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The androgen receptor (AR) plays a critical role in the development of prostate cancer (PCa) through the activation of androgen-induced cellular proliferation genes. Thus, blocking AR-mediated transcriptional activation is expected to inhibit the growth and spread of PCa. Using tailor-made splice-switching locked nucleic acid (LNA) oligonucleotides (SSOs), we successfully redirected splicing of the AR precursor (pre-)mRNA and destabilized the transcripts via the introduction of premature stop codons. Furthermore, the SSOs simultaneously favored production of the AR45 mRNA in lieu of the full-length AR. AR45 is an AR isoform that can attenuate the activity of both full-length and oncogenic forms of AR by binding to their common N-terminal domain (NTD), thereby blocking their transactivation potential. A large screen was subsequently used to identify individual SSOs that could best perform this dual function. The selected SSOs powerfully silence AR expression and modulate the expression of AR-responsive cellular genes. This bi-functional strategy that uses a single therapeutic molecule can be the basis for novel PCa treatments. It might also be customized to other types of therapies that require the silencing of one gene and the simultaneous expression of a different isoform.
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Well-validated strategies for discovering potent and efficacious antisense oligonucleotides are central to realize the full therapeutic potential of RNA therapy. In this study, we focus on RNA targets where the same sequence of 16-20 nt is found in several regions across the RNA, and not in any other RNA. Targeting such unique repeated regions with oligonucleotides designed as gapmers and capable of recruiting RNase H has previously been proposed as a strategy for identifying potent gapmers. By sequence analysis of the human and monkey transcriptomes, we find that such unique repeated regions in RNA are often conserved between humans and monkeys, which allow pharmacodynamic effects to be evaluated in non-human primates before testing in humans. For eight potential RNA targets chosen in an unbiased fashion, we targeted their unique repeated regions with locked nucleic acid (LNA)-modified gapmers, and for six of them we identified gapmers that were significantly more potent and efficacious in vitro than non-repeat-targeting gapmer controls. We suggest a stochastic model for repeat-targeting gapmers that explains all effects observed so far and can help guide future work. Our results support the targeting of repeated regions as an effective strategy for discovering gapmer antisense oligonucleotides suitable for therapeutic development.
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Methods for the quantification of antisense oligonucleotides (AONs) provide insightful information on biodistribution and intracellular trafficking. However, the established methods have not provided information on the absolute number of molecules in subcellular compartments or about how many AONs are needed for target gene reduction for unconjugated AONs. We have developed a new method for nuclear AON quantification that enables us to determine the absolute number of AONs per nucleus without relying on AON conjugates such as fluorophores that may alter AON distribution. This study describes an alternative and label-free method using subcellular fractionation, nucleus counting, and locked nucleic acid (LNA) sandwich enzyme-linked immunosorbent assay to quantify absolute numbers of oligonucleotides in nuclei. Our findings show compound variability (diversity) by which 247,000-693,000 LNAs/nuclei results in similar target reduction for different compounds. This method can be applied to any antisense drug discovery platform providing information on specific and clinically relevant AONs. Finally, this method can directly compare nuclear entry of AON with target gene knockdown for any compound design and nucleobase sequence, gene target, and phosphorothioate stereochemistry.
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Terapia Molecular Dirigida , Oligonucleótidos Antisentido/aislamiento & purificación , Oligonucleótidos/aislamiento & purificación , Distribución Tisular/genética , Núcleo Celular/genética , Ensayo de Inmunoadsorción Enzimática/métodos , Humanos , Oligonucleótidos/uso terapéutico , Oligonucleótidos Antisentido/uso terapéutico , Distribución Tisular/efectos de los fármacosRESUMEN
Somatostatin (SST) analogues are used to control the proliferation and symptoms of neuroendocrine tumors (NETs). MicroRNAs (miRNA) are small non-coding RNAs that modulate posttranscriptional gene expression. We wanted to characterize the miRNAs operating under the control of SST to elucidate to what extent they mediate STT actions. NCI-H727 carcinoid cell line was treated with either a chimeric SST/dopamine analogue; a SST or dopamine analogue for proliferation assays and for identifying differentially expressed miRNAs using miRNA microarray. The miRNAs induced by SST analogue treatment are investigated in carcinoid cell lines NCI-H727 and CNDT2 using in situ hybridization, qPCR and proliferation assays. SST analogues inhibited the growth of carcinoid cells more potently compared to the dopamine analogue. Principal Component Analysis (PCA) of the samples based on miRNA expression clearly separated the samples based on treatment. Two miRNAs which were highly induced by SST analogues, miR-7 and miR-148a, were shown to inhibit the proliferation of NCI-H727 and CNDT2 cells. SST analogues also produced a general up-regulation of the let-7 family members. SST analogues control and induce distinct miRNA expression patterns among which miR-7 and miR-148a both have growth inhibitory properties.
