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1.
Tumour Biol ; 41(8): 1010428319869101, 2019 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-31423948

RESUMO

Stemness phenotype mammospheres established from cell lines and tissues taken from autopsy can be used to test and to identify the most sensitive drugs for chemotherapy. Therefore, the aim of the present study was isolation and characterization of cancer stem cells derived from MCF7, MDA-MB231, and SKBR3 breast cancer cell lines to demonstrate the stemness phenotypes of mammospheres generated for further their applications in therapeutic approaches. In this study, two luminal subtypes of cell lines, MCF7 and SKBR3 and a basal subtype cell line, MDA-MB-231, were chosen. Mammosphere culturing was implemented for breast cancer stem cells isolation and mammosphere formation efficiency. At the next step, CD44+/CD24- cell ratio, Oct4 and Nanog mRNA levels, proliferation rate, migration rate of mammospheres, and drug resistance (in third passage) were evaluated. In addition, tumorigenicity of mammospheres in the chick embryo model was evaluated and compared through the chick chorioallantoic membrane assay. Among mammospheres formed in all three cell lines, MCF7 had the highest mammosphere formation efficiency. CD24 marker (a differentiation marker for the breast cancer cells) was significantly reduced in the mammospheres generated from MCF7 and SKBR3, during three passages. Also, Oct4 and Nanog transcript levels were significantly higher in all three types of mammospheres, as compared with their cell lines. Proliferation, migration rate, and drug resistance of mammospheres generated from all three cell lines were found to be significantly higher. Tumorigenicity of MCF7 mammospheres was confirmed through tumor size measurement. Also, tumorigenicity of MCF7 and SKBR3 mammospheres was confirmed through more migration from ectoderm to mesoderm and endoderm. We succeeded to establish the technology that can be extended to tissue in the future. We have demonstrated a number of mammospheres can be generated from cell lines. Also, cells with different molecular features showed different stemness phenotypes.


Assuntos
Biomarcadores Tumorais/genética , Transformação Celular Neoplásica/patologia , Células-Tronco Neoplásicas/patologia , Esferoides Celulares/patologia , Animais , Antígeno CD24/metabolismo , Movimento Celular/fisiologia , Proliferação de Células/fisiologia , Embrião de Galinha , Membrana Corioalantoide/citologia , Resistencia a Medicamentos Antineoplásicos , Humanos , Receptores de Hialuronatos/metabolismo , Células MCF-7 , Proteína Homeobox Nanog/genética , Fator 3 de Transcrição de Octâmero/genética , RNA Mensageiro/genética , Células Tumorais Cultivadas
2.
BMC Cancer ; 19(1): 518, 2019 May 30.
Artigo em Inglês | MEDLINE | ID: mdl-31146720

RESUMO

BACKGROUND: Prostate cancer displays different morphologies which, in turn, affect patient outcome. This fact prompted questions about the lineage relationship between differentiated, more treatable prostate adenocarcinoma and poorly differentiated, less treatable non-adenocarcinoma including small cell carcinoma, and the molecular mechanism underlying prostate cancer differentiation. METHODS: Newly available non-adenocarcinoma/small cell carcinoma PDX LuCaP lines were analyzed for expression of stem cell transcription factors (scTF) LIN28A, NANOG, POU5F1, SOX2, which are responsible for reprogramming or de-differentiation. cDNA of these genes were cloned from small cell carcinoma LuCaP 145.1 into expression vectors to determine if they could function in reprogramming. RESULTS: Expression of scTF was detected in small cell carcinoma LuCaP 93, 145.1, 145.2, and non-adenocarcinoma LuCaP 173.1, 173.2A. Transfection of scTF from LuCaP 145.1 altered the gene expression of prostate non-small cell carcinoma cells, as well as fibroblasts. The resultant cells grew in stem-like colonies. Of note was a 10-fold lower expression of B2M in the transfected cells. Low B2M was also characteristic of LuCaP 145.1. Conversely, B2M was increased when stem cells were induced to differentiate. CONCLUSIONS: This work suggested a pathway in the emergence of non-adenocarcinoma/small cell carcinoma from adenocarcinoma through activation of scTF genes that produced cancer de-differentiation.


