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1.
J Cancer Res Ther ; 16(4): 804-810, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32930122

RESUMO

Background: The promising improvement in the clinical outcome of lung cancer can be possibly achieved by identification of the molecular events that underlie its pathogenesis. Cancer stem cell (CSC) being one of the subsets of tumor majorly participates in drug resistance and treatment failure because of the moderate cell cycle, lower proliferation, and increased expression of DNA repair and anti-apoptosis genes. Although many putative CSC markers exist, a precise characterization for non-small cell lung cancer is of utmost importance due to increased mortality rate and lack of targeted therapies. Hence, the article focuses on the expression of stemness-associated markers, namely octamer-binding transcription factor 4 (OCT4), NANOG, and sex-determining region Y-box 2 (SOX2) in non-small cell lung cancer (NSCLC) patients. Methods: The expression of OCT4, NANOG, and SOX2 were evaluated in 32 histopathologically confirmed NSCLC tissues using real-time polymerase chain reaction. The obtained expression was correlated with clinical and pathological manifestations using the statistical test such as Student's t-test and Pearson correlation in varied statistical software. Results: Results showed a significantly higher expression of OCT4 and NANOG compared to SOX2 in the tumor tissues. When the expression of these markers was correlated with the clinical parameters, higher expression was seen in males, patients with age above 60 years, and in adenocarcinoma subtype. In correlation with the habit, higher expression of OCT4 and SOX2 was observed in habituated patients. Expression of NANOG and OCT4 was higher even in patients with poor differentiation. Conclusion: The expression and prognostic significance of CSC markers obviously vary depending on histological NSCLC subtype. Importantly, our findings suggest that OCT4, SOX2, and NANOG network together may be promising for ongoing targeted therapies in specific NSCLC subgroups.


Assuntos
Biomarcadores Tumorais/genética , Carcinoma Pulmonar de Células não Pequenas/patologia , Regulação Neoplásica da Expressão Gênica , Proteína Homeobox Nanog/genética , Células-Tronco Neoplásicas/patologia , Fator 3 de Transcrição de Octâmero/genética , Fatores de Transcrição SOXB1/genética , Carcinoma Pulmonar de Células não Pequenas/genética , Carcinoma Pulmonar de Células não Pequenas/metabolismo , Feminino , Humanos , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/patologia , Masculino , Pessoa de Meia-Idade , Gradação de Tumores , Células-Tronco Neoplásicas/metabolismo , Prognóstico
2.
Anticancer Res ; 40(10): 5481-5487, 2020 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-32988870

RESUMO

BACKGROUND/AIM: γδ T cells mediate cytotoxicity against prostate cancer (PCa) cells in vitro; however, the clinical efficacy of γδ T cell-targeted immunotherapy for recurrent and metastatic PCa is unsatisfactory. We hypothesized that the resistance of recurrent and metastatic PCa to γδ T cells is related to the presence of prostate cancer stem cells (PCSCs), and we examined their relationship. MATERIALS AND METHODS: PCa spheres (prostaspheres) were generated from five PCa cell lines, and their susceptibility to cytotoxicity by γδ T cells was investigated. Expression of stemness-related markers was evaluated by qRT-PCR. RESULTS: Prostasphere-derived cancer cells were resistant to lysis by γδ T cells and expressed higher levels of several stemness markers, including CD133, NANOG, SOX2, and OCT4, than the parental PCa cell lines. CONCLUSION: Ex vivo-expanded γδ T cells are not effective against PCSCs.


Assuntos
Linfócitos Intraepiteliais/imunologia , Células-Tronco Neoplásicas/imunologia , Neoplasias da Próstata/imunologia , Receptores de Antígenos de Linfócitos T gama-delta/genética , Antígeno AC133/genética , Linhagem Celular Tumoral , Proliferação de Células/genética , Humanos , Masculino , Proteína Homeobox Nanog/genética , Células-Tronco Neoplásicas/patologia , Fator 3 de Transcrição de Octâmero/genética , Neoplasias da Próstata/genética , Neoplasias da Próstata/patologia , Receptores de Antígenos de Linfócitos T gama-delta/imunologia , Fatores de Transcrição SOXB1/genética , Linfócitos T
3.
Zhongguo Shi Yan Xue Ye Xue Za Zhi ; 28(4): 1086-1095, 2020 Aug.
Artigo em Chinês | MEDLINE | ID: mdl-32798382

RESUMO

OBJECTIVE: To explore the effect of OCT4 over-expression on the expression of induced pluripotent stem cell (iPSC)-related transcription factors (cMYC,KLF4,LIN28,NANOG and SOX2) in human bone marrow derived mesenchymal stem cells (hBMMSCs), so as to provide fundamental basis for exploring the pathogenesis of hematological diseases (leukemia, aplastic anemia, etc.) from the perspective of hemopoietic microenvironment in the future. METHODS: Recombinant plasmid pcDNA3.1-OCT4 was constructed and transferred into the optimal generation P3-4 hBMMSCs by liposome transfection. The cells with stable and high expression of OCT4(hBMMSCs-OCT4)were screened by G418 resistance screening (limited dilution) and subcloning, the expression of OCT4 mRNA and OCT4 protein was verified by RT-PCR and FCM, respectively. The expression of iPSC-related transcription factors (cMYC, KLF4, LIN28, NANOG and SOX2) were also determined by FCM and RT-PCR, so as to evaluate the effect of ectopic high expression of OCT4 on the expression of iPSC related transcription factors in hBMMSCs. RESULTS: Recombinant plasmid pcDNA3.1-OCT4 was successfully constructed and cells with stable and high expression of OCT4 were successfully screened from hBMMSCs by limited dilution and subcloning. The result of flow cytometry showed that the mean expression level of OCT4 protein increased from (3.03±1.49)% to (95.46±1.40)% compared with the untransfected parental MSCs, which was also confirmed by RT-PCR analysis. At the same time, the expression levels of OCT4 protein and mRNA were compared between transient transfection (day 4) and stable expression cells (day 96), respectively, it was showed that the OCT4 protein level increased from (36.36±0.28)% at day 4 to (96.25±1.38)% at day 96, and the OCT4 mRNA level increased from 2.75-folds to 6.23-folds, respectively. Compared with the untransfected parental MSCs, the average expression levels of stemness transcription factors increased from (1.12±0.47)% (cMYC), (0.84±0.30)% (KLF4), (2.14±0.79)% (LIN28), (0.63±0.37)% (NANOG) and (14.34±2.44)% (SOX2) to (80.65±4.75)%, (73.03±4.70)%, (68.08±3.05)%, (39.39±1.85)%and (91.45±4.56)% in hBMMSCs-OCT4, respectively, which were consistent with results of RT-PCR analysis. Moreover, the expression levels of NANOG and SOX2 positively correlated with the mean expression of OCT4 (OCT4 vs NANOG: r=0.7802,OCT4 vs SOX2: r=0.4981;NANOG vs SOX2: r=0.7426). CONCLUSION: Cells with stable and high expression of OCT4 have been successfully established from hBMMSCs. Ectopic high expression of transcription factor OCT4 in hBMMSCs can up-regulate the expression of other iPSC-related transcription factors such as cMYC, KLF4, LIN28, NANOG and SOX2.