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Hematology analyzers sometimes generate spurious results. A patient had EDTA-induced pseudothrombocytosis and platelet agglutination in citrate blood samples. This case verifies that addition of 1% paraformaldehyde to the citrate tubes can prevent platelet clumping. Further, it illustrates the advantages of having access to more than one platelet count method.
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INTRODUCTION: Recent studies suggest that the human endolymphatic sac (ES) may have multiple functions, including an ion-transport capacity comparable to the kidney, an immunological capacity and a possible natriuretic capacity. Further, there have been speculations of a yet undefined role in intracranial pressure homeostasis. The anatomical location towards the sigmoid sinus would suggest a possible endo- and/or paracrine signaling. However, neuronal connections may also apply, but it remains very scarcely explored in the human ES. STUDY DESIGN: DNA micro-arrays and immunohistochemistry were used for analyses of fresh human ES tissue samples. METHODS: A total of 30 tissue samples from the human ES were obtained during translabyrinthine surgery for vestibular schwannoma. Microarray technology was used to investigate tissue sample gene expression, using adjacent dura mater as control. The expression of genes specific for neuronal signaling was determined and results for selected key molecules verified by immunohistochemistry. Transmission electron microscopy was used for ultrastructural analysis. RESULTS: For the transmission electron microscopy analysis, a direct innervation of the ES was observed with unmyelinated fibers imbedded in the ES epithelial lining. The microarrays confirmed, that several molecules involved in neuronal signaling were found expressed significantly in the ES DNA profile, such as the Cholecystokinin peptide and related receptors, Dopamine receptors 2 and 5, vesicular monoamine transporter 2 (VMAT2), plasma monoamine transporter (PMAT), and Serotonin 1D. All peptides were verified by immunohistochemistry. CONCLUSIONS: Based on global gene expression profiling and immuno-histochemical labeling, we conclude that the human ES expresses neuropeptide receptors and monoamine transporters. Combined with the ultrastructural demonstration of unmyelinated axons imbedded within the epithelial lining, the findings suggest that neuro-signaling mechanisms are involved in functions exerted by the ES.
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Saco Endolinfático/inervación , Saco Endolinfático/metabolismo , Saco Endolinfático/ultraestructura , Perfilación de la Expresión Génica , Humanos , Inmunohistoquímica , Microscopía Electrónica de Transmisión , Fibras Nerviosas/ultraestructura , Receptores de Neurotransmisores/biosíntesis , TranscriptomaRESUMEN
OBJECTIVES/HYPOTHESIS: The function of the human endolymphatic sac (ES) has been enigmatic for decades. Hypotheses include controlling endolymphatic fluid homeostasis and inner ear immunological defense. Additionally, several studies indicate a possible endocrine capacity and a yet undefined role in intracranial pressure homeostasis. However, no direct evidence of such capacity exists. This study aims to explore and identify the hypothesized endocrine capacity of the human ES. STUDY DESIGN: DNA microarrays and immunohistochemistry were used for analyses of fresh human ES tissue samples. METHODS: Twelve tissue samples from the human ES were obtained during translabyrinthine surgery for vestibular schwannoma. Microarray technology was used to investigate tissue sample gene expression. Genes specific for an endocrine function were determined, and results were verified by immunohistochemistry. RESULTS: Several natriuretic peptides were found expressed significantly in the ES, including uroguanylin and brain natriuretic peptide, but also peptides regulating vascular tone, including adrenomedullin 2. In addition, both neurophysin and oxytocin (OXT) were found significantly expressed. All peptides were verified by immunohistochemistry. CONCLUSION: The present data support the hypothesis that the human ES may have an endocrine/paracrine capacity through expression of several peptides with potent natriuretic activity. Furthermore, the ES may influence the hypothalamo-pituitary-adrenal axis and may regulate vasopressin receptors and aquaporin-2 channels in the inner ear via OXT expression. We hypothesize that the ES is likely to regulate inner ear endolymphatic homeostasis, possibly through secretion of several peptides, but it may also influence systemic and/or intracranial blood pressure through direct and indirect action on the vascular system and the kidney. LEVEL OF EVIDENCE: NA. Laryngoscope, 127:E201-E208, 2017.