Assuntos
Adenocarcinoma/patologia , Carcinoma de Células Pequenas/patologia , Linhagem da Célula/genética , Neoplasias da Próstata/patologia , Adenocarcinoma/genética , Carcinoma de Células Pequenas/genética , Desdiferenciação Celular/genética , Linhagem Celular , Perfilação da Expressão Gênica , Regulação Neoplásica da Expressão Gênica , Humanos , Masculino , Proteína Homeobox Nanog/genética , Células-Tronco Neoplásicas/metabolismo , Células-Tronco Neoplásicas/patologia , Fator 3 de Transcrição de Octâmero/genética , Neoplasias da Próstata/genética , Proteínas de Ligação a RNA/genética , Fatores de Transcrição SOXB1/genética , Microglobulina beta-2/genética
3.
Mol Cell ; 74(3): 622-633.e4, 2019 05 02.
Artigo em Inglês | MEDLINE | ID: mdl-31051141

RESUMO

The control of gene expression by transcription factor binding sites frequently determines phenotype. However, it is difficult to determine the function of single transcription factor binding sites within larger transcription networks. Here, we use deactivated Cas9 (dCas9) to disrupt binding to specific sites, a method we term CRISPRd. Since CRISPR guide RNAs are longer than transcription factor binding sites, flanking sequence can be used to target specific sites. Targeting dCas9 to an Oct4 site in the Nanog promoter displaced Oct4 from this site, reduced Nanog expression, and slowed division. In contrast, disrupting the Oct4 binding site adjacent to Pax6 upregulated Pax6 transcription and disrupting Nanog binding its own promoter upregulated its transcription. Thus, we can easily distinguish between activating and repressing binding sites and examine autoregulation. Finally, multiple guide RNA expression allows simultaneous inhibition of multiple binding sites, and conditionally destabilized dCas9 allows rapid reversibility.


Assuntos
Sistemas CRISPR-Cas/genética , Proteína Homeobox Nanog/genética , Fator 3 de Transcrição de Octâmero/genética , Fator de Transcrição PAX6/genética , Animais , Sítios de Ligação/genética , Proteína 9 Associada à CRISPR/genética , Regulação da Expressão Gênica/genética , Redes Reguladoras de Genes , Humanos , Camundongos , Células-Tronco Embrionárias Murinas/citologia , Células-Tronco Embrionárias Murinas/metabolismo , Regiões Promotoras Genéticas , RNA Guia/genética , Fatores de Transcrição/genética , Ativação Transcricional/genética
4.
J Exp Clin Cancer Res ; 38(1): 182, 2019 May 02.
Artigo em Inglês | MEDLINE | ID: mdl-31046781

RESUMO

BACKGROUND: MicroRNA-139-5p (miR-139) has been shown to play important roles in hepatocellular carcinoma (HCC) development. However, the exact mechanism of miR-139 in HCC remains largely unknown. METHODS: We investigated the function in human cell lines and patient tissue samples by experimental techniques in molecular biology including Co-IP assay, cell viability assay, quantitative real-time-PCR, et al. In addition, datasets were used to verify the results by database analysis. Statistical analysis was performed by using the GraphPad Prism 6 (GraphPad Software Inc., USA). A P value < 0.05 was defined as statistically significant. RESULTS: In this study, we found that miR-139 was significantly down-regulated in HCC. MiR-139 level was negatively associated with the stage of HCC, and HCC patients with higher miR-139 level had longer overall survival (OS) than these having lower miR-139 expression. Overexpression of miR-139 led to reduced cell viability, elevated apoptosis, and decreased colony forming, migratory and invasive capacities in HCC cells, while down-regulation of miR-139 led to opposite phenotypes. MiR-139 also inhibited HCC growth in a xenograft mouse model. We identified karyopherin alpha 2 (KPNA2) as a direct target of miR-139. KPNA2 is up-regulated in HCC and higher KPNA2 level is associated with poor patient prognosis. Silencing of KPNA2 expression led to similar phenotypic changes as miR-139 overexpression. Restoration of KPNA2 attenuated the suppressive effects of miR-139 overexpression on cell viability, apoptosis, colony formation, migration and invasion. In addition, miR-139 overexpression and KPNA2 depletion led to decreased nucleus level of POU class 5 homeobox 1 (POU5F1) and c-myc, two well-known pro-oncogenes. CONCLUSION: In together, these data revealed the essential roles of the miR-139/KPNA2 axis in HCC.


Assuntos
Carcinoma Hepatocelular/genética , Neoplasias Hepáticas/genética , MicroRNAs/genética , alfa Carioferinas/genética , Apoptose/genética , Carcinoma Hepatocelular/patologia , Movimento Celular/genética , Proliferação de Células/genética , Transição Epitelial-Mesenquimal/genética , Feminino , Regulação Neoplásica da Expressão Gênica/genética , Células Hep G2 , Humanos , Neoplasias Hepáticas/patologia , Masculino , Invasividade Neoplásica/genética , Invasividade Neoplásica/patologia , Fator 3 de Transcrição de Octâmero/genética , Proteínas Proto-Oncogênicas c-myc/genética , Transdução de Sinais/genética
5.
Artif Cells Nanomed Biotechnol ; 47(1): 1772-1781, 2019 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-31131631

