RESUMEN
Long non-coding RNAs (lncRNAs) are nucleotide sequences that participate in different biological processes and are associated with different pathologies, including cancer. Long intergenic non-protein-coding RNA 662 (LINC00662) has been reported to be involved in different cancers, including colorectal, prostate, and breast cancer. However, its role in gallbladder cancer has not yet been described. In this article, we hypothesize that LINC00662 has an important role in the acquisition of aggressiveness traits such as a stem-like phenotype, invasion, and chemoresistance in gallbladder cancer. Here, we show that LINC00662 is associated with larger tumor size and lymph node metastasis in patients with gallbladder cancer. Furthermore, we show that the overexpression of LINC00662 promotes an increase in CD133+/CD44+ cell populations and the expression of stemness-associated genes. LINC00662 promotes greater invasive capacity and the expression of genes associated with epithelial-mesenchymal transition. In addition, the expression of LINC00662 promotes resistance to cisplatin and 5-fluorouracil, associated with increased expression of chemoresistance-related ATP-binding cassette (ABC) transporters in gallbladder cancer (GBC) cell lines. Finally, we show that the mechanism by which LINC00662 exerts its function is through a decrease in microRNA 335-5p (miR-335-5p) and an increase in octamer-binding transcription factor 4 (OCT4) in GBC cells. Thus, our data allow us to propose LINC00662 as a biomarker of poor prognosis and a potential therapeutic target for patients with GBC.
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Neoplasias de la Vesícula Biliar , Regulación Neoplásica de la Expresión Génica , MicroARNs , Factor 3 de Transcripción de Unión a Octámeros , ARN Largo no Codificante , Humanos , Neoplasias de la Vesícula Biliar/genética , Neoplasias de la Vesícula Biliar/patología , Neoplasias de la Vesícula Biliar/metabolismo , MicroARNs/genética , ARN Largo no Codificante/genética , ARN Largo no Codificante/metabolismo , Línea Celular Tumoral , Factor 3 de Transcripción de Unión a Octámeros/genética , Factor 3 de Transcripción de Unión a Octámeros/metabolismo , Femenino , Transición Epitelial-Mesenquimal/genética , Resistencia a Antineoplásicos/genética , Masculino , Invasividad Neoplásica , Cisplatino/farmacología , Persona de Mediana Edad , Células Madre Neoplásicas/metabolismo , Células Madre Neoplásicas/patología , Fluorouracilo/farmacología , Metástasis LinfáticaRESUMEN
In birds, primordial germ cells (PGCs) use the bloodstream to travel to a specific region, where the cells undergo extravasation followed by intrastromal migration to the gonadal crest for further colonization. Currently, DDX4, SSEA1, and Oct4 are used to identify germ cells. Other germline cell-associated molecules are N-cadherin, GnRHR, and 3ß hydroxysteroid dehydrogenase (3ßHSD), which have been used in mice and birds during gonadal development; however, its role in early gonadogenesis in birds is poorly described. This study aimed to evaluate the differential immunodetection of N-cadherin binding molecule, Oct4 pluripotency protein, GnRHR receptor, and 3ßHSD enzyme in Columba livia embryos during migration colonization of PGCs in the gonadal crest and early gonadogenesis. These markers were revealed by immunohistochemistry in histological preparations of C. livia corresponding to stages (S)15 to S40. Immunodetection of N-cadherin, Oct4, GnRHR, and 3ßHSD in the germ line of C. livia allowed the identification of PGCs in the yolk sac membrane at the level of the splanchnic mesoderm during migration to the genital crest and its colonization. In the same way, it was possible to characterize and localize PGCs during early gonadogenesis. This study in C. livia demonstrates that Oct4, N-cadherin, GNRHR, and 3ßHSD are immunodetected in PGCs and could be used as potential germline cell markers during cell migration out of blood vessels, colonization in the genital crest, and early gonadogenesis. Furthermore, this study could be used as a novel general model to understand the early gonadogenesis in altricial species.