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Endolinfa/metabolismo , Saco Endolinfático/metabolismo , Expresión Génica , Péptidos Natriuréticos/metabolismo , Oído Interno/cirugía , Saco Endolinfático/patología , Humanos , Sistema Hipotálamo-Hipofisario/metabolismo , Inmunohistoquímica , Péptido Natriurético Encefálico/metabolismo , Neuroma Acústico/patología , Neuroma Acústico/cirugía , Neurofisinas/metabolismo , Análisis de Secuencia por Matrices de Oligonucleótidos , Oxitocina/metabolismo , Hormonas Peptídicas/metabolismo , Sistema Hipófiso-Suprarrenal/metabolismoRESUMEN
A thorough understanding of the idiopathic hypereosinophilic syndrome (IHES) and further optimization of diagnostic work-up procedures are warranted. We analyzed purified eosinophils from patients with IHES by next-generation whole-exome sequencing and compared DNA methylation profiles from reactive eosinophilic conditions to known clonal and suspected clonal eosinophilia. Somatic missense mutations in cancer-related genes were detected in three IHES patients. These included the spliceosome gene PUF60 and the cadherin gene CDH17. Furthermore, reactive eosinophilia samples could be differentiated from known- and suspected clonal eosinophilia samples based on 285 differentially methylated CpG sites corresponding to 128 differentially methylated genes. Using Ingenuity pathway analysis, we found that differentially methylated genes were highly enriched in functional pathways such as cancer, cell death and survival, and hematological disease. Our data show that a subset of IHES may be of clonal origin not related to the classical molecular aberrations of FGFR, PDGFRA/B, or T-cells, and that the initiating hits could be point mutations in a variety of genes, including spliceosome mutations or hypermethylated tumor suppressor genes. In addition, we identified a DNA methylation signature that is relevant for distinguishing clonal and suspected clonal eosinophilia from reactive eosinophilia per se, which may be useful in daily clinical work.
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Biomarcadores/metabolismo , Metilación de ADN , Exoma/genética , Genoma Humano , Secuenciación de Nucleótidos de Alto Rendimiento/métodos , Síndrome Hipereosinofílico/genética , Mutación/genética , Diferenciación Celular , Femenino , Estudio de Asociación del Genoma Completo , Humanos , Síndrome Hipereosinofílico/patología , Masculino , ARN Mensajero/genética , Reacción en Cadena en Tiempo Real de la Polimerasa , Reacción en Cadena de la Polimerasa de Transcriptasa InversaRESUMEN
BACKGROUND: Cancers of unknown primary (CUPs) constitute ~5% of all cancers. The tumors have an aggressive biological and clinical behavior. The aim of the present study has been to uncover whether CUPs exhibit distinct molecular features compared to metastases of known origin. METHODS: Employing genome wide transcriptome analysis, Linear Discriminant Analysis (LDA) and Quadratic Discriminant Analysis (QDA), we defined the putative origins of a large series of CUP and how closely related a particular CUP was to corresponding metastases of known origin. LDA predictions were subsequently used to define a universal CUP core set of differentially expressed genes, that by means of gene set enrichment analysis was exploited to depict molecular pathways characterizing CUP. RESULTS: The analyses show that CUPs are distinct from metastases of known origin. CUPs exhibit inconsistent expression of conventional cancer biomarkers and QDA derived outlier scores show that CUPs are more distantly related to their primary tumor class than corresponding metastases of known origin. Gene set enrichment analysis showed that CUPs display increased expression of genes involved in DNA damage repair and mRNA signatures of chromosome instability (CIN), indicating that CUPs are chromosome unstable compared to metastases of known origin. CONCLUSIONS: CIN may account for the uncommon clinical presentation, chemoresistance and poor outcome in patients with CUP and warrant selective diagnostic strategies and treatment.