RESUMO

Since proliferation and differentiation of spermatogonial stem cells (SSCs) in culture system provide successful transplantation in this study, culture of human SSCs was compared to SACS (soft agar culture system), gelatin and control groups. The cells were isolated from seminiferous tubules of non-azoospermia patients (NOA) and cultured in DMEM for 3 weeks. The presence of SSCs in culture system was confirmed by immunocytochemistry of GFR-α1 and ITGα6 antibodies. The proliferated cells were cultured in three mentioned groups in the presence of retinoic acid and Sertoli cells conditioned medium for another 2 weeks. The number of colonies in the SACS group was significantly higher than two other groups. Before 2 weeks of culture, only Oct4 expression was observed in testicular cells (2.32 ± 0.25). After 2 weeks, the expression of Oct4 in the gelatin group was higher than that of the SACS group on day 7. The maximum expression of Stra8 was observed in SACS and gelatin groups after 7 days, but its expression was significantly decreased after 14 days of culture (p < .05). The expression of Scp3 and Acrosin genes were higher after 14 days in the SACS group compared to other groups. SACS has positive effects on proliferation and differentiation of hSSCs.


Assuntos
Células-Tronco Germinativas Adultas/citologia , Células-Tronco Germinativas Adultas/efeitos dos fármacos , Ágar/farmacologia , Técnicas de Cultura de Células/métodos , Diferenciação Celular/efeitos dos fármacos , Proteínas Adaptadoras de Transdução de Sinal/genética , Adulto , Células-Tronco Germinativas Adultas/metabolismo , Regulação da Expressão Gênica/efeitos dos fármacos , Humanos , Masculino , Proteínas Nucleares/genética , Fator 3 de Transcrição de Octâmero/genética , Fatores de Tempo , Adulto Jovem
6.
Intervirology ; 62(1): 9-14, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31104062

RESUMO

OBJECTIVE: The human endogenous retroviruses (HERVs) are endogenous retroviruses that were inserted into the germ cell DNA of humans over 30 million years ago. Insertion of HERVs into the chromosomal DNA can influence a number of host genes in various modes during human evolution and their proviral long terminal repeats can participate in the transcriptional regulation of various cellular genes. Our aim was to evaluate the pol gene expression of HERV-K and HERV-H in mesenchymal stem cells (MSCs) in relation with the expression of stemness genes such as NANOG, OCT-4, and SOX-2. METHODS: MSCs were isolated from bone marrow of healthy donors and expanded until the 5th passage in α-MEM with 10% fetal bovine serum. HERV-K, HERV-H pol gene, NANOG, OCT-4, SOX-2, and GAPDH expression was quantified by real-time PCR in MSCs during the expansion. RESULTS: HERV-K and HERV-H expression was always higher at p1 compared to other passages and this difference reached a high statistical significance when passage p1 was compared with passage 3. In addition, NANOG, OCT-4, and SOX-2 expression at p1 was significantly higher than their expression at p3. Pearson's test demonstrated a strong correlation between the expression of HERV-K and HERV-H and the expression of NANOG, OCT-4, and SOX-2. CONCLUSIONS: Our findings showed that HERV-K and H were concurrently expressed with pluripotency biomarkers NANOG, OCT-4, and SOX-2.


Assuntos
Retrovirus Endógenos/genética , Expressão Gênica , Genes pol , Células-Tronco Mesenquimais/virologia , Biomarcadores , Humanos , Proteína Homeobox Nanog/genética , Fator 3 de Transcrição de Octâmero/genética , Fatores de Transcrição SOXB1/genética
7.
Mol Cell ; 74(4): 651-663.e8, 2019 05 16.
Artigo em Inglês | MEDLINE | ID: mdl-30954402

RESUMO

Accumulating evidence supports the role of the DNA damage response (DDR) in the negative regulation of tumorigenesis. Here, we found that DDR signaling poises a series of epigenetic events, resulting in activation of pro-tumorigenic genes but can go as far as reactivation of the pluripotency gene OCT4. Loss of DNA methylation appears to be a key initiating event in DDR-dependent OCT4 locus reactivation although full reactivation required the presence of a driving oncogene, such as Myc and macroH2A downregulation. Using genetic-lineage-tracing experiments and an in situ labeling approach, we show that DDR-induced epigenetic reactivation of OCT4 regulates the resistance to chemotherapy and contributes to tumor relapse both in mouse and primary human cancers. In turn, deletion of OCT4 reverses chemoresistance and delays the relapse. Here, we uncovered an unexpected tumor-promoting role of DDR in cancer cell reprogramming, providing novel therapeutic entry points for cancer intervention strategies.