Assuntos
Células-Tronco Pluripotentes Induzidas , Células-Tronco Mesenquimais , Medula Óssea , Humanos , Proteína Homeobox Nanog/genética , Fator 3 de Transcrição de Octâmero/genética , Fatores de Transcrição , Regulação para Cima
4.
PLoS One ; 15(7): e0235852, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32628717

RESUMO

The medulloblastoma (MB) microenvironment is diverse, and cell-cell interactions within this milieu is of prime importance. Astrocytes, a major component of the microenvironment, have been shown to impact primary tumor cell phenotypes and metastasis. Based on proximity of MB cells and astrocytes in the brain microenvironment, we investigated whether astrocytes may influence MB cell phenotypes directly. Astrocyte conditioned media (ACM) increased Daoy MB cell invasion, adhesion, and in vivo cellular protrusion formation. ACM conditioning of MB cells also increased CD133 surface expression, a key cancer stem cell marker of MB. Additional neural stem cell markers, Nestin and Oct-4A, were also increased by ACM conditioning, as well as neurosphere formation. By knocking down CD133 using short interfering RNA (siRNA), we showed that ACM upregulated CD133 expression in MB plays an important role in invasion, adhesion and neurosphere formation. Collectively, our data suggests that astrocytes influence MB cell phenotypes by regulating CD133 expression, a key protein with defined roles in MB tumorgenicity and survival.


Assuntos
Antígeno AC133/genética , Astrócitos/metabolismo , Meduloblastoma/metabolismo , Fenótipo , Antígeno AC133/metabolismo , Animais , Adesão Celular , Linhagem Celular Tumoral , Movimento Celular , Células Cultivadas , Meios de Cultivo Condicionados , Humanos , Células-Tronco Neoplásicas/efeitos dos fármacos , Células-Tronco Neoplásicas/metabolismo , Células-Tronco Neoplásicas/fisiologia , Nestina/genética , Nestina/metabolismo , Fator 3 de Transcrição de Octâmero/genética , Fator 3 de Transcrição de Octâmero/metabolismo , Microambiente Tumoral , Peixe-Zebra
5.
PLoS One ; 15(7): e0235617, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32634160

RESUMO

Low egg quality and embryonic survival are critical challenges in aquaculture, where assisted reproduction procedures and other factors may impact egg quality. This includes European eel (Anguilla anguilla), where pituitary extract from carp (CPE) or salmon (SPE) is applied to override a dopaminergic inhibition of the neuroendocrine system, preventing gonadotropin secretion and gonadal development. The present study used either CPE or SPE to induce vitellogenesis in female European eel and compared impacts on egg quality and offspring developmental competence with emphasis on the maternal-to-zygotic transition (MZT). Females treated with SPE produced significantly higher proportions of floating eggs with fewer cleavage abnormalities and higher embryonic survival. These findings related successful embryogenesis to higher abundance of mRNA transcripts of genes involved in cell adhesion, activation of MZT, and immune response (dcbld1, epcam, oct4, igm) throughout embryonic development. The abundance of mRNA transcripts of cldnd, foxr1, cea, ccna1, ccnb1, ccnb2, zar1, oct4, and npm2 was relatively stable during the first eight hours, followed by a drop during MZT and low levels thereafter, indicating transfer and subsequent clearance of maternal mRNA. mRNA abundance of zar1, epcam, and dicer1 was associated with cleavage abnormalities, while mRNA abundance of zar1, sox2, foxr1, cldnd, phb2, neurod4, and neurog1 (before MZT) was associated with subsequent embryonic survival. In a second pattern, low initial mRNA abundance with an increase during MZT and higher levels persisting thereafter indicating the activation of zygotic transcription. mRNA abundance of ccna1, npm2, oct4, neurod4, and neurog1 during later embryonic development was associated with hatch success. A deviating pattern was observed for dcbld1, which mRNA levels followed the maternal-effect gene pattern but only for embryos from SPE treated females. Together, the differences in offspring production and performance reported in this study show that PE composition impacts egg quality and embryogenesis and in particular, the transition from initial maternal transcripts to zygotic transcription.