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Inestabilidad Cromosómica/genética , Regulación Neoplásica de la Expresión Génica , Metástasis de la Neoplasia/genética , Neoplasias Primarias Desconocidas/genética , Femenino , Perfilación de la Expresión Génica , Humanos , Masculino , Metástasis de la Neoplasia/patología , Proteínas de Neoplasias/biosíntesis , Proteínas de Neoplasias/genética , Neoplasias Primarias Desconocidas/clasificación , Neoplasias Primarias Desconocidas/patología , PronósticoRESUMEN
OBJECTIVES/HYPOTHESIS: The purpose of the present study is to explore, demonstrate, and describe the expression of genes related to the innate immune system in the human endolymphatic sac. It is hypothesized that the endolymphatic sac has a significant immunological function in the human inner ear. STUDY DESIGN: DNA microarrays and immunohistochemistry were used for analyses of fresh human endolymphatic-sac tissue samples. METHODS: Twelve tissue samples from the human endolymphatic sac were obtained during translabyrinthine surgery for vestibular schwannoma. Microarray technology was used to investigate tissue sample gene expression using adjacent dura mater as control. The expression of genes specific for the innate immune system was determined and results for selected key molecules verified by immunohistochemistry. RESULTS: A comprehensive overview of expressed genes of the innate immune system was obtained. Multiple key elements of both the cellular and humoral innate immune system were expressed, including Toll-like receptors 4 and 7, as well as beta-defensin and lactoferrin. CONCLUSIONS: The present data provides the first direct evidence of an immunological capacity of the human endolymphatic sac. At the molecular level, the endolymphatic sac is capable of antigen recognition and processing for initiation of an immune response. In addition, potent molecules directly toxic to invading pathogens are expressed by the sac epithelium. This evidence strongly supports the endolymphatic sac as a significant immunological entity of the inner ear. LEVEL OF EVIDENCE: N/A.
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Biomarcadores de Tumor/genética , ADN de Neoplasias/genética , Saco Endolinfático/inmunología , Regulación Neoplásica de la Expresión Génica , Inmunidad Innata/genética , Adulto , Anciano , Biomarcadores de Tumor/biosíntesis , Neoplasias del Oído/genética , Neoplasias del Oído/metabolismo , Neoplasias del Oído/patología , Saco Endolinfático/metabolismo , Saco Endolinfático/patología , Humanos , Inmunohistoquímica , Persona de Mediana Edad , Neurilemoma/genética , Neurilemoma/metabolismo , Neurilemoma/patología , Análisis de Secuencia por Matrices de Oligonucleótidos/métodosRESUMEN
Because tissue regeneration deteriorates with age, it is generally assumed that the younger the animal, the better it compensates for tissue damage. We have examined the effect of young age on compensatory proliferation of pancreatic ß cells in vivo. Surprisingly, ß cells in suckling mice fail to enter the cell division cycle in response to a diabetogenic injury or increased glycolysis. The potential of ß cells for compensatory proliferation is acquired following premature weaning to normal chow, but not to a diet mimicking maternal milk. In addition, weaning coincides with enhanced glucose-stimulated oxidative phosphorylation and insulin secretion from islets. Transcriptome analysis reveals that weaning increases the expression of genes involved in replication licensing, suggesting a mechanism for increased responsiveness to the mitogenic activity of high glucose. We propose that weaning triggers a discrete maturation step of ß cells, elevating both the mitogenic and secretory response to glucose.