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Columbidae , Columbiformes , Animales , Ratones , Células Germinativas/metabolismo , Diferenciación Celular , Movimiento Celular , Cadherinas/metabolismoRESUMEN
Introducción: El cáncer escamocelular de cavidad oral es una patología con bajas tasas de sobrevivencia. Cuando no es tratado adecuadamente es un tumor de alta recurrencia y resistente al tratamiento. Nuevas hipótesis plantean que las células tumorales progenitoras por sus propiedades de auto renovación, iniciación tumoral, migración y metástasis pueden ser responsables de la manutención y renovación de este tumor. Sin embargo, aún no existe un consenso sobre la verdadera participación de ellas, debido a que su identificación y caracterización es aún un reto experimental. Objetivo: En este trabajo se busca detectar células con expresión de marcadores de células tumorales Progenitoras en muestras cáncer escamocelular de cavidad oral y relacionarlo con los estadios de diferenciación del tumor. Metodología: En esta investigación se tomaron 32 muestras de pacientes con carcinoma escamocelular de cavidad oral. Se logró detectar in situ, mediante la técnica de inmunofluorescencia, cuatro reconocidos marcadores de células tumorales progenitoras. Resultados: Se identificaron los marcadores OCT4, SSEA4, NANOG y TRA-1-60 en los diferentes estadios de diferenciación tumoral, lo que sugiere la participación de las células progenitoras tumorales en la evolución de esta patología. Conclusiones: El establecimiento y correcta identificación de las células tumorales progenitoras abre nuevas vías terapéuticas para el abordaje de este tumor, en busca de mejorar el pronóstico, tasa de sobrevivencia y calidad de vida del paciente.
Introduction: Squamous cell carcinoma of the oral cavity is a pathology with poor survival rates. When it is not adequately treated, it is a tumor with high recurrence and resistance to treatment. According to new hypotheses, progenitor tumor cells, due to their properties of self-renewal, tumor initiation, migration, and metastasis, could be responsible for the maintenance and renewal of this tumor. However, there is still no consensus on their true participation, subsequent to difficult in their identification and characterization. Materials and methods: In this research, 32 samples provided from patients diagnosis with squamous cell carcinoma of the oral cavity were used. To detect specific markers progenitor tumor cells were used immunofluorescence microscopy. Results: The cells markers OCT4, SSEA4, NANOG and TRA-1-60 were identified in the different stages of the tumor samples, all these findings suggest the role of tumor progenitor cells in the evolution of this pathology. Conclusions: The establishment and correct identification of the progenitor tumor cells provide new therapeutic options for the approach of this tumor seeking to improve the prognosis, survival rate and quality of life of the patient.
RESUMEN
Mechanisms of cell reprogramming by pluripotency-related transcription factors or nuclear transfer seem to be mediated by similar pathways, and the study of the contribution of OCT4 and SOX2 in both processes may help elucidate the mechanisms responsible for pluripotency. Bovine fibroblasts expressing exogenous OCT4 or SOX2, or both, were analyzed regarding the expression of pluripotency factors and imprinted genes H19 and IGF2R, and used for in vitro reprogramming. The expression of the H19 gene was increased in the control sorted group, and putative iPSC-like cells were obtained when cells were not submitted to cell sorting. When sorted cells expressing OCT4, SOX2, or none (control) were used as donor cells for somatic cell nuclear transfer, fusion rates were 60.0% vs. 64.95% and 70.53% vs. 67.24% for SOX2 vs. control and OCT4 vs. control groups, respectively; cleavage rates were 66.66% vs. 81.68% and 86.47% vs. 85.18%, respectively; blastocyst rates were 33.05% vs. 44.15% and 52.06% vs. 44.78%, respectively. These results show that the production of embryos by NT resulted in similar rates of in vitro developmental competence compared to control cells regardless of different profiles of pluripotency-related gene expression presented by donor cells; however, induced reprogramming was compromised after cell sorting.
RESUMEN
The transcription factors Oct4, Sox2, Klf4, and c-Myc enable the reprogramming of somatic cells into induced pluripotent cells. Reprogramming generates newly differentiated cells for potential therapies in cancer, neurodegenerative diseases, and rejuvenation processes. In cancer therapies, these transcription factors lead to a reduction in the size and aggressiveness of certain tumors, such as sarcomas, and in neurodegenerative diseases, they enable the production of dopaminergic cells in Parkinson's disease, the replacement of affected neuronal cells in olivopontocerebellar atrophy, and the regeneration of the optic nerve. However, there are limitations, such as an increased risk of cancer development when using Klf4 and c-Myc and the occurrence of abnormal dyskinesias in the medium term, possibly generated by the uncontrolled growth of differentiated dopaminergic cells and the impairment of the survival of the new cells. Therefore, the Yamanaka transcription factors have shown therapeutic potential through cell reprogramming for some carcinomas, neurodegenerative diseases, and rejuvenation. However, the limitations found in the studies require further investigation before the use of these transcription factors in humans.