Assuntos
Anguilla/fisiologia , Carpas/metabolismo , Desenvolvimento Embrionário , Oogênese , Hipófise/metabolismo , Salmão/metabolismo , Anguilla/crescimento & desenvolvimento , Animais , Ciclina A1/genética , Desenvolvimento Embrionário/efeitos dos fármacos , Desenvolvimento Embrionário/genética , Feminino , Proteínas de Peixes/genética , Regulação da Expressão Gênica no Desenvolvimento/efeitos dos fármacos , Fator 3 de Transcrição de Octâmero/genética , Oogênese/efeitos dos fármacos , Hipófise/química , Hormônios Hipofisários/farmacologia , RNA Mensageiro/metabolismo , Zigoto/efeitos dos fármacos , Zigoto/crescimento & desenvolvimento , Zigoto/metabolismo
7.
Anticancer Res ; 40(7): 3801-3809, 2020 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-32620619

RESUMO

AIM: Cancer stem-like cell (CSC) markers and the role of CSCs derived from papillary thyroid carcinoma (PTC) in pathogenesis are unclear. This study aimed to investigate CSC properties using tumor spheres from passaged PTC cells but without sorting CSCs. MATERIALS AND METHODS: To identify the properties of CSCs derived from PTC, the expression of SRY-box transcription factor 2(SOX2), octamer-binding transcription factor 4 (OCT4), Nanog homeobox (NANOG), thyroglobulin (TG), thyroid-stimulating hormone receptor (TSHR), E-cadherin, YES-associated protein 1 (YAP1), and signal transducer and activator of transcription 3 (STAT3) was investigated in tumor spheres serially passaged without sorting CSCs. RESULTS: The cultured tumor spheres had cancer stemness; high expression of OCT4, SOX2, NANOG, and YAP1; low expression of E-cadherin; and varied expression of TG, TSHR, and STAT3. PTC tumor spheres transfected with small interfering RNA targeting YAP1 had fewer CSC properties than the non-transfected tumor spheres did. CONCLUSION: Tumor spheres derived from PTC cells by passaging without sorting CSCs have more stem-like cell properties, and less differentiation potential. Thus, this simple and cost-effective method can be used for the enrichment of PTC stemness for employment in cell-based models, reducing the need for use of animal models.


Assuntos
Células-Tronco Neoplásicas/patologia , Câncer Papilífero da Tireoide/patologia , Neoplasias da Glândula Tireoide/patologia , Proteínas Adaptadoras de Transdução de Sinal/biossíntese , Proteínas Adaptadoras de Transdução de Sinal/genética , Antígenos CD/biossíntese , Antígenos CD/genética , Biomarcadores Tumorais/biossíntese , Biomarcadores Tumorais/genética , Caderinas/biossíntese , Caderinas/genética , Linhagem Celular Tumoral , Proliferação de Células/fisiologia , Humanos , Células-Tronco Neoplásicas/metabolismo , Fator 3 de Transcrição de Octâmero/biossíntese , Fator 3 de Transcrição de Octâmero/genética , Fator de Transcrição STAT3/biossíntese , Fator de Transcrição STAT3/genética , Esferoides Celulares , Câncer Papilífero da Tireoide/genética , Câncer Papilífero da Tireoide/metabolismo , Neoplasias da Glândula Tireoide/genética , Neoplasias da Glândula Tireoide/metabolismo , Fatores de Transcrição/biossíntese , Fatores de Transcrição/genética
8.
Sci Rep ; 10(1): 8875, 2020 06 01.
Artigo em Inglês | MEDLINE | ID: mdl-32483269

RESUMO

Cancer stem cells (CSCs) play a critical role in cancer development and growth. The aim of this study was to identify and isolate CSCs from populations of primary oral squamous cell carcinoma (OSCC) cells, which were obtained from OSCC specimens and identified by cell morphology and immunohistochemical staining for keratin. CD133+ cells detected by flow cytometry comprised 0.41 ± 0.06% of primary OSCC cells and were isolated from primary OSCC cell populations using magnetic-activated cell sorting, revealing that 93.39% of high-purity CD133+ cells were in the G0/G1 phase of the cell cycle. Additionally, the growth rate of CD133+ cells was higher than that of CD133- cells, and in vivo tumourigenesis experiments showed that the tumourigenic ability of CD133+ cells was markedly stronger than that of CD133- cells. Moreover, CD133+ cells showed increased chemotherapeutic resistance to cisplatin and higher self-renewal ability according to sphere-formation assay, as well as higher mRNA levels of stemness-associated genes, including NANOG, SOX2, ALDH1A1, and OCT4. These results indicated that OSCC cells, which share certain characteristics of CSCs, harbour CD133+ cells potentially responsible for OSCC aggressiveness, suggesting CD133 as a potential prognostic marker and therapeutic target.


Assuntos
Antígeno AC133/metabolismo , Carcinoma de Células Escamosas/patologia , Neoplasias Bucais/patologia , Células-Tronco Neoplásicas/metabolismo , Animais , Antineoplásicos/farmacologia , Antineoplásicos/uso terapêutico , Grupo com Ancestrais do Continente Asiático , Carcinoma de Células Escamosas/diagnóstico , Carcinoma de Células Escamosas/tratamento farmacológico , Proliferação de Células/efeitos dos fármacos , Autorrenovação Celular , China , Cisplatino/farmacologia , Cisplatino/uso terapêutico , Resistencia a Medicamentos Antineoplásicos , Fase G1 , Humanos , Camundongos , Camundongos Endogâmicos BALB C , Neoplasias Bucais/diagnóstico , Neoplasias Bucais/tratamento farmacológico , Proteína Homeobox Nanog/genética , Proteína Homeobox Nanog/metabolismo , Células-Tronco Neoplásicas/citologia , Fator 3 de Transcrição de Octâmero/genética , Fator 3 de Transcrição de Octâmero/metabolismo , RNA Mensageiro/metabolismo , Transplante Heterólogo
9.
Oncogene ; 39(26): 4970-4982, 2020 06.
Artigo em Inglês | MEDLINE | ID: mdl-32507861