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Biomarcadores/metabolismo , Proliferación Celular , Glucosa/farmacología , Insulina/farmacología , Islotes Pancreáticos/citología , Destete , Animales , Apoptosis , Western Blotting , Células Cultivadas , Femenino , Perfilación de la Expresión Génica , Hipoglucemiantes/farmacología , Técnicas para Inmunoenzimas , Células Secretoras de Insulina/citología , Células Secretoras de Insulina/efectos de los fármacos , Células Secretoras de Insulina/metabolismo , Islotes Pancreáticos/efectos de los fármacos , Islotes Pancreáticos/metabolismo , Masculino , Ratones , Ratones Endogámicos ICR , Ratones Transgénicos , Análisis de Secuencia por Matrices de Oligonucleótidos , ARN Mensajero/genética , Reacción en Cadena en Tiempo Real de la Polimerasa , Reacción en Cadena de la Polimerasa de Transcriptasa InversaRESUMEN
OBJECTIVES/HYPOTHESIS: The purpose of the present study is to explore, demonstrate, and describe the expression of genes related to the solute carrier (SLC) molecules of ion transporters in the human endolymphatic sac. STUDY DESIGN: cDNA microarrays and immunohistochemistry were used for analyses of fresh human endolymphatic sac tissue samples. METHODS: Twelve tissue samples of the human endolymphatic sac were obtained during translabyrinthine surgery for vestibular schwannoma. Microarray technology was used to investigate tissue sample expression of solute carrier family genes, using adjacent dura mater as control. Immunohistochemistry was used for verification of translation of selected genes, as well as localization of the specific protein within the sac. RESULTS: An extensive representation of the SLC family genes were upregulated in the human endolymphatic sac, including SLC26a4 Pendrin, SLC4a1 sodium-bicarbonate transporter, SLC9a2 sodium-hydrogen transporter, SLC12a3 thiazide-sensitive Na-Cl transporter, and SLC34a2 sodium-phosphate transporter. CONCLUSIONS: Several important ion transporters of the SLC family are expressed in the human endolymphatic sac, including Pendrin, the thiazide-sensitive Na-Cl transporter, and the Na-phosphate transporter SLC34a2. The data provide a new knowledge base considering the ion-dependent metabolic mechanisms maintaining inner ear homeostasis. More specifically, the results indicate a strong similarity with the ion transportation occurring in the kidney collecting ducts. In addition, the findings prompt a revision of the theories behind contemporary pharmacological treatment of Ménière's disease and may broaden the understanding of the pathogenesis of BPPV.
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Líquidos Corporales/metabolismo , Saco Endolinfático/metabolismo , Expresión Génica , Homeostasis/fisiología , Proteínas de Transporte de Membrana/metabolismo , Adulto , Femenino , Humanos , Inmunohistoquímica , Masculino , Persona de Mediana Edad , Análisis de Secuencia por Matrices de OligonucleótidosRESUMEN
The IMP3 RNA-binding protein is associated with metastasis and poor outcome in human cancer. Using solid cancer transcriptome data, we found that IMP3 correlates with HMGA2 mRNA expression. Cytoplasmic IMP3 granules contain HMGA2, and IMP3 dose-dependently increases HMGA2 mRNA. HMGA2 is regulated by let-7, and let-7 antagomiRs make HMGA2 refractory to IMP3. Removal of let-7 target sites eliminates IMP3-dependent stabilization, and IMP3-containing bodies are depleted of Ago1-4 and miRNAs. The relationship between Hmga2 mRNA and IMPs also exists in the developing limb bud, where IMP1-deficient embryos show dose-dependent Hmga2 mRNA downregulation. Finally, IMP3 ribonucleoproteins (RNPs) contain other let-7 target mRNAs, including LIN28B, and a global gene set enrichment analysis demonstrates that miRNA-regulated transcripts in general are upregulated following IMP3 induction. We conclude that IMP3 RNPs may function as cytoplasmic safe houses and prevent miRNA-directed mRNA decay of oncogenes during tumor progression.