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Carcinoma , Sarcoma , Humanos , Agresión , Diferenciación Celular/genética , Laboratorios , Factor 3 de Transcripción de Unión a Octámeros/genética , Factor 4 Similar a Kruppel , Factores de Transcripción SOXB1 , Proteínas Proto-Oncogénicas c-mycRESUMEN
Rectal cancer (RC) is one of the most common malignant neoplasms, and cancer stem cells (CSCs) of the intestinal tract have been implicated in its origin. The oncofetal protein OCT4 has been linked to neoplastic processes, but its role and clinical significance in RC are unknown. This study investigates the expression of the stem cell marker OCT4 related to clinical-pathological characteristics and its clinical significance in RC patients. The expression level of stem cell marker OCT4 was analyzed in 22 primary rectal tumors by western blot. The association between OCT4 protein expression and the clinical-pathological features of tumors was evaluated by χ2 test and Fisher's exact test. We demonstrated that the expression of the stem cell marker OCT4 was observed in tumor tissue but not adjacent non-tumor tissue. High expression of the stem cell marker OCT4 was significantly associated with histological differentiation grade (p = 0.039), tumor invasion level (p = 0.004), lymph node involvement (p = 0.044), tumor-node-metastasis (TNM) stage (p = 0.002), and clinical stage (p = 0.021). These findings suggest that high OCT4 expression is associated with a more aggressive RC phenotype, with a greater likelihood of progression and metastasis. These results shed light on the importance of targeting this CSC marker to attenuate RC progression.
RESUMEN
Quantitative real-time polymerase chain reaction (qRT-PCR) flexibility, robustness and reproducibility have rapidly extended the scope of the method. Cancer stem cells are gaining increasing importance since their role in cancer initiation, treatment resistance and recurrence give rise to a wide range of potential diagnostic and therapeutic applications. The expression of several characteristic markers is proven a reliable method to assess stem-like-phenotype of cancer cells. Here, we provided a thorough protocol for the study of cancer stem cells in hepatocellular carcinoma mouse models and cell cultures using qRT-PCR.
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Carcinoma Hepatocelular , Neoplasias Hepáticas , Antígeno AC133/genética , Antígeno AC133/metabolismo , Animales , Carcinoma Hepatocelular/genética , Carcinoma Hepatocelular/metabolismo , Carcinoma Hepatocelular/patología , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/patología , Ratones , Células Madre Neoplásicas/patología , Reacción en Cadena en Tiempo Real de la Polimerasa , Reproducibilidad de los ResultadosRESUMEN
BACKGROUND: The cytoskeleton is a key component of the system responsible for transmitting mechanical cues from the cellular environment to the nucleus, where they trigger downstream responses. This communication is particularly relevant in embryonic stem (ES) cells since forces can regulate cell fate and guide developmental processes. However, little is known regarding cytoskeleton organization in ES cells, and thus, relevant aspects of nuclear-cytoskeletal interactions remain elusive. RESULTS: We explored the three-dimensional distribution of the cytoskeleton in live ES cells and show that these filaments affect the shape of the nucleus. Next, we evaluated if cytoskeletal components indirectly modulate the binding of the pluripotency transcription factor OCT4 to chromatin targets. We show that actin depolymerization triggers OCT4 binding to chromatin sites whereas vimentin disruption produces the opposite effect. In contrast to actin, vimentin contributes to the preservation of OCT4-chromatin interactions and, consequently, may have a pro-stemness role. CONCLUSIONS: Our results suggest roles of components of the cytoskeleton in shaping the nucleus of ES cells, influencing the interactions of the transcription factor OCT4 with the chromatin and potentially affecting pluripotency and cell fate.
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Actinas , Cromatina , Actinas/metabolismo , Diferenciación Celular , Cromatina/metabolismo , Citoesqueleto/metabolismo , Células Madre Embrionarias/metabolismo , Factor 3 de Transcripción de Unión a Octámeros/genética , Factor 3 de Transcripción de Unión a Octámeros/metabolismo , Vimentina/metabolismoRESUMEN
BACKGROUND: Human endometrium harbors stem/progenitor cells (SPCs) that may contribute to the establishment of endometriosis when seeded outside the uterus. Oct-4, C-kit and Musashi-1 are some of the many proteins used to characterize SPCs, but their association with endometriosis is uncertain. OBJECTIVE AND DESIGN: In this study, specimens of normal endometrium (n = 12), eutopic endometrium from women with endometriosis (n = 9), superficial peritoneal endometriosis (SUP, n = 12) and deep endometriosis (DE, n = 13) lesions were evaluated for localization and intensity of immunostaining for Oct-4, C-kit and Musashi-1. RESULTS: The three markers were abundantly expressed in normal endometrium, eutopic endometrium from endometriosis patients, SUP and DE specimens. Oct-4 and C-kit expression did not vary across groups as regards intensity or frequency. C-kit staining signal was seldom detected in vascular endothelium of normal or eutopic endometrium from endometriosis patients; however, it was positive in 67% of the SUP lesions and in 25% of the DE lesions (p = 0.042). Musashi-1 was expressed in some endometriotic glands as cell clusters, but its signal was similar between the four types of tissue (p = 0.971) CONCLUSION: The wide distribution of Oct-4, C-kit and Musashi-1 in endometria of patients with and without endometriosis and in SUP and DE endometriotic lesions suggests that these markers are not suitable for the in situ characterization of endometrial SPCs and should not be taken as surrogates for the study of SPCs in the pathogenesis of endometriosis.