RESUMO

Cancer stem cells (CSCs) are cells within tumors that maintain the ability to self-renew, drive tumor growth, and contribute to therapeutic resistance and cancer recurrence. In this study, we investigate the role of Zinc finger and SCAN domain containing 4 (ZSCAN4) in human head and neck squamous cell carcinoma (HNSCC). The murine Zscan4 is involved in telomere maintenance and genomic stability of mouse embryonic stem cells. Our data indicate that the human ZSCAN4 is enriched for, marks and is co-expressed with CSC markers in HNSCC. We show that transient ZSCAN4 induction for just 2 days increases CSC frequency both in vitro and in vivo and leads to upregulation of pluripotency and CSC factors. Importantly, we define for the first time the role of ZSCAN4 in altering the epigenetic profile and regulating the chromatin state. Our data show that ZSCAN4 leads to a functional histone 3 hyperacetylation at the promoters of OCT3/4 and NANOG, leading to an upregulation of CSC factors. Consistently, ZSCAN4 depletion leads to downregulation of CSC markers, decreased ability to form tumorspheres and severely affects tumor growth. Our study suggests that ZSCAN4 plays an important role in the maintenance of the CSC phenotype, indicating it is a potential therapeutic target in HNSCC.


Assuntos
Carcinoma de Células Escamosas/genética , Montagem e Desmontagem da Cromatina/genética , Proteínas de Ligação a DNA/genética , Regulação Neoplásica da Expressão Gênica , Neoplasias de Cabeça e Pescoço/genética , Células-Tronco Neoplásicas/metabolismo , Fatores de Transcrição/genética , Acetilação , Animais , Carcinoma de Células Escamosas/metabolismo , Carcinoma de Células Escamosas/terapia , Linhagem Celular Tumoral , Proteínas de Ligação a DNA/metabolismo , Neoplasias de Cabeça e Pescoço/metabolismo , Neoplasias de Cabeça e Pescoço/terapia , Histonas/metabolismo , Humanos , Camundongos Endogâmicos NOD , Camundongos Knockout , Camundongos SCID , Proteína Homeobox Nanog/genética , Proteína Homeobox Nanog/metabolismo , Fator 3 de Transcrição de Octâmero/genética , Fator 3 de Transcrição de Octâmero/metabolismo , Fenótipo , Interferência de RNA , Fatores de Transcrição/metabolismo , Ensaios Antitumorais Modelo de Xenoenxerto/métodos
10.
Anim Sci J ; 91(1): e13408, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32578338

RESUMO

We examined the allelic expression and positioning of two pluripotency-associated genes, OCT4 and SOX2, and two housekeeping genes, ACTB and TUBA, in 4- and 8-cell porcine embryos utilizing RNA and DNA fluorescence in situ hybridization (FISH) in single blastomeres. The proportion of blastomeres expressing SOX2 bi-allelically increased from 45% at the 4-cell stage to 60% at the 8-cell stage. Moreover, in 8-cell embryos, SOX2 was expressed bi-allelically in significantly more blastomeres than was the case for OCT4, and this was associated with a tendency for SOX2 alleles to move toward the nuclear interior during 4- to 8-cell transition. However, the radial location of OCT4 alleles did not change significantly during this transition. The locations of active and inactive alleles based on DNA and RNA FISH signals were also calculated. Inactive OCT4 alleles were located in very close proximity to the nuclear membrane, whereas active OCT4 alleles were more centrally disposed in the nucleus. Nevertheless, the nuclear location of active and inactive SOX2 alleles did not change in either 4- or 8-cell blastomeres. Our RNA and DNA FISH data provide novel information on the allelic expression patterns and positioning of pluripotency-associated genes, OCT4 and SOX2, during embryonic genome activation in pigs.


Assuntos
Blastômeros/citologia , Blastômeros/metabolismo , Desenvolvimento Embrionário/genética , Regulação da Expressão Gênica no Desenvolvimento , Expressão Gênica , Suínos/embriologia , Suínos/genética , Alelos , Animais , Proteínas do Citoesqueleto/genética , Proteínas do Citoesqueleto/metabolismo , Fertilização In Vitro , Hibridização in Situ Fluorescente , Técnicas de Maturação in Vitro de Oócitos , Técnicas In Vitro , Fator 3 de Transcrição de Octâmero/genética , Fator 3 de Transcrição de Octâmero/metabolismo , Fatores de Transcrição SOXB1/genética , Fatores de Transcrição SOXB1/metabolismo
11.
Sci Rep ; 10(1): 5195, 2020 03 23.
Artigo em Inglês | MEDLINE | ID: mdl-32251342

RESUMO

Pluripotency maintenance requires transcription factors (TFs) that induce genes necessary to preserve the undifferentiated state and repress others involved in differentiation. Recent observations support that the heterogeneous distribution of TFs in the nucleus impacts on gene expression. Thus, it is essential to explore how TFs dynamically organize to fully understand their role in transcription regulation. Here, we examine the distribution of pluripotency TFs Oct4 and Sox2 in the nucleus of embryonic stem (ES) cells and inquire whether their organization changes during early differentiation stages preceding their downregulation. Using ES cells expressing Oct4-YPet or Sox2-YPet, we show that Oct4 and Sox2 partition between nucleoplasm and a few chromatin-dense foci which restructure after inducing differentiation by 2i/LIF withdrawal. Fluorescence correlation spectroscopy showed distinct changes in Oct4 and Sox2 dynamics after differentiation induction. Specifically, we detected an impairment of Oct4-chromatin interactions whereas Sox2 only showed slight variations in its short-lived, and probably more unspecific, interactions with chromatin. Our results reveal that differentiation cues trigger early changes of Oct4 and Sox2 nuclear distributions that also include modifications in TF-chromatin interactions. This dynamical reorganization precedes Oct4 and Sox2 downregulation and may contribute to modulate their function at early differentiation stages.