Assuntos
Carcinogênese/genética , Metilação de DNA/genética , Neoplasias/genética , Fator 3 de Transcrição de Octâmero/genética , Animais , Reprogramação Celular/genética , Dano ao DNA/genética , Epigênese Genética/genética , Regulação Neoplásica da Expressão Gênica , Histonas/genética , Humanos , Camundongos , Recidiva Local de Neoplasia/genética , Recidiva Local de Neoplasia/patologia , Neoplasias/patologia , Proteínas Proto-Oncogênicas c-myc/genética , Recidiva , Transdução de Sinais/genética
8.
Oncol Rep ; 41(6): 3393-3403, 2019 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-31002352

RESUMO

Scientific evidence linking vitamin D with various cancer types is growing, but the effects of vitamin D on ovarian cancer stem cell­like cells (CSCs) are largely unknown. The present study aimed to examine whether vitamin D was able to restrain the stemness of ovarian cancer. A side population (SP) from malignant ovarian surface epithelial cells was identified as CSCs, in vitro and in vivo. Furthermore, 1α,25­dihydroxyvitamin D3 [1α,25(OH)2D3] treatment inhibited the self­renewal capacity of SP cells by decreasing the sphere formation rate and by suppressing the mRNA expression levels of cluster of differentiation CD44, NANOG, OCT4, SOX2, Krüppel­like factor 4 and adenosine triphosphate binding cassette subfamily G member 2. Additionally, 1α,25(OH)2D3 treatment decreased the expression of Cyclin D1, whereas it increased the expression of ß­catenin and vitamin D receptor (VDR). Notably, immunofluorescence staining verified that 1α,25(OH)2D3 promoted the expression of ß­catenin in the cytoplasm. Furthermore, vitamin D3 delayed the onset of tumor formation derived from injection of ovarian CSCs to nude mice, by reducing CD44 and enhancing ß­catenin expressions in vivo. In conclusion, 1α,25(OH)2D3 restrains the stem cell­like properties of ovarian cancer cells by enhancing the expression of VDR, by promoting the expression of ß­catenin in the cytoplasm, and by suppressing the expression of CD44. These findings provide a novel insight into the functions of vitamin D in diminishing the stemness of cancer CSCs.


Assuntos
Neoplasias Ovarianas/tratamento farmacológico , Receptores de Calcitriol/genética , Vitamina D/análogos & derivados , Vitamina D/genética , Autorrenovação Celular/efeitos dos fármacos , Feminino , Regulação Neoplásica da Expressão Gênica , Humanos , Receptores de Hialuronatos/genética , Fatores de Transcrição Kruppel-Like/genética , Proteína Homeobox Nanog/genética , Células-Tronco Neoplásicas/efeitos dos fármacos , Células-Tronco Neoplásicas/patologia , Fator 3 de Transcrição de Octâmero/genética , Neoplasias Ovarianas/genética , Neoplasias Ovarianas/patologia , Fatores de Transcrição SOXB1/genética , Vitamina D/metabolismo , Vitamina D/farmacologia , beta Catenina/genética
9.
Cell Prolif ; 52(4): e12612, 2019 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-31012189

RESUMO

OBJECTIVE: POU5F1 (OCT4) is implicated in cancer stem cell self-renewal. Currently, some studies have shown that OCT4 has a dual function in suppressing or promoting cancer progression. However, the precise molecular mechanism of OCT4 in breast cancer progression remains unclear. MATERIALS AND METHODS: RT-PCR and Western blot were utilized to investigate OCT4 expression in breast cancer tissues and cells. Cell proliferation assays and mouse models were applied to determine the effects of OCT4 on breast cancer cell proliferation. DNMT1 inhibitors, ChIP, CoIP, IHC and ERα inhibitors were used to explore the molecular mechanism of OCT4 in breast cancer. RESULTS: OCT4 was down-regulated in breast cancer tissues, and the overexpression of OCT4 promoted MDA-MB-231 cell proliferation and inhibited the proliferation of MCF-7 cells in vitro and in vivo, respectively. Two DNMT1 inhibitors (5-aza-dC and zebularine) suppressed OCT4-induced MDA-MB-231 cell proliferation through Ras/Raf1/ERK inactivation by targeting ISL1, which is the downstream of DNMT1. In contrast, OCT4 interacted with ERα, decreased DNMT1 expression and inactivated the Ras/Raf1/ERK signalling pathway in MCF-7 cells. Moreover, ERα inhibitor (AZD9496) reversed the suppression of OCT4-induced proliferation in MCF-7 cells via the activation of ERK signalling pathway. CONCLUSIONS: OCT4 is dependent on ERα to suppress the proliferation of breast cancer cells through DNMT1/ISL1/ERK axis.


Assuntos
Neoplasias da Mama/genética , Proliferação de Células/genética , DNA (Citosina-5-)-Metiltransferase 1/genética , Receptor alfa de Estrogênio/genética , Proteínas com Homeodomínio LIM/genética , Sistema de Sinalização das MAP Quinases/genética , Fator 3 de Transcrição de Octâmero/genética , Fatores de Transcrição/genética , Animais , Linhagem Celular Tumoral , Regulação para Baixo/genética , Feminino , Regulação Neoplásica da Expressão Gênica/genética , Células HEK293 , Humanos , Células MCF-7 , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Transdução de Sinais/genética
10.
DNA Cell Biol ; 38(5): 410-422, 2019 May.
Artigo em Inglês | MEDLINE | ID: mdl-30896984