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Endometriosis/metabolismo , Proteínas del Tejido Nervioso/metabolismo , Factor 3 de Transcripción de Unión a Octámeros/metabolismo , Proteínas Proto-Oncogénicas c-kit/metabolismo , Proteínas de Unión al ARN/metabolismo , Células Madre/metabolismo , Adulto , Biomarcadores/metabolismo , Biopsia , Endometriosis/patología , Femenino , Humanos , Inmunohistoquímica , Persona de Mediana EdadRESUMEN
In embryonic stem (ES) cells, oxidative stress control is crucial for genomic stability, self-renewal, and cell differentiation. Heme oxygenase-1 (HO-1) is a key player of the antioxidant system and is also involved in stem cell differentiation and pluripotency acquisition. We found that the HO-1 gene is expressed in ES cells and induced after promoting differentiation. Moreover, downregulation of the pluripotency transcription factor (TF) OCT4 increased HO-1 mRNA levels in ES cells, and analysis of ChIP-seq public data revealed that this TF binds to the HO-1 gene locus in pluripotent cells. Finally, ectopic expression of OCT4 in heterologous systems repressed a reporter carrying the HO-1 gene promoter and the endogenous gene. Hence, this work highlights the connection between pluripotency and redox homeostasis.
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Regulación de la Expresión Génica , Hemo-Oxigenasa 1/genética , Proteínas de la Membrana/genética , Células Madre Embrionarias de Ratones/metabolismo , Factor 3 de Transcripción de Unión a Octámeros/genética , Células Madre Pluripotentes/metabolismo , ARN Mensajero/genética , Animales , Benzamidas/farmacología , Diferenciación Celular/efectos de los fármacos , Difenilamina/análogos & derivados , Difenilamina/farmacología , Embrión de Mamíferos , Proteínas Fluorescentes Verdes/genética , Proteínas Fluorescentes Verdes/metabolismo , Hemo-Oxigenasa 1/metabolismo , Luciferasas/genética , Luciferasas/metabolismo , Proteínas de la Membrana/metabolismo , Ratones , Células Madre Embrionarias de Ratones/citología , Células Madre Embrionarias de Ratones/efectos de los fármacos , Células 3T3 NIH , Proteína Homeótica Nanog/antagonistas & inhibidores , Proteína Homeótica Nanog/genética , Proteína Homeótica Nanog/metabolismo , Factor 3 de Transcripción de Unión a Octámeros/antagonistas & inhibidores , Factor 3 de Transcripción de Unión a Octámeros/metabolismo , Células Madre Pluripotentes/citología , Células Madre Pluripotentes/efectos de los fármacos , Regiones Promotoras Genéticas , Piridinas/farmacología , Pirimidinas/farmacología , ARN Mensajero/metabolismo , ARN Interferente Pequeño/genética , ARN Interferente Pequeño/metabolismo , Factores de Transcripción SOXB1/antagonistas & inhibidores , Factores de Transcripción SOXB1/genética , Factores de Transcripción SOXB1/metabolismo , Transducción de Señal , Transcripción GenéticaRESUMEN
Somatic cell nuclear transfer or cytoplasm microinjection have been used to generate genome-edited farm animals; however, these methods have several drawbacks that reduce their efficiency. This study aimed to develop electroporation conditions that allow delivery of CRISPR/Cas9 system to bovine zygotes for efficient gene knock-out. We optimized electroporation conditions to deliver Cas9:sgRNA ribonucleoproteins to bovine zygotes without compromising embryo development. Higher electroporation pulse voltage resulted in increased membrane permeability; however, voltages above 15 V/mm decreased embryo developmental potential. The zona pellucida of bovine embryos was not a barrier to efficient RNP electroporation. Using parameters optimized for maximal membrane permeability while maintaining developmental competence we achieved high rates of gene editing when targeting bovine OCT4, which resulted in absence of OCT4 protein in 100% of the evaluated embryos and the expected arrest of embryonic development at the morula stage. In conclusion, Cas9:sgRNA ribonucleoproteins can be delivered efficiently by electroporation to zona-intact bovine zygotes, resulting in efficient gene knockouts.