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Regulación Neoplásica de la Expresión Génica , Proteína HMGA2/metabolismo , MicroARNs/metabolismo , Estabilidad del ARN , ARN Mensajero/metabolismo , Proteínas de Unión al ARN/metabolismo , Línea Celular Tumoral , Células Cultivadas , Proteína HMGA2/genética , Humanos , MicroARNs/genética , Neoplasias/metabolismo , ARN Mensajero/genética , Proteínas de Unión al ARN/genéticaRESUMEN
Matrix metalloproteinases (MMPs), in particular MMP-2, MMP-9 and MMP-14, play a key role in various aspects of cancer pathology. Likewise, ADAMs (a disintegrin and metalloproteinases), including ADAM12, are upregulated in malignant tumors and contribute to the pathology of cancers. Here, we show that there is a positive correlation between MMP-14 and ADAM12 expression in human breast cancer. We demonstrated that in 293-VnR and human breast cancer cells expressing ADAM12 at the cell surface, endogenous MMP-14 was recruited to the cell surface, resulting in its activation. Subsequent to this activation, gelatin degradation was stimulated and tumor cell apoptosis was decreased, with reduced expression of the pro-apoptotic proteins BCL2L11 and BIK. The effect on gelatin degradation was abrogated by inhibition of the MMP-14 activity and appeared to be dependent on cell surface αVß3 integrin localization, but neither the catalytic activity of ADAM12 nor the cytoplasmic tail of ADAM12 were required. The significance of ADAM12-induced activation of MMP-14 was underscored by a reduction in MMP-14-mediated gelatin degradation and abolition of apoptosis-protective effects by specific monoclonal antibodies against ADAM12. Furthermore, orthotopic implantation of ADAM12-expressing MCF7 cells in nude mice produced tumors with increased levels of activated MMP-14 and confirmed that ADAM12 protects tumor cells against apoptosis, leading to increased tumor progression. In conclusion, our data suggest that a ternary protein complex composed of ADAM12, αVß3 integrin and MMP-14 at the tumor cell surface regulates the function of MMP-14. This interaction might point to a novel concept for the development of MMP-14-targeting drugs in treating cancer.
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Proteínas ADAM/metabolismo , Neoplasias de la Mama/enzimología , Neoplasias de la Mama/patología , Gelatina/metabolismo , Metaloproteinasa 14 de la Matriz/metabolismo , Proteínas de la Membrana/metabolismo , Proteínas ADAM/inmunología , Proteína ADAM12 , Animales , Anticuerpos Monoclonales/inmunología , Anticuerpos Monoclonales/farmacología , Apoptosis/efectos de los fármacos , Apoptosis/fisiología , Procesos de Crecimiento Celular/fisiología , Línea Celular Tumoral , Femenino , Células HEK293 , Xenoinjertos , Humanos , Integrina alfaVbeta3/metabolismo , Células MCF-7 , Metaloproteinasa 2 de la Matriz/metabolismo , Proteínas de la Membrana/inmunología , Ratones , Ratones Endogámicos NODRESUMEN
BACKGROUND DATA: microRNAs (miRNAs) are short â¼22 nucleotide RNA sequences that regulate messengerRNA translation. miRNAs have shown to play a role in synthesis of inflammatory mediators. Since inflammation play a role in intervertebral disk (IVD) degeneration, the objective was to isolate miRNA from human lumbar intervertebral disks and subsequently characterize the difference in miRNA expression between the annulus fibrosus (AF) and nucleus pulposus (NP). METHODS: Fourteen patients undergoing anterior interbody fusion for degenerative disk disease of the lumbar spine were included. During surgery biopsies from the intervertebral disks were obtained and immediately placed in RNAlater. The RNAlater was decanted and the samples frozen at -80ËC until RNA extraction. This was performed using the Trizol method. Global miRNA expression analysis was performed using the Affymetrix GeneChip® miRNA array. RESULTS: We developed a method allowing the extraction of miRNA from human intervertebral disks usually yielding 1-4 µg of total RNA pr. 100 mg of disk. Twenty-seven miRNAs had a higher expression in the AF and 10 had the highest expression in the NP. Among the top 15 signaling pathways most likely to be controlled by these miRNAs were the transforming growth factor ß (TGFß), platelet-derived growth factor (PDGF), insulin-like growth factor (IGF) epidermal growth factor (EGF), and actin cytoskeletal pathway. CONCLUSION: We have demonstrated the presence of miRNA in the human IVD. The miRNA expression differs from muscle tissue and there are differences between the miRNA expressed in the NP and AF. The miRNAs identified control signaling pathways important for maintenance of the IVD. Future studies may determine the importance of miRNA in the development of IVD disease.