Assuntos
Núcleo Celular/metabolismo , Montagem e Desmontagem da Cromatina , Células-Tronco Embrionárias/citologia , Fator 3 de Transcrição de Octâmero/metabolismo , Células-Tronco Pluripotentes/metabolismo , Fatores de Transcrição SOXB1/metabolismo , Transcrição Genética , Animais , Ciclo Celular , Diferenciação Celular , Núcleo Celular/ultraestrutura , Células Cultivadas , Doxiciclina/farmacologia , Células-Tronco Embrionárias/metabolismo , Regulação da Expressão Gênica no Desenvolvimento/efeitos dos fármacos , Genes Reporter , Camundongos , Microscopia de Fluorescência , Fator 3 de Transcrição de Octâmero/genética , Células-Tronco Pluripotentes/citologia , Proteínas Recombinantes de Fusão/metabolismo , Fatores de Transcrição SOXB1/genética , Transfecção
12.
Biochem Pharmacol ; 177: 113984, 2020 07.
Artigo em Inglês | MEDLINE | ID: mdl-32311348

RESUMO

Pluripotent stem cells are have therapeutic applications in regenerative medicine and drug discovery. However, the differentiation of stem cells in vitro hinders their large-scale production and clinical applications. The maintenance of cell pluripotency relies on a complex network of transcription factors; of these, octamer-binding transcription factor-4 (Oct4) plays a key role. This study aimed to construct an Oct4 gene promoter-driven firefly luciferase reporter and screen small-molecule compounds could maintain cell self-renewal and pluripotency. The results showed that ethyl-p-methoxycinnamate (EPMC) enhance the promoter activity of the Oct4 gene, increased the expression of Oct4 at both mRNA and protein levels, and significantly promoted the colony formation of P19 cells. These findings suggesting that EPMC could reinforce the self-renewal capacity of P19 cells. The pluripotency markers Oct4, SRY-related high-mobility-group-box protein-2, and Nanog were expressed at higher levels in EPMC-induced colonies. EPMC could promote teratoma formation and differentiation potential of P19 cells in vivo. It also enhanced self-renewal and pluripotency of human umbilical cord mesenchymal stem cells and mouse embryonic stem cells. Moreover, it significantly activated the nuclear factor kappa B (NF-κB) signaling pathway via the myeloid differentiation factor 88-dependent pathway. The expression level of Oct4 decreased after blocking the NF-κB signaling pathway, suggesting that EPMC promoted the expression of Oct4 partially through the NF-κB signaling pathway. This study indicated that EPMC could maintain self-renewal and pluripotency of stem cells.


Assuntos
Autorrenovação Celular/efeitos dos fármacos , Cinamatos/farmacologia , NF-kappa B/genética , Fator 3 de Transcrição de Octâmero/genética , Células-Tronco Pluripotentes/efeitos dos fármacos , Transdução de Sinais/efeitos dos fármacos , Animais , Diferenciação Celular/efeitos dos fármacos , Autorrenovação Celular/genética , Células-Tronco Embrionárias/citologia , Células-Tronco Embrionárias/efeitos dos fármacos , Células-Tronco Embrionárias/metabolismo , Fibroblastos/citologia , Fibroblastos/efeitos dos fármacos , Fibroblastos/metabolismo , Regulação da Expressão Gênica , Genes Reporter , Humanos , Luciferases/genética , Luciferases/metabolismo , Células-Tronco Mesenquimais/citologia , Células-Tronco Mesenquimais/efeitos dos fármacos , Células-Tronco Mesenquimais/metabolismo , Camundongos , Fator 88 de Diferenciação Mieloide/genética , Fator 88 de Diferenciação Mieloide/metabolismo , Inibidor de NF-kappaB alfa/genética , Inibidor de NF-kappaB alfa/metabolismo , NF-kappa B/agonistas , NF-kappa B/metabolismo , Proteína Homeobox Nanog/genética , Proteína Homeobox Nanog/metabolismo , Fator 3 de Transcrição de Octâmero/agonistas , Fator 3 de Transcrição de Octâmero/metabolismo , Células-Tronco Pluripotentes/citologia , Células-Tronco Pluripotentes/metabolismo , Regiões Promotoras Genéticas , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Fatores de Transcrição SOXB1/genética , Fatores de Transcrição SOXB1/metabolismo , Transdução de Sinais/genética
13.
Comp Biochem Physiol B Biochem Mol Biol ; 243-244: 110436, 2020 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-32247058

RESUMO

The wood frog (Rana sylvatica) is a remarkable species. These frogs can endure prolonged oxygen deprivation as well as dehydration to ~60% of total body water lost and, combining these two abilities, they survive whole body freezing for weeks at a time during the winter. Episodes of anoxia/reoxygenation or freeze/thaw can trigger elevated production of reactive oxygen species (ROS) causing cellular damage, especially when oxygen is reintroduced during reoxygenation or thawing. To mitigate ROS damage, stress-responsive transcription factors such as the Octamer Binding Transcription factor (OCT4) and Nuclear factor (erythroid-derived 2)-like 2 transcription factor (Nrf2) were postulated to be involved in enhancing pro-survival pathways and antioxidant defenses. The present study used immunoblotting to analyze OCT4 and Nrf2 responses (and downstream factors under their control) to 24 h anoxia and 4 h reoxygenation in liver and skeletal muscle of wood frogs, with an emphasis on antioxidant systems. Surprisingly, no change was observed in relative total protein expression of either of the two transcription factors in liver. Furthermore, a significant decrease in total protein levels of OCT4 and Nrf2 occurred in skeletal muscle after 4 h recovery. However, essential cofactors of OCT4 and Nrf2 were significantly upregulated during anoxia and/or recovery. Downstream targets of the Nrf2-ARE pathway were evaluated, including glutathione-S-transferases (GSTs) and aldo-keto reductases (AKRs). Significant increases in GSTT1 and GSTP1 were observed in liver and muscle whereas AKRs showed a tissue specific response to both anoxia and recovery from anoxia. This study demonstrates activation of antioxidants as a cell protective mechanism against generation of reactive oxygen species during anoxia in wood frogs.