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Understanding the effects of Bisphenol A (BPA) on early germ cell differentiation and their consequences in adult life is an area of growing interest in the field of endocrine disruption. Herein, we investigate whether perinatal exposure to BPA affects the differentiation of male germ cells in early life using a transgenic mouse expressing the GFP reporter protein under the Oct4 promoter. In this model, the expression of GFP reflects the expression of the Oct4 gene. This pluripotency gene is required to maintain the spermatogonial stem cells in an undifferentiated stage. Thus, GFP expression was used as a parameter to evaluate the effect of BPA on early germ cell development. Female pregnant transgenic mice were exposed to BPA by oral gavage, from embryonic day 5.5 to postnatal day 7 (PND7). The effects of BPA on male germ cell differentiation were evaluated at PND7, while sperm quality, testicular morphology, and protein expression of androgen receptor and proliferating cell nuclear antigen were studied at PND130. We found that perinatal/lactational exposure to BPA up-regulates the expression of Oct4-driven GFP in testicular cells at PND7. This finding suggests a higher proportion of undifferentiated spermatogonia in BPA-treated animals compared with non-exposed mice. Moreover, in adulthood, the number of spermatozoa per epididymis was reduced in those animals perinatally exposed to BPA. This work shows that developmental exposure to BPA disturbed the normal differentiation of male germ cells early in life, mainly by altering the expression of Oct4 and exerted long-lasting sequelae at the adult stage, affecting sperm count and testis.
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Compuestos de Bencidrilo/toxicidad , Disruptores Endocrinos/toxicidad , Células Germinativas/efectos de los fármacos , Fenoles/toxicidad , Efectos Tardíos de la Exposición Prenatal/inducido químicamente , Animales , Diferenciación Celular , Proliferación Celular/efectos de los fármacos , Femenino , Células Germinativas/citología , Células Germinativas/crecimiento & desarrollo , Células Germinativas/metabolismo , Masculino , Intercambio Materno-Fetal , Ratones Transgénicos , Factor 3 de Transcripción de Unión a Octámeros/genética , Factor 3 de Transcripción de Unión a Octámeros/metabolismo , Embarazo , Receptores Androgénicos/metabolismo , Factores de Transcripción SOXB1/genética , Recuento de Espermatozoides , Motilidad Espermática/efectos de los fármacos , Testículo/efectos de los fármacos , Testículo/crecimiento & desarrollo , Testículo/metabolismoRESUMEN
Primary central nervous system (CNS) tumors are the most common deadly childhood cancer. Several patients with medulloblastoma experience local or metastatic recurrences after standard treatment, a condition associated with very poor prognosis. Current neuroimaging techniques do not accurately detect residual stem-like medulloblastoma cells promoting tumor relapses. In attempt to identify candidate tumor markers that could be circulating in blood or cerebrospinal (CSF) fluid of patients, we evaluated the proteome and miRNome content of extracellular microvesicles (MVs) released by highly-aggressive stem-like medulloblastoma cells overexpressing the pluripotent factor OCT4A. These cells display enhanced tumor initiating capability and resistance to chemotherapeutic agents. A common set of 464 proteins and 10 microRNAs were exclusively detected in MVs of OCT4A-overexpressing cells from four distinct medulloblastoma cell lines, DAOY, CHLA-01-MED, D283-MED, and USP13-MED. The interactome mapping of these exclusive proteins and miRNAs revealed ERK, PI3K/AKT/mTOR, EGF/EGFR, and stem cell self-renewal as the main oncogenic signaling pathways altered in these aggressive medulloblastoma cells. Of these MV cargos, four proteins (UBE2M, HNRNPCL2, HNRNPCL3, HNRNPCL4) and five miRNAs (miR-4449, miR-500b, miR-3648, miR-1291, miR-3607) have not been previously reported in MVs from normal tissues and in CSF. These proteins and miRNAs carried within MVs might serve as biomarkers of aggressive stem-like medulloblastoma cells to improve clinical benefit by helping refining diagnosis, patient stratification, and early detection of relapsed disease.