RESUMO

Trophoblast stem cells (TSCs), the precursors of placental cells, are effective for studying placental formation in vitro. Using a dual inhibition (2i) medium and mixed L-Wnt3a/mouse embryonic fibroblast feeder cells, we previously established the bovine trophoblast cell line BTS-1. In this study, we used bovine fetal fibroblasts and added Wnt3a to the 2i medium to establish another bovine TSC line (BTSW). BTSW cells expressed pluripotency markers, including NANOG, SOX2, OCT4, TRA-1-60, TRA-1-81, SSEA4, CDH1, and KRT18, and TSC markers CDX2, TEAD4, and ESRRB. Methylation sequencing of the promoter regions of NANOG, OCT4, and CDX2 revealed no significant differences between BTS-1 and BTSW cells. Removal of Wnt3a from the culture medium resulted in downregulation (p < 0.05) of NANOG, OCT4, CDX2, and TSC marker genes, and upregulation of TSC differentiation markers, including MASH2, GCM1, and PAG. Western blotting indicated activation of the WNT-YAP/TAZ signaling pathway in BTS-1 and BTSW cells, consequently activating TEAD4 transcription. However, this pathway was not activated in BCFF cells, an established bovine embryonic stem-like cell line that expresses OCT4, SOX2, and NANOG, but not CDX2. Thus, Wnt3a may play a critical role in bovine TSC maintenance by activating and regulating CDX2 expression through the WNT-YAP/TAZ signaling pathway.


Assuntos
Fator de Transcrição CDX2/metabolismo , Células-Tronco Embrionárias/citologia , Fator 3 de Transcrição de Octâmero/genética , Trofoblastos/citologia , Via de Sinalização Wnt/fisiologia , Proteína Wnt3/metabolismo , Animais , Fator de Transcrição CDX2/genética , Bovinos , Linhagem Celular , Linhagem da Célula
11.
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
12.
Nat Commun ; 10(1): 1368, 2019 03 25.
Artigo em Inglês | MEDLINE | ID: mdl-30911006

RESUMO

Long noncoding RNAs (lncRNAs) have emerged as important components of gene regulatory network in embryonic stem cells (ESCs). However, the function and molecular mechanism of lncRNAs are still largely unknown. Here we identifies Trincr1 (TRIM71 interacting long noncoding RNA 1) lncRNA that regulates the FGF/ERK signaling and self-renewal of ESCs. Trincr1 is exported by THOC complex to cytoplasm where it binds and represses TRIM71, leading to the downregulation of SHCBP1 protein. Knocking out Trincr1 leads to the upregulation of phosphorylated ERK and ERK pathway target genes and the decrease of ESC self-renewal, while knocking down Trim71 completely rescues the defects of Trincr1 knockout. Furthermore, ectopic expression of Trincr1 represses FGF/ERK signaling and the self-renewal of neural progenitor cells (NPCs). Together, this study highlights lncRNA as an important player in cell signaling network to coordinate cell fate specification.


Assuntos
Fatores de Crescimento de Fibroblastos/genética , Proteína Quinase 1 Ativada por Mitógeno/genética , Proteína Quinase 3 Ativada por Mitógeno/genética , Células-Tronco Embrionárias Murinas/metabolismo , RNA Longo não Codificante/genética , Fatores de Transcrição/genética , Animais , Diferenciação Celular , Proliferação de Células , Células Cultivadas , Embrião de Mamíferos , Fatores de Crescimento de Fibroblastos/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Redes Reguladoras de Genes , Sistema de Sinalização das MAP Quinases , Camundongos , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Proteína Quinase 3 Ativada por Mitógeno/metabolismo , Células-Tronco Embrionárias Murinas/citologia , Células-Tronco Neurais/citologia , Células-Tronco Neurais/metabolismo , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Fator 3 de Transcrição de Octâmero/genética , Fator 3 de Transcrição de Octâmero/metabolismo , Fosforilação , Ligação Proteica , RNA Longo não Codificante/metabolismo , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , Proteínas Adaptadoras da Sinalização Shc/genética , Proteínas Adaptadoras da Sinalização Shc/metabolismo , Fatores de Transcrição/antagonistas & inibidores , Fatores de Transcrição/metabolismo
13.
Nat Commun ; 10(1): 967, 2019 02 27.
Artigo em Inglês | MEDLINE | ID: mdl-30814500

RESUMO

The stem cell pluripotency factor Oct4 serves a critical protective role during atherosclerotic plaque development by promoting smooth muscle cell (SMC) investment. Here, we show using Myh11-CreERT2 lineage-tracing with inducible SMC and pericyte (SMC-P) knockout of Oct4 that Oct4 regulates perivascular cell migration and recruitment during angiogenesis. Knockout of Oct4 in perivascular cells significantly impairs perivascular cell migration, increases perivascular cell death, delays endothelial cell migration, and promotes vascular leakage following corneal angiogenic stimulus. Knockout of Oct4 in perivascular cells also impairs perfusion recovery and decreases angiogenesis following hindlimb ischemia. Transcriptomic analyses demonstrate that expression of the migratory gene Slit3 is reduced following loss of Oct4 in cultured SMCs, and in Oct4-deficient perivascular cells in ischemic hindlimb muscle. Together, these results provide evidence that Oct4 plays an essential role within perivascular cells in injury- and hypoxia-induced angiogenesis.