Assuntos
Antioxidantes/metabolismo , Desidratação/metabolismo , Hipóxia/veterinária , Oxigênio/metabolismo , Ranidae/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Adaptação Fisiológica , Aldo-Ceto Redutases/metabolismo , Animais , Congelamento , Glutationa Transferase/metabolismo , Hipóxia/genética , Hipóxia/metabolismo , Fígado/metabolismo , Músculo Esquelético/metabolismo , Fator 2 Relacionado a NF-E2/genética , Fator 2 Relacionado a NF-E2/metabolismo , Fator 3 de Transcrição de Octâmero/genética , Fator 3 de Transcrição de Octâmero/metabolismo , Estresse Fisiológico
14.
Cancer Res ; 80(11): 2125-2137, 2020 06 01.
Artigo em Inglês | MEDLINE | ID: mdl-32265227

RESUMO

Peptidylarginine deiminases (PADI) catalyze posttranslational modification of many target proteins and have been suggested to play a role in carcinogenesis. Citrullination of histones by PADI4 was recently implicated in regulating embryonic stem and hematopoietic progenitor cells. Here, we investigated a possible role for PADI4 in regulating breast cancer stem cells. PADI4 activity limited the number of cancer stem cells (CSC) in multiple breast cancer models in vitro and in vivo. Mechanistically, PADI4 inhibition resulted in a widespread redistribution of histone H3, with increased accumulation around transcriptional start sites. Interestingly, epigenetic effects of PADI4 on the bulk tumor cell population did not explain the CSC phenotype. However, in sorted tumor cell populations, PADI4 downregulated expression of master transcription factors of stemness, NANOG and OCT4, specifically in the cancer stem cell compartment, by reducing the transcriptionally activating H3R17me2a histone mark at those loci; this effect was not seen in the non-stem cells. A gene signature reflecting tumor cell-autonomous PADI4 inhibition was associated with poor outcome in human breast cancer datasets, consistent with a tumor-suppressive role for PADI4 in estrogen receptor-positive tumors. These results contrast with known tumor-promoting effects of PADI4 on the tumor stroma and suggest that the balance between opposing tumor cell-autonomous and stromal effects may determine net outcome. Our findings reveal a novel role for PADI4 as a tumor suppressor in regulating breast cancer stem cells and provide insight into context-specific effects of PADI4 in epigenetic modulation. SIGNIFICANCE: These findings demonstrate a novel activity of the citrullinating enzyme PADI4 in suppressing breast cancer stem cells through epigenetic repression of stemness master transcription factors NANOG and OCT4.


Assuntos
Neoplasias da Mama/enzimologia , Neoplasias da Mama/patologia , Células-Tronco Neoplásicas/enzimologia , Células-Tronco Neoplásicas/patologia , Proteína-Arginina Desiminase do Tipo 4/metabolismo , Animais , Neoplasias da Mama/genética , Linhagem Celular Tumoral , Progressão da Doença , Feminino , Técnicas de Silenciamento de Genes , Humanos , Isoenzimas , Células MCF-7 , Camundongos , Proteína Homeobox Nanog/genética , Proteína Homeobox Nanog/metabolismo , Fator 3 de Transcrição de Octâmero/genética , Fator 3 de Transcrição de Octâmero/metabolismo , Proteína-Arginina Desiminase do Tipo 4/antagonistas & inibidores , Proteína-Arginina Desiminase do Tipo 4/genética , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Transcriptoma
15.
Toxicology ; 438: 152444, 2020 05 30.
Artigo em Inglês | MEDLINE | ID: mdl-32283119

RESUMO

As a common birth defect, Cleft palate can be caused by the disturbance during the developmental process of the palatal shelves. The 2,3,7,8-tetrachlorodibenzo- p-dioxin (TCDD) is a well-known environmental teratogenic agent for cleft palate and Aryl hydrocarbon receptor (AhR) pathway can be activated by dioxins. Oct4 as a pluripotent stem cell transcription factor is also involved in the process of embryonic development. The AHR and retinoid receptors have cross-talk at CYP1A1 (cytochrome P450, family 1, subfamily A, polypeptide 1) promoter. There are also bidirectional talk between AhR and Oct4. In this study, we used C57/BL6 N mice and TCDD (64 µg/Kg body weight) to establish a model of fetal cleft palate to observe the effects of dioxin on fetal mesenchymal proliferation and apoptosis, and explore the role of Oct4 in inducing cleft palate. The results showed that dioxin inhibited mesenchymal proliferation and promoted apoptosis. In addition, dioxin inhibited Oct4 expression, and preliminary data suggest that hypermethylation of the Oct4 promoter may be a putative mechanism, suggesting that TCDD might induce cleft palate by inhibiting the proliferation of palatal mesenchymal cells mediated by Oct4.


Assuntos
Proliferação de Células , Fissura Palatina/metabolismo , Mesoderma/metabolismo , Fator 3 de Transcrição de Octâmero/metabolismo , Palato/metabolismo , Dibenzodioxinas Policloradas , Animais , Apoptose , Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Fissura Palatina/induzido quimicamente , Fissura Palatina/patologia , Metilação de DNA , Modelos Animais de Doenças , Feminino , Masculino , Mesoderma/anormalidades , Camundongos Endogâmicos C57BL , Fator 3 de Transcrição de Octâmero/genética , Palato/anormalidades , Gravidez , Regiões Promotoras Genéticas , Receptores de Hidrocarboneto Arílico/genética , Receptores de Hidrocarboneto Arílico/metabolismo , Transdução de Sinais
16.
PLoS Pathog ; 16(4): e1008468, 2020 04.
Artigo em Inglês | MEDLINE | ID: mdl-32298395