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Neoplasias Cerebelosas/diagnóstico , Vesículas Extracelulares/metabolismo , Meduloblastoma/diagnóstico , MicroARNs/análisis , Proteoma/análisis , Biomarcadores de Tumor/sangre , Biomarcadores de Tumor/líquido cefalorraquídeo , Línea Celular Tumoral , Neoplasias Cerebelosas/sangre , Neoplasias Cerebelosas/líquido cefalorraquídeo , Humanos , Meduloblastoma/sangre , Meduloblastoma/líquido cefalorraquídeo , Pronóstico , ProteómicaRESUMEN
Aberrant expression of the pluripotency factor OCT4A in embryonal tumors of the central nervous system (CNS) is a key factor that contributes to tumor aggressiveness and correlates with poor patient survival. OCT4A overexpression has been shown to up-regulate miR-367, a microRNA (miRNA) that regulates pluripotency in embryonic stem cells and stem-like aggressive traits in cancer cells. Here, we show that (a) miR-367 is carried in microvesicles derived from embryonal CNS tumor cells expressing OCT4A; and (b) inhibition of miR-367 in these cells attenuates their aggressive traits. miR-367 silencing in OCT4A-overexpressing tumor cells significantly reduced their proliferative and invasive behavior, clonogenic activity, and tumorsphere generation capability. In vivo, targeting of miR-367 through direct injections of a specific inhibitor into the cerebrospinal fluid of Balb/C nude mice bearing OCT4A-overexpressing tumor xenografts inhibited tumor development and improved overall survival. miR-367 was also shown to target SUZ12, one of the core components of the polycomb repressive complex 2 known to be involved in epigenetic silencing of pluripotency-related genes, including POU5F1, which encodes OCT4A. Our findings reveal possible clinical applications of a cancer stemness pathway, highlighting miR-367 as a putative liquid biopsy biomarker that could be further explored to improve early diagnosis and prognosis prediction, and potentially serve as a therapeutic target in aggressive embryonal CNS tumors.
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Biomarcadores de Tumor , Neoplasias del Sistema Nervioso Central , Silenciador del Gen , MicroARNs , Neoplasias de Células Germinales y Embrionarias , Células Madre Neoplásicas , ARN Neoplásico , Animales , Biomarcadores de Tumor/antagonistas & inhibidores , Biomarcadores de Tumor/genética , Biomarcadores de Tumor/metabolismo , Línea Celular Tumoral , Neoplasias del Sistema Nervioso Central/tratamiento farmacológico , Neoplasias del Sistema Nervioso Central/genética , Neoplasias del Sistema Nervioso Central/metabolismo , Neoplasias del Sistema Nervioso Central/patología , Células HEK293 , Humanos , Ratones , Ratones Endogámicos BALB C , Ratones Desnudos , MicroARNs/antagonistas & inhibidores , MicroARNs/genética , MicroARNs/metabolismo , Neoplasias de Células Germinales y Embrionarias/tratamiento farmacológico , Neoplasias de Células Germinales y Embrionarias/genética , Neoplasias de Células Germinales y Embrionarias/metabolismo , Neoplasias de Células Germinales y Embrionarias/patología , Células Madre Neoplásicas/metabolismo , Células Madre Neoplásicas/patología , ARN Neoplásico/antagonistas & inhibidores , ARN Neoplásico/genética , ARN Neoplásico/metabolismo , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Mechanisms mediating mesenchymal stromal/stem cells' (MSCs) multipotency are unclear. Although the expression of the pluripotency factor OCT4 has been detected in MSCs, whether it has a functional role in adult stem cells is still controversial. We hypothesized that a physiological expression level of OCT4 is important to regulate MSCs' multipotency and trigger differentiation in response to environmental signals. Here, we specifically suppressed OCT4 in MSCs by using siRNA technology before directed differentiation. OCT4 expression levels were reduced by 82% in siOCT4-MSCs, compared with controls. Interestingly, siOCT4-MSCs also presented a hypermethylated OCT4 promoter. OCT4 silencing significantly impaired the ability of MSCs to differentiate into osteoblasts. Histologic and macroscopic analysis showed a lower degree of mineralization in siOCT4-MSCs than in controls. Moreover, OCT4 silencing prevented the up-regulation of osteoblast lineage-associated genes during differentiation. Similarly, OCT4 silencing resulted in decreased MSC differentiation potential towards the adipogenic lineage. The accumulation of lipids was reduced 3.0-fold in siOCT4-MSCs, compared with controls. The up-regulation of genes engaged in the early stages of adipogenesis was also suppressed in siOCT4-MSCs. Our findings provide evidence of a functional role for OCT4 in MSCs and indicate that a basal expression of this transcription factor is essential for their multipotent capacity.