Assuntos
Neovascularização Fisiológica , Fator 3 de Transcrição de Octâmero/deficiência , Células-Tronco Pluripotentes/metabolismo , Animais , Morte Celular , Linhagem da Célula , Movimento Celular , Células Cultivadas , Neovascularização da Córnea/metabolismo , Neovascularização da Córnea/patologia , Feminino , Membro Posterior , Isquemia/metabolismo , Isquemia/patologia , Masculino , Proteínas de Membrana/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Miócitos de Músculo Liso/metabolismo , Neovascularização Patológica , Fator 3 de Transcrição de Octâmero/genética , Pericitos/metabolismo , Pericitos/patologia , Células-Tronco Pluripotentes/patologia
14.
EBioMedicine ; 41: 427-442, 2019 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-30827930

RESUMO

BACKGROUND: Transcriptional dysregulation drives cancer formation but the underlying mechanisms are still poorly understood. Renal cell carcinoma (RCC) is the most common malignant kidney tumor which canonically activates the hypoxia-inducible transcription factor (HIF) pathway. Despite intensive study, novel therapeutic strategies to target RCC have been difficult to develop. Since the RCC epigenome is relatively understudied, we sought to elucidate key mechanisms underpinning the tumor phenotype and its clinical behavior. METHODS: We performed genome-wide chromatin accessibility (DNase-seq) and transcriptome profiling (RNA-seq) on paired tumor/normal samples from 3 patients undergoing nephrectomy for removal of RCC. We incorporated publicly available data on HIF binding (ChIP-seq) in a RCC cell line. We performed integrated analyses of these high-resolution, genome-scale datasets together with larger transcriptomic data available through The Cancer Genome Atlas (TCGA). FINDINGS: Though HIF transcription factors play a cardinal role in RCC oncogenesis, we found that numerous transcription factors with a RCC-selective expression pattern also demonstrated evidence of HIF binding near their gene body. Examination of chromatin accessibility profiles revealed that some of these transcription factors influenced the tumor's regulatory landscape, notably the stem cell transcription factor POU5F1 (OCT4). Elevated POU5F1 transcript levels were correlated with advanced tumor stage and poorer overall survival in RCC patients. Unexpectedly, we discovered a HIF-pathway-responsive promoter embedded within a endogenous retroviral long terminal repeat (LTR) element at the transcriptional start site of the PSOR1C3 long non-coding RNA gene upstream of POU5F1. RNA transcripts are induced from this promoter and read through PSOR1C3 into POU5F1 producing a novel POU5F1 transcript isoform. Rather than being unique to the POU5F1 locus, we found that HIF binds to several other transcriptionally active LTR elements genome-wide correlating with broad gene expression changes in RCC. INTERPRETATION: Integrated transcriptomic and epigenomic analysis of matched tumor and normal tissues from even a small number of primary patient samples revealed remarkably convergent shared regulatory landscapes. Several transcription factors appear to act downstream of HIF including the potent stem cell transcription factor POU5F1. Dysregulated expression of POU5F1 is part of a larger pattern of gene expression changes in RCC that may be induced by HIF-dependent reactivation of dormant promoters embedded within endogenous retroviral LTRs.


Assuntos
Retrovirus Endógenos/genética , Epigenômica , Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Sítios de Ligação , Carcinoma de Células Renais/genética , Carcinoma de Células Renais/mortalidade , Carcinoma de Células Renais/patologia , Linhagem Celular Tumoral , Redutases do Citocromo/genética , Retrovirus Endógenos/fisiologia , Regulação Neoplásica da Expressão Gênica , Humanos , Fator 1 Induzível por Hipóxia/genética , Neoplasias Renais/genética , Neoplasias Renais/mortalidade , Neoplasias Renais/patologia , Fator 3 de Transcrição de Octâmero/genética , Fator 3 de Transcrição de Octâmero/metabolismo , Diester Fosfórico Hidrolases/genética , Regiões Promotoras Genéticas , Proteínas/genética , Pirofosfatases/genética , Taxa de Sobrevida , Sequências Repetidas Terminais/genética , Enzimas de Conjugação de Ubiquitina/genética
15.
Int J Mol Sci ; 20(5)2019 Mar 04.
Artigo em Inglês | MEDLINE | ID: mdl-30836626