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Perfilación de la Expresión Génica , Regulación de la Expresión Génica , Degeneración del Disco Intervertebral/genética , Disco Intervertebral/patología , Vértebras Lumbares/patología , MicroARNs/genética , Humanos , Hibridación in Situ , MicroARNs/metabolismo , Músculos/metabolismo , Músculos/patología , Reacción en Cadena de la Polimerasa , Análisis de Componente Principal , Reproducibilidad de los Resultados , Transducción de Señal/genéticaRESUMEN
Oocytes become enclosed in primordial follicles during fetal life and remain dormant there until activation followed by growth and meiotic resumption. Current knowledge about the molecular pathways involved in oogenesis is incomplete. This study identifies the specific transcriptome of the human oocyte in the quiescent state and at the pinnacle of maturity at ovulation. In silico bioinformatic comparisons were made between the transcriptome of human oocytes from dormant primordial follicles and that of human metaphase II (MII) oocytes and granulosa cells and unique gene expression profiles were identified as well as functional and pathway enrichments associated with the oocytes from the two developmental hallmarks. A total of 729 genes were highly enriched in oocytes from primodial follicles and 1456 genes were highly enriched in MII oocytes (>10-fold, P < 0.001) representing functional categories such as cell cycle regulation, DNA protection and epigenetics, with representative genes validated by qPCR analysis. Dominating canonical pathways in the oocytes from primordial follicles were androgen, estrogen receptor, glucocorticoid receptor and PI3K/AKT signaling (P < 0.001). In the MII, mitotic roles of polo-like kinases, estrogen receptor, JAK/Stat signaling (P < 0.001) and the ERK/MAPK (P < 0.01) signaling were enriched. Some of the highly differentially expressed genes were completely new in human reproduction (CDR1, TLC1A, UHRF2) while other genes [ABO, FOLR1 (folate receptor), CHRNA3 (nicotine receptor)] may relate to clinical observations as diverse as premature ovarian failure, folic acid deficiency and smoking affecting female fertility. The in silico analysis identified novel reproduction-associated genes and highlighted molecular mechanisms and pathways associated with the unique functions of the human oocyte in its two extremes during folliculogenesis. The data provides a fundamental basis for future functional studies in regulation of human oogenesis.
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Regulación del Desarrollo de la Expresión Génica , Células de la Granulosa/metabolismo , Metafase , Oocitos/metabolismo , Oogénesis/genética , Transcriptoma , Ciclo Celular/genética , Epigénesis Genética , Quinasas MAP Reguladas por Señal Extracelular/genética , Quinasas MAP Reguladas por Señal Extracelular/metabolismo , Femenino , Perfilación de la Expresión Génica , Células de la Granulosa/citología , Humanos , Redes y Vías Metabólicas , Oocitos/citología , Receptores Androgénicos/genética , Receptores Androgénicos/metabolismo , Receptores de Estrógenos/genética , Receptores de Estrógenos/metabolismo , Receptores de Glucocorticoides/genética , Receptores de Glucocorticoides/metabolismo , Transducción de Señal , Factores de TiempoRESUMEN
AIMS: Uraemia is a strong risk factor for cardiovascular disease. Osteopontin (OPN) is highly expressed in aortas of uraemic apolipoprotein E knockout (E KO) mice. OPN affects key atherogenic processes, i.e. inflammation and phenotypic modulation of smooth muscle cells (SMCs). We explored the role of OPN on vascular pathology in uraemic mice. METHODS AND RESULTS: Uraemia was induced by 5/6 nephrectomy in E KO and in OPN and E double KO mice (E/OPN KO). In E KO mice, uraemia increased the relative surface plaque area in the aortic arch (from 28 ± 2% [n = 15], to 37 ± 3% [n = 20] of the aortic arch area, P < 0.05). A positive correlation was observed between plasma OPN and aortic atherosclerosis in uraemic E KO mice (r(2) = 0.48, P = 0.001). In contrast, aortic atherosclerosis was not increased by uraemia in E/OPN KO mice. OPN deficiency in haematopoietic cells (including macrophages) did not affect development of uraemic atherosclerosis, even though OPN-deficient foam cells had decreased inflammatory capacity. Gene expression analyses indicated that uraemia de-differentiates SMCs in the arterial wall. This effect was dampened in whole-body OPN-deficient mice. CONCLUSION: The data suggest that OPN promotes development of uraemic atherosclerosis possibly by changing the phenotype of vascular smooth muscle cells.