RESUMO

Octamer binding transcription factor-4 (Oct4), is highly expressed in stem cells and has indispensable roles in pluripotency and cellular reprogramming. In contrast to other factors used for cellular reprogramming, a role for Oct4 outside embryonic stem cells has been elusive and highly controversial. Emerging evidence implicates Oct4 in the carcinogenic process, but the mechanism through which Oct4 may be functioning in cancers is not fully appreciated. Here, we provide evidence that Oct4 is expressed in human cervical cancer and this expression correlates with the presence of the human papillomavirus (HPV) oncogenes E6 and E7. Surprisingly, the viral oncogenes can complement exogenously provided Oct4 in reprogramming assays, providing functional validation for their ability to activate Oct4 transcription in Mouse Embryonic Fibroblasts (MEFs). To interrogate potential roles of Oct4 in cervical cancers we knocked-down Oct4 in HPV(+) (HeLa & CaSki) and HPV(-) (C33A) cervical cancer cell lines and found that Oct4 knockdown attenuated clonogenesis, only in the HPV(+) cells. More unexpectedly, cell proliferation and migration, were differentially affected in HPV(+) and HPV(-) cell lines. We provide evidence that Oct4 interacts with HPV E7 specifically at the CR3 region of the E7 protein and that introduction of the HPV oncogenes in C33A cells and human immortalised keratinocytes generates Oct4-associated transcriptional and phenotypic patterns, which mimic those seen in HPV(+) cells. We propose that a physical interaction of Oct4 with E7 regulates its activity in HPV(+) cervical cancers in a manner not seen in other cancer types.


Assuntos
Fator 3 de Transcrição de Octâmero/metabolismo , Proteínas E7 de Papillomavirus/metabolismo , Infecções por Papillomavirus/metabolismo , Neoplasias do Colo do Útero/metabolismo , Neoplasias do Colo do Útero/virologia , Carcinogênese/metabolismo , Linhagem Celular Tumoral , Movimento Celular/fisiologia , Proliferação de Células/fisiologia , Feminino , Células HeLa , Humanos , Fator 3 de Transcrição de Octâmero/biossíntese , Fator 3 de Transcrição de Octâmero/genética , Proteínas Oncogênicas Virais/metabolismo , Oncogenes/fisiologia , Papillomaviridae/genética , Papillomaviridae/metabolismo , Proteínas E7 de Papillomavirus/genética , Infecções por Papillomavirus/patologia , Infecções por Papillomavirus/virologia , Proteínas Repressoras/metabolismo , Neoplasias do Colo do Útero/genética , Neoplasias do Colo do Útero/patologia
17.
PLoS One ; 15(3): e0230253, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32176716

RESUMO

Autologous transplantation of spermatogonial stem cells is a promising new avenue to restore fertility in infertile recipients. Expansion of the initial spermatogonial stem cell pool through cell culturing is a necessary step to obtain enough cells for effective repopulation of the testis after transplantation. Since in vitro propagation can lead to (epi-)genetic mutations and possibly malignant transformation of the starting cell population, we set out to investigate genome-wide DNA methylation status in uncultured and cultured primary testicular ITGA6+ sorted cells and compare them with germ cell tumor samples of the seminoma subtype. Seminomas displayed a severely global hypomethylated profile, including loss of genomic imprinting, which we did not detect in cultured primary testicular ITGA6+ cells. Differential methylation analysis revealed altered regulation of gamete formation and meiotic processes in cultured primary testicular ITGA6+ cells but not in seminomas. The pivotal POU5F1 marker was hypomethylated in seminomas but not in uncultured or cultured primary testicular ITGA6+ cells, which is reflected in the POU5F1 mRNA expression levels. Lastly, seminomas displayed a number of characteristic copy number variations that were not detectable in primary testicular ITGA6+ cells, either before or after culture. Together, the data show a distinct DNA methylation patterns in cultured primary testicular ITGA6+ cells that does not resemble the pattern found in seminomas, but also highlight the need for more sensitive methods to fully exclude the presence of malignant cells after culture and to further study the epigenetic events that take place during in vitro culture.


Assuntos
Variações do Número de Cópias de DNA/genética , Metilação de DNA/genética , Instabilidade Genômica/genética , Integrina alfa6/genética , Seminoma/genética , Idoso , Idoso de 80 Anos ou mais , Biomarcadores/metabolismo , Células Cultivadas , Epigênese Genética/genética , Impressão Genômica/genética , Humanos , Masculino , Pessoa de Meia-Idade , Mutação/genética , Neoplasias Embrionárias de Células Germinativas , Fator 3 de Transcrição de Octâmero/genética , Neoplasias Testiculares/genética , Testículo/metabolismo
18.
Life Sci ; 248: 117461, 2020 May 01.
Artigo em Inglês | MEDLINE | ID: mdl-32097665

RESUMO

AIMS: To compare how OCT4A proteins interact with and regulate multiple OCT4A-octamer motifs (OMs) in different regions of the FOS gene expressed in somatic cancer cells versus pluripotent stem cells. MATERIALS AND METHODS: Two FOS reporter gene systems harboring predicted OMs or their mutational counterparts were introduced into HeLa and NCCIT cells with varying OCT4A protein levels. The transcription of dsGFP reflecting FOS expression was quantitated by RT-qPCR, the OCT4A-OMs binding and the correlation between OCT4A and FOS transcription was determined by ChIP-PCR and RNA-Seq, respectively. KEY FINDINGS: In NCCIT cells, abundant OCT4A proteins bound to and inhibited OM1 and OM2 at the promoter of the FOS gene. RA-induced OCT4A down-regulation transiently increased FOS transcription. In contrast, in HeLa cells that contain much lower levels of endogenous OCT4A proteins, OCT4A primarily bound to and activate OM1 thereby promoting FOS transcription. OCT4A KO significantly reduced FOS expression. Ectopically introduced OCT4A, at its leaked or induced expression level, promoted FOS transcription by binding to OM2/OM3 or OM1/OM3, respectively. Thus, the interaction of OCT4A proteins with different OMs is cellular context- and protein level-dependent, and such complicated OCT4A binding mode can only be reflected by a dsGFP-based reporter harboring the full-length FOS gene but not by that merely having the FOS promoter. SIGNIFICANCE: Our findings unravel an additional layer of regulatory mechanisms that account for the cellular context- and dose-related versatile functions of OCT4A protein, and further underscore the importance of precise modulation of OCT4A in the regenerative medicine and anticancer therapies.