RESUMO

A deeper understanding of the detailed mechanism of in vivo tissue healing is necessary for the development of novel regenerative therapies. Among several external factors, environmental pH is one of the crucial parameters that greatly affects enzyme activity and cellular biochemical reactions involving tissue repair and homeostasis. In this study, in order to analyze the microenvironmental conditions during bone healing, we first measured the pH in vivo at the bone healing site using a high-resolution fiber optic pH microsensor directly in femur defects and tooth extraction sockets. The pH was shown to decrease from physiological 7.4 to 6.8 during the initial two days of healing (inflammatory phase). In the same initial stages of the inflammatory phase of the bone healing process, mesenchymal stem cells (MSCs) are known to migrate to the healing site to contribute to tissue repair. Therefore, we investigated the effect of a short-term acidic (pH 6.8) pre-treatment on the stemness of bone marrow-derived MSCs (BMSCs). Interestingly, the results showed that pre-treatment of BMSCs with acidic pH enhances the expression of stem cell markers (OCT-4, NANOG, SSEA-4), as well as cell viability and proliferation. On the other hand, acidic pH decreased BMSC migration ability. These results indicate that acidic pH during the initial stages of bone healing is important to enhance the stem cell properties of BMSCs. These findings may enable the development of novel methods for optimization of stem cell function towards tissue engineering or regenerative medicine.


Assuntos
Ácidos/farmacologia , Regeneração Óssea/genética , Osteogênese/efeitos dos fármacos , Engenharia Tecidual/métodos , Animais , Células da Medula Óssea/citologia , Células da Medula Óssea/efeitos dos fármacos , Regeneração Óssea/efeitos dos fármacos , Diferenciação Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Microambiente Celular/efeitos dos fármacos , Humanos , Concentração de Íons de Hidrogênio/efeitos dos fármacos , Transplante de Células-Tronco Mesenquimais , Células-Tronco Mesenquimais/citologia , Células-Tronco Mesenquimais/efeitos dos fármacos , Camundongos , Proteína Homeobox Nanog/genética , Fator 3 de Transcrição de Octâmero/genética , Medicina Regenerativa , Antígenos Embrionários Estágio-Específicos/genética , Células-Tronco/citologia , Células-Tronco/efeitos dos fármacos , Cicatrização/genética
16.
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
17.
Int J Pharm ; 562: 151-161, 2019 May 01.
Artigo em Inglês | MEDLINE | ID: mdl-30853482

RESUMO

Bone tissue engineering is an emerging medical field that has been developed in recent years to address pathologies with limited ability of bones to regenerate. Here we report the fabrication and characterization of microbial transglutaminase crosslinked gelatin-based scaffolds designed for serving as both cell substrate and growth factor release system. In particular, morphological, biomechanical and biological features have been analyzed. The enzyme ratio applied during the fabrication of the scaffolds affects the swelling capacity and the mechanical properties of the final structure. The developed systems are not cytotoxic according to the biocompatibility tests. The biological performance of selected formulations was studied using L-929 fibroblasts, D1 MSC and MG63 osteoblasts. Moreover, scaffolds allowed efficient osteogenic differentiation and signaling of MSCs. MSC cultured on the scaffolds not only presented lower proliferative and stemness profile, but also increased expression of osteoblast-related genes (Col1a1, Runx2, Osx). Furthermore, the in vitro release kinetics of vascular endothelial growth factor (VEGF) and bone morphogenetic protein -2 (BMP-2) from the scaffolds were also investigated. The release of the growth factors produced from the scaffolds followed a first order kinetics. These results highlight that the scaffolds designed and developed in this work may be suitable candidates for bone tissue regeneration purposes.


Assuntos
Gelatina/química , Tecidos Suporte , Transglutaminases/química , Animais , Proteína Morfogenética Óssea 2/química , Osso e Ossos , Linhagem Celular , Liberação Controlada de Fármacos , Humanos , Camundongos , Proteína Homeobox Nanog/genética , Fator 3 de Transcrição de Octâmero/genética , Fator de Transcrição Sp7/genética , Engenharia Tecidual , Fator A de Crescimento do Endotélio Vascular/química
18.
Nucleic Acids Res ; 47(9): 4449-4461, 2019 05 21.
Artigo em Inglês | MEDLINE | ID: mdl-30838422

RESUMO

HMGN proteins localize to chromatin regulatory sites and modulate the cell-type specific transcription profile; however, the molecular mechanism whereby these ubiquitous nucleosome binding proteins affect gene expression is not fully understood. Here, we show that HMGNs regulate the expression of Rex1, one of the most highly transcribed genes in mouse embryonic stem cells (ESCs), by recruiting the transcription factors NANOG, OCT4 and SOX2 to an ESC-specific super enhancer located in the 5' region of Rex1. HMGNs facilitate the establishment of an epigenetic landscape characteristic of active chromatin and enhancer promoter interactions, as seen by chromatin conformation capture. Loss of HMGNs alters the local epigenetic profile, increases histone H1 occupancy, decreases transcription factors binding and reduces enhancer promoter interactions, thereby downregulating, but not abolishing Rex1 expression. ChIP-seq analyses show high colocalization of HMGNs and of REX1, a zinc finger protein, at promoters and enhancers. Loss of HMGNs preferentially reduces the specific binding of REX1 to these chromatin regulatory sites. Thus, HMGNs affects both the expression and the chromatin binding specificity of REX1. We suggest that HMGNs affect cell-type specific gene expression by modulating the binding specificity of transcription factors to chromatin.


