proto-Oncogênicas/metabolismo
20.
Development ; 145(12)2018 06 18.
Artigo em Inglês | MEDLINE | ID: mdl-29915126

RESUMO

Lineage segregation in the mouse embryo is a finely controlled process dependent upon coordination of signalling pathways and transcriptional responses. Here we employ a conditional deletion system to investigate embryonic patterning and lineage specification in response to loss of Oct4. We first observe ectopic expression of Nanog in Oct4-negative postimplantation epiblast cells. The expression domains of lineage markers are subsequently disrupted. Definitive endoderm expands at the expense of mesoderm; the anterior-posterior axis is positioned more distally and an ectopic posterior-like domain appears anteriorly, suggesting a role for Oct4 in maintaining the embryonic axis. Although primitive streak forms in the presumptive proximal-posterior region, epithelial-to-mesenchymal transition is impeded by an increase of E-cadherin, leading to complete tissue disorganisation and failure to generate germ layers. In explant and in vitro differentiation assays, Oct4 mutants also show upregulation of E-cadherin and Foxa2, suggesting a cell-autonomous phenotype. We confirm requirement for Oct4 in self-renewal of postimplantation epiblast ex vivo Our results indicate a role for Oct4 in orchestrating multiple fates and enabling expansion, correct patterning and lineage choice in the postimplantation epiblast.


Assuntos
Padronização Corporal , Embrião de Mamíferos/metabolismo , Camadas Germinativas/citologia , Fator 3 de Transcrição de Octâmero/metabolismo , Células-Tronco Pluripotentes/citologia , Animais , Biomarcadores/metabolismo , Diferenciação Celular , Linhagem da Célula , Implantação do Embrião , Embrião de Mamíferos/citologia , Endoderma/citologia , Endoderma/metabolismo , Feminino , Gastrulação , Deleção de Genes , Regulação da Expressão Gênica no Desenvolvimento , Genótipo , Camadas Germinativas/metabolismo , Imagem Tridimensional , Masculino , Camundongos , Mutação/genética , Proteína Homeobox Nanog/metabolismo , Fenótipo , Células-Tronco Pluripotentes/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Fatores de Transcrição SOXB1/metabolismo , Transdução de Sinais
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