Assuntos
Regulação da Expressão Gênica , Fator 3 de Transcrição de Octâmero/genética , Células-Tronco Pluripotentes/metabolismo , Proteínas Proto-Oncogênicas c-fos/genética , Motivos de Aminoácidos , Linhagem Celular Tumoral , Genes Reporter , Vetores Genéticos/química , Vetores Genéticos/metabolismo , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Células HeLa , Humanos , Fator 3 de Transcrição de Octâmero/metabolismo , Especificidade de Órgãos , Células-Tronco Pluripotentes/citologia , Regiões Promotoras Genéticas , Ligação Proteica , Proteínas Proto-Oncogênicas c-fos/metabolismo , Transdução de Sinais , Transcrição Genética
19.
Mol Biol Rep ; 47(3): 1859-1869, 2020 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-32016633

RESUMO

Cancer stem cells are commonly tolerant toward chemotherapy and radiotherapy. Oct4 and Sox2 transcription factors are shown to be overexpressed in various cancers. At the current research, inhibition of Oct4 and Sox2 transcription factors was performed through application of decoy oligodeoxynucleotides (ODNs) strategy via repressing stemness properties in HT29-ShE cells encompassing enriched cancer stem-like cells. Designed Oct4-Sox2 complex decoy ODNs were transfected into HT29-ShE cells with Lipofectamine reagent. At the next step, ODNs efficiency transfection and subcellular localization were determined via flow cytometry and fluorescence microscopy, respectively. Further investigations such as cell proliferation and apoptosis analysis, colonosphere formation, invasion and migration, and real-time PCR assays were also carried out. Obtained results shed light on the fact that the designed complex decoys were effectively transfected into HT29-ShE cells, and they were found to be localized in subcellular compartments. Oct4-Sox2 decoy ODNs led to decreased cell viability, arresting the cell cycle in G0/G1 phases, increasing apoptosis, inhibition of migration/invasion and colonosphere formation ability of HT29-ShE cells in comparison with control and scramble groups. Furthermore, Oct4-Sox2 complex decoy could modulate the MET process via alteration of mRNA expression of downstream genes. It could be concluded that application of Oct4-Sox2 transcription factor decoy strategy in cells with stemness potential could lead to inhibiting the cell growth and triggering differentiation. Therefore, this technique could be applied along with usual remedies (chemotherapy and radiotherapy) as high potential method for treating cancer.


Assuntos
Células-Tronco Neoplásicas/metabolismo , Fator 3 de Transcrição de Octâmero/genética , Oligodesoxirribonucleotídeos/farmacologia , Fatores de Transcrição SOXB1/genética , Movimento Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Transição Epitelial-Mesenquimal/efeitos dos fármacos , Citometria de Fluxo , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Redes Reguladoras de Genes/efeitos dos fármacos , Células HT29 , Humanos , Microscopia de Fluorescência , Complexos Multiproteicos/antagonistas & inibidores , Células-Tronco Neoplásicas/efeitos dos fármacos , Fator 3 de Transcrição de Octâmero/metabolismo , Fatores de Transcrição SOXB1/metabolismo
20.
Nucleic Acids Res ; 48(7): 3935-3948, 2020 04 17.
Artigo em Inglês | MEDLINE | ID: mdl-32055844

RESUMO

Formation of a pluripotency-specific chromatin network is a critical event in reprogramming somatic cells into pluripotent status. To characterize the regulatory components in this process, we used 'chromatin RNA in situ reverse transcription sequencing' (CRIST-seq) to profile RNA components that interact with the pluripotency master gene Oct4. Using this approach, we identified a novel nuclear lncRNA Oplr16 that was closely involved in the initiation of reprogramming. Oplr16 not only interacted with the Oct4 promoter and regulated its activity, but it was also specifically activated during reprogramming to pluripotency. Active expression of Oplr16 was required for optimal maintenance of pluripotency in embryonic stem cells. Oplr16 was also able to enhance reprogramming of fibroblasts into pluripotent cells. RNA reverse transcription-associated trap sequencing (RAT-seq) indicated that Oplr16 interacted with multiple target genes related to stem cell self-renewal. Of note, Oplr16 utilized its 3'-fragment to recruit the chromatin factor SMC1 to orchestrate pluripotency-specific intrachromosomal looping. After binding to the Oct4 promoter, Oplr16 recruited TET2 to induce DNA demethylation and activate Oct4 in fibroblasts, leading to enhanced reprogramming. These data suggest that Oplr16 may act as a pivotal chromatin factor to control stem cell fate by modulating chromatin architecture and DNA demethylation.


Assuntos
Reprogramação Celular , Cromatina/química , Proteínas de Ligação a DNA/metabolismo , Células-Tronco Pluripotentes Induzidas/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , RNA Longo não Codificante/fisiologia , Animais , Proteínas de Ciclo Celular/metabolismo , Células Cultivadas , Proteínas Cromossômicas não Histona/metabolismo , DNA/metabolismo , Fibroblastos/metabolismo , Camundongos , Fator 3 de Transcrição de Octâmero/genética , Regiões Promotoras Genéticas , RNA Longo não Codificante/química , RNA Longo não Codificante/metabolismo , Análise de Sequência de RNA
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