Assuntos
Cromatina/genética , Epigênese Genética , Proteínas HMGN/genética , Fatores de Transcrição/genética , Animais , Sítios de Ligação/genética , Regulação da Expressão Gênica/genética , Proteínas HMGN/química , Histonas/genética , Camundongos , Células-Tronco Embrionárias Murinas , Proteína Homeobox Nanog/genética , Nucleossomos/genética , Fator 3 de Transcrição de Octâmero/genética , Regiões Promotoras Genéticas , Ligação Proteica/genética , Sequências Reguladoras de Ácido Nucleico/genética , Fatores de Transcrição SOXB1/genética
19.
Chem Commun (Camb) ; 55(18): 2672-2675, 2019 Feb 26.
Artigo em Inglês | MEDLINE | ID: mdl-30746545

RESUMO

Replacing expensive and thermally unstable growth factors with synthetic alternatives has been an important issue in stem cell-based regenerative medicines. Here we developed DNA aptamer-assemblies that act as functional mimics of basic fibroblast growth factor (bFGF), one of the essential factors for stem cell culture. The most potent aptamer assembly named TD0, composed solely of 76-mer single-stranded DNA, could support the self-renewal and pluripotency of induced pluripotent stem cells (iPSCs). This work presents the first application of DNA aptamer in the maintenance of iPSCs.


Assuntos
Aptâmeros de Nucleotídeos/química , Materiais Biocompatíveis/química , Aptâmeros de Nucleotídeos/metabolismo , Aptâmeros de Nucleotídeos/farmacologia , Materiais Biocompatíveis/metabolismo , Materiais Biocompatíveis/farmacologia , Proliferação de Células/efeitos dos fármacos , Células Cultivadas , Fatores de Crescimento de Fibroblastos/química , Fatores de Crescimento de Fibroblastos/farmacologia , Humanos , Células-Tronco Pluripotentes Induzidas/citologia , Células-Tronco Pluripotentes Induzidas/efeitos dos fármacos , Células-Tronco Pluripotentes Induzidas/metabolismo , Fator 3 de Transcrição de Octâmero/genética , Fator 3 de Transcrição de Octâmero/metabolismo , Fosforilação , Ligação Proteica , Receptor Tipo 1 de Fator de Crescimento de Fibroblastos/agonistas , Receptor Tipo 1 de Fator de Crescimento de Fibroblastos/metabolismo , Regulação para Cima/genética
20.
Cell Prolif ; 52(3): e12574, 2019 May.
Artigo em Inglês | MEDLINE | ID: mdl-30724402

RESUMO

Induced pluripotent stem cells (iPSCs) are reprogrammed somatic cells that gained self-renewal and differentiation capacity similar to embryonic stem cells. Taking the precious opportunity of the TianZhou-1 spacecraft mission, we studied the effect of space microgravity (µg) on the self-renewal capacity of iPSCs. Murine iPSCs carrying pluripotency reporter Oct4-GFP were used. The Oct4-EGFP-iPSCs clones were loaded into the bioreactor and exposed to µg in outer space for 14 days. The control experiment was performed in identical device but on the ground in earth gravity (1 g). iPSCs clones were compact and highly expressed Oct4 before launch. In µg condition, cells in iPSC clones spread out more rapidly than those in ground 1 g condition during the first 3 days after launch. However, in 1 g condition, as the cell density increases, the Oct4-GFP signal dropped significantly during the following 3 days. Interestingly, in µg condition, iPSCs originated from the spread-out clones during the first 3 days appeared to cluster together and reform colonies that activated strong Oct4 expression. On the other hand, iPSC clones in 1 g condition were not able to recover Oct4 expression after overgrown. Our study for the first time performed real-time imaging on the proliferation process of iPSCs in space and found that in µg condition, cell behaviour appeared to be more dynamic than on the ground.


Assuntos
Células-Tronco Pluripotentes Induzidas/citologia , Células-Tronco Pluripotentes Induzidas/fisiologia , Voo Espacial , Ausência de Peso , Animais , Reatores Biológicos , Proliferação de Células , Autorrenovação Celular , Células Clonais , Sistemas de Computação , Camundongos , Camundongos Transgênicos , Fator 3 de Transcrição de Octâmero/genética , Fator 3 de Transcrição de Octâmero/metabolismo , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Regeneração
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