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Adipogénesis , Represión Epigenética , Células Madre Mesenquimatosas/metabolismo , Factor 3 de Transcripción de Unión a Octámeros/genética , Osteogénesis , Animales , Metilación de ADN , Células Madre Mesenquimatosas/fisiología , Ratones , Factor 3 de Transcripción de Unión a Octámeros/metabolismo , Factor 3 de Transcripción de Unión a Octámeros/fisiología , Regiones Promotoras GenéticasRESUMEN
Lab on a Chip (LOC) farming systems have emerged as a powerful tool for single cell studies combined with a non-adherent cell culture substrate and single cell capture chips for the study of single cell derived tumor spheres. Cancer is characterized by its cellular heterogeneity where only a small population of cancer stem cells (CSCs) are responsible for tumor metastases and recurrences. Thus, the in vitro strategy to the formation of a single cell-derived sphere is an attractive alternative to identify CSCs. In this study, we test the effectiveness of microdevices for analysis of heterogeneity within CSC populations and its interaction with different components of the extracellular matrix. CSC could be identify using specific markers related to its pluripotency and self-renewal characteristics such as the transcription factor Oct-4 or the surface protein CD44. The results confirm the usefulness of LOC as an effective method for quantification of CSC, through the formation of spheres under conditions of low adhesion or growing on components of the extracellular matrix. The device used is also a good alternative for evaluating the individual growth of each sphere and further identification of these CSC markers by immunofluorescence. In conclusion, LOC devices have not only the already known advantages, but they are also a promising tool since they use small amounts of reagents and are under specific culture parameters. LOC devices could be considered as a novel technology to be used as a complement or replacement of traditional studies on culture plates.
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Proliferación Celular/fisiología , Esferoides Celulares/patología , Animales , Biomarcadores de Tumor/metabolismo , Técnicas de Cultivo de Célula/métodos , Línea Celular Tumoral , Matriz Extracelular/metabolismo , Matriz Extracelular/patología , Receptores de Hialuranos/metabolismo , Dispositivos Laboratorio en un Chip , Ratones , Células Madre Neoplásicas/metabolismo , Células Madre Neoplásicas/patología , Factor 3 de Transcripción de Unión a Octámeros/metabolismo , Esferoides Celulares/metabolismoRESUMEN
Resumen El teratoma inmaduro se describió por primera vez en 1960 y puede ser puro o estar mezclado con un componente maduro. Es la segunda neoplasia maligna más común de células germinales de todos los cánceres de ovario (representa <1%). Alrededor del 50% de los teratomas inmaduros puros del ovario ocurren en mujeres entre las edades de 10 y 20 años. Se debe considerar el tratamiento para preservar su fertilidad futura porque la mayoría de los tumores de células germinales de ovario son curables con la cirugía conservadora y la quimioterapia combinada de seguimiento. La mayoría de los pacientes diagnosticados con un teratoma inmaduro se curan de su enfermedad. Sin embargo en todas las pacientes, se recomienda un seguimiento cercano, particularmente en los primeros 2 años después del diagnóstico, donde existe una mayor probabilidad de recurrencia.
Abstract The immature teratoma was first described in 1960 and cay be pure or mixed with a mature component. It is the second most common malignant germ cell neoplasm of all ovarian cancers (representing <1%). About 50% of pure immature teratomas of the ovary occur in women between the ages of 10 and 20 years. Treatment should be considered to preserve future fertility because the majority of ovarian germ cell tumors are curable with conservative surgery and combined combination chemotherapy. Most patients diagnosed with an immature teratoma are cured of their disease. However, in all patients, we recommend close follow-up, particularly in the first 2 years after diagnosis, where there is a greater chance of recurrence.
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Humanos , Femenino , Neoplasias Ováricas/diagnóstico , Ovario , Teratoma/diagnóstico , Encefalitis LímbicaRESUMEN
PURPOSE: Circulating tumor cells (CTCs) with epithelial-to-mesenchymal transition (EMT) phenotypes might be related to tumor progression while OCT4 expression is involved in tumor metastasis and poor prognosis. But the possible clinical significance of EMT phenotypes of CTCs from non-small-cell lung cancer (NSCLC) patients has still to be demonstrated. Furthermore, none has been investigated the expression of OCT4 in CTCs. We therefore identified the EMT phenotype-based subsets of CTCs and determined the OCT4 expression status of CTCs in NSCLC patients, to explore their possible clinical relevance. METHODS: 37 NSCLC patients and ten healthy volunteers were enrolled, respectively. The Canpatrol™ CTC enrichment technique was used to isolate and identify the EMT phenotype-based subsets of CTCs. OCT4 expression in each CTC was also determined. Results were correlated with patients' clinico-pathological features. RESULTS: CTCs were detected in 33 of 37 (89.2%) NSCLC patients, and no CTCs were identified in ten healthy volunteers. Three CTCs phenotypes, including epithelial, biophenotypic, and mesenchymal CTCs were identified based on the expression of EMT markers. Mesenchymal CTCs were more commonly found in patients with distant metastasis. Patients with distant metastasis tended to have a higher median CTCs number. OCT4-positive was observed in 21 of 28 (75.0%) patients. High expression of OCT4 tended to occur in advanced patients as well as in distant metastatic patients. CONCLUSIONS: The findings suggest that identification of CTCs by EMT markers as well as evaluation of OCT4 expression status by assessment of OCT4 expression in CTCs could serve as potential adjuncts for evaluating metastasis and prognosis in NSCLC patients.
