RESUMEN
LIN28 function is fundamental to the activity and behavior of human embryonic stem cells (hESCs) and induced pluripotent stem cells. Its main roles in these cell types are the regulation of translational efficiency and let-7 miRNA maturation. However, LIN28-associated mRNA cargo shifting and resultant regulation of translational efficiency upon the initiation of differentiation remain unknown. An RNA-immunoprecipitation and microarray analysis protocol, eRIP, that has high specificity and sensitivity was developed to test endogenous LIN28-associated mRNA cargo shifting. A combined eRIP and polysome analysis of early stage differentiation of hESCs with two distinct differentiation cues revealed close similarities between the dynamics of LIN28 association and translational modulation of genes involved in the Wnt signaling, cell cycle, RNA metabolism and proteasomal pathways. Our data demonstrate that change in translational efficiency is a major contributor to early stages of differentiation of hESCs, in which LIN28 plays a central role. This implies that eRIP analysis of LIN28-associated RNA cargoes may be used for rapid functional quality control of pluripotent stem cells under manufacture for therapeutic applications.
Asunto(s)
Diferenciación Celular/genética , Células Madre Embrionarias/metabolismo , Biosíntesis de Proteínas , ARN Mensajero/metabolismo , Proteínas de Unión al ARN/metabolismo , Células Cultivadas , Células Madre Embrionarias/citología , Humanos , Polirribosomas/metabolismoRESUMEN
The gene expression networks governing embryonic stem cell (ESC) pluripotency are complex and finely regulated during differentiation toward specific lineages. We describe a new role for Amd1 (adenosyl methionine decarboxylase), a key enzyme in the polyamine synthesis pathway, in regulating both ESC self-renewal and differentiation to the neural lineage. Amd1 is highly expressed in ESCs and is translationally down-regulated by the neural precursor cell (NPC)-enriched microRNA miR-762 during NPC differentiation. Overexpression of Amd1 or addition of the polyamine spermine blocks ESC-to-NPC conversion, suggesting Amd1 must be down-regulated to decrease the levels of inhibitory spermine during differentiation. In addition, we demonstrate that high levels of Amd1 are required for maintenance of the ESC state. We show that forced overexpression of Amd1 in ESCs results in maintenance of high Myc levels and a delay in differentiation on removal of LIF. We propose that Amd1 is a major regulator of ESC self-renewal and that its essential role lies in its regulation of Myc levels within the cell.
Asunto(s)
Adenosilmetionina Descarboxilasa/genética , Adenosilmetionina Descarboxilasa/metabolismo , Diferenciación Celular/genética , Regulación hacia Abajo , Células Madre Embrionarias/citología , Células Madre Embrionarias/enzimología , Animales , Regulación del Desarrollo de la Expresión Génica , Ratones , MicroARNs/metabolismo , Neuronas/citología , Neuronas/enzimología , Proteínas Proto-Oncogénicas c-myc/genética , Proteínas Proto-Oncogénicas c-myc/metabolismoRESUMEN
Identification of the factors critical to the tumor-initiating cell (TIC) state may open new avenues in cancer therapy. Here we show that the metabolic enzyme glycine decarboxylase (GLDC) is critical for TICs in non-small cell lung cancer (NSCLC). TICs from primary NSCLC tumors express high levels of the oncogenic stem cell factor LIN28B and GLDC, which are both required for TIC growth and tumorigenesis. Overexpression of GLDC and other glycine/serine enzymes, but not catalytically inactive GLDC, promotes cellular transformation and tumorigenesis. We found that GLDC induces dramatic changes in glycolysis and glycine/serine metabolism, leading to changes in pyrimidine metabolism to regulate cancer cell proliferation. In the clinic, aberrant activation of GLDC correlates with poorer survival in lung cancer patients, and aberrant GLDC expression is observed in multiple cancer types. This link between glycine metabolism and tumorigenesis may provide novel targets for advancing anticancer therapy.
Asunto(s)
Carcinoma de Pulmón de Células no Pequeñas/enzimología , Transformación Celular Neoplásica , Glicina-Deshidrogenasa (Descarboxilante)/metabolismo , Neoplasias Pulmonares/metabolismo , Secuencia de Aminoácidos , Antígenos CD/metabolismo , Carcinoma de Pulmón de Células no Pequeñas/genética , Carcinoma de Pulmón de Células no Pequeñas/patología , Moléculas de Adhesión Celular Neuronal/metabolismo , Línea Celular Tumoral , Proteínas de Unión al ADN/metabolismo , Proteínas Fetales/metabolismo , Glicina/metabolismo , Humanos , Datos de Secuencia Molecular , Neoplasias/enzimología , Neoplasias/genética , Proteínas de Unión al ARN , Alineación de Secuencia , Serina/metabolismo , Thermus thermophilus/enzimología , Trasplante HeterólogoRESUMEN
AIMS: We aimed to develop an image analysis software that enabled measurement of glomerular basement membrane (GBM) thickness. METHODS: With this software, we evaluated the range of GBM widths found in a cohort of Asian patients diagnosed with a spectrum of renal diseases including minimal change/IgM nephropathy, focal and segmental glomerulosclerosis, IgA nephropathy, systemic lupus erythematosus nephritis, diabetic nephropathy, pauci-immune crescentic glomerulonephritis, thin basement membrane disease, and tubulointerstitial nephritis. Measurements were taken from a minimum of five glomerular capillary loops of each glomerulus. For each loop, at least 10 different points of the GBM were measured. RESULTS: The average GBM width measured for minimal change disease was 347.4 +/- 9.0 nm, with the highest value being 403.9 nm and lowest being 214.7 nm. No association was found between GBM width and gender. We found a significant increase in GBM width in pathological states like lupus nephropathy (p < 0.0001), diabetic nephritis (p < 0.001) and tubulointerstitial nephritis (p < 0.01) as compared with minimal change disease. Only one case of thin membrane nephropathy (198.7 nm) was available for analysis and we found a significant thinning of the GBM. CONCLUSIONS: These observations provide insights into the range of GBM thickness in several disease states and support the use of this novel software in the daily diagnostic laboratory setting.
Asunto(s)
Membrana Basal Glomerular/ultraestructura , Interpretación de Imagen Asistida por Computador/métodos , Enfermedades Renales/patología , Programas Informáticos , Adolescente , Adulto , Anciano , Pueblo Asiatico , Niño , Femenino , Humanos , Masculino , Microscopía Electrónica de Transmisión , Persona de Mediana Edad , Adulto JovenRESUMEN
Stem cells self-renew or differentiate under the governance of a stem-cell-specific transcriptional program, with each transcription factor orchestrating the activities of a particular set of genes. Here we demonstrate that a single transcription factor is able to regulate distinct core circuitries in two different blastocyst-derived stem cell lines, embryonic stem cells (ESCs) and extraembryonic endoderm (XEN) cells. The transcription factor Sall4 is required for early embryonic development and for ESC pluripotency. Sall4 is also expressed in XEN cells, and depletion of Sall4 disrupts self-renewal and induces differentiation. Genome-wide analysis reveals that Sall4 is regulating different gene sets in ESCs and XEN cells, and depletion of Sall4 targets in the respective cell types induces differentiation. With Oct4, Sox2, and Nanog, Sall4 forms a crucial interconnected autoregulatory network in ESCs. In XEN cells, Sall4 regulates the key XEN lineage-associated genes Gata4, Gata6, Sox7, and Sox17. Our findings demonstrate how Sall4 functions as an essential stemness factor for two different stem cell lines.