Asunto(s)
Biomarcadores de Tumor/análisis , Carcinoma de Pulmón de Células no Pequeñas/patología , Neoplasias Pulmonares/patología , Células Neoplásicas Circulantes/patología , Factor 3 de Transcripción de Unión a Octámeros/biosíntesis , Adulto , Anciano , Carcinoma de Pulmón de Células no Pequeñas/mortalidad , Transición Epitelial-Mesenquimal/fisiología , Femenino , Humanos , Neoplasias Pulmonares/mortalidad , Masculino , Persona de Mediana Edad , Factor 3 de Transcripción de Unión a Octámeros/análisis , PronósticoRESUMEN
Medulloblastoma is a highly aggressive pediatric brain tumor, in which sporadic expression of the pluripotency factor OCT4 has been recently correlated with poor patient survival. However the contribution of specific OCT4 isoforms to tumor aggressiveness is still poorly understood. Here, we report that medulloblastoma cells stably overexpressing the OCT4A isoform displayed enhanced clonogenic, tumorsphere generation, and invasion capabilities. Moreover, in an orthotopic metastatic model of medulloblastoma, OCT4A overexpressing cells generated more developed, aggressive and infiltrative tumors, with tumor-bearing mice attaining advanced metastatic disease and shorter survival rates. Pro-oncogenic OCT4A effects were expression-level dependent and accompanied by distinct chromosomal aberrations. OCT4A overexpression in medulloblastoma cells also induced a marked differential expression of non-coding RNAs, including poorly characterized long non-coding RNAs and small nucleolar RNAs. Altogether, our findings support the relevance of pluripotency-related factors in the aggravation of medulloblastoma traits classically associated with poor clinical outcome, and underscore the prognostic and therapeutic value of OCT4A in this challenging type of pediatric brain cancer.
Asunto(s)
Biomarcadores de Tumor/metabolismo , Neoplasias Encefálicas/secundario , Neoplasias Cerebelosas/patología , Meduloblastoma/patología , Factor 3 de Transcripción de Unión a Octámeros/metabolismo , Animales , Apoptosis , Biomarcadores de Tumor/genética , Neoplasias Encefálicas/genética , Neoplasias Encefálicas/metabolismo , Movimiento Celular , Proliferación Celular , Neoplasias Cerebelosas/genética , Neoplasias Cerebelosas/metabolismo , Femenino , Humanos , Meduloblastoma/genética , Meduloblastoma/metabolismo , Ratones , Ratones Endogámicos BALB C , Ratones Desnudos , Factor 3 de Transcripción de Unión a Octámeros/genética , Pronóstico , Células Tumorales Cultivadas , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
PURPOSE: Cisplatin is commonly used in non-small-cell lung cancer (NSCLC) chemotherapy; however, chemoresistance to cisplatin remains a great clinical challenge. Octamer-binding protein 4 (OCT4) has been reported to be overexpressed in NSCLC. In this study, we aimed to investigate the potential role of OCT4 in NSCLC with chemoresistance to cisplatin. METHODS: Expressions of OCT4 was detected in NSCLC tissues and cell lines. We utilized siRNA to knock down OCT4 expression in human NSCLC cells and analyzed their phenotypic changes. RESULTS: We found that the difference of OCT4 expression between NSCLC and the adjacent non-tumourous tissues was statistically significant. Knockdown of OCT4 in NSCLC cells could decrease cell proliferation, and potentiate apoptosis induced by cisplatin, suggesting OCT4 may contribute to cisplatin resistance in NSCLC. CONCLUSION: Our findings indicate that targeting OCT4 could improve cisplatin effect in NSCLC, confirming their role in modulating cisplatin sensitivity.