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Enfermedades de la Aorta/prevención & control , Aterosclerosis/prevención & control , Músculo Liso Vascular/metabolismo , Miocitos del Músculo Liso/metabolismo , Osteopontina/deficiencia , Uremia/complicaciones , Animales , Aorta Torácica/metabolismo , Aorta Torácica/patología , Enfermedades de la Aorta/etiología , Enfermedades de la Aorta/genética , Enfermedades de la Aorta/metabolismo , Enfermedades de la Aorta/mortalidad , Apolipoproteínas E/deficiencia , Apolipoproteínas E/genética , Aterosclerosis/etiología , Aterosclerosis/genética , Aterosclerosis/metabolismo , Aterosclerosis/patología , Desdiferenciación Celular , Células Cultivadas , Modelos Animales de Enfermedad , Genotipo , Inflamación/genética , Inflamación/metabolismo , Inflamación/prevención & control , Macrófagos/metabolismo , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Músculo Liso Vascular/patología , Miocitos del Músculo Liso/patología , Nefrectomía , Osteopontina/genética , Fenotipo , Placa Aterosclerótica , Uremia/genética , Uremia/metabolismoRESUMEN
Most adult mammalian tissues are quiescent, with rare cell divisions serving to maintain homeostasis. At present, the isolation and study of replicating cells from their in vivo niche typically involves immunostaining for intracellular markers of proliferation, causing the loss of sensitive biological material. We describe a transgenic mouse strain, expressing a CyclinB1-GFP fusion reporter, that marks replicating cells in the S/G2/M phases of the cell cycle. Using flow cytometry, we isolate live replicating cells from the liver and compare their transcriptome to that of quiescent cells to reveal gene expression programs associated with cell proliferation in vivo. We find that replicating hepatocytes have reduced expression of genes characteristic of liver differentiation. This reporter system provides a powerful platform for gene expression and metabolic and functional studies of replicating cells in their in vivo niche.
Asunto(s)
Proliferación Celular , Hepatocitos/citología , Transcripción Genética/genética , Transcriptoma , Animales , Biomarcadores/análisis , Biomarcadores/metabolismo , Ciclo Celular , Diferenciación Celular , Supervivencia Celular , Ciclina B1/genética , Ciclina B1/metabolismo , Citometría de Flujo , Proteínas Fluorescentes Verdes/genética , Proteínas Fluorescentes Verdes/metabolismo , Hepatocitos/metabolismo , Ratones , Ratones Transgénicos , Células 3T3 NIH , Análisis de Secuencia por Matrices de Oligonucleótidos , Reacción en Cadena en Tiempo Real de la PolimerasaRESUMEN
BACKGROUND: MYG1 (Melanocyte proliferating gene 1, also C12orf10 in human) is a ubiquitous nucleo-mitochondrial protein, involved in early developmental processes and in adult stress/illness conditions. We recently showed that MYG1 mRNA expression is elevated in the skin of vitiligo patients. Our aim was to examine nine known polymorphisms in the MYG1 gene, to investigate their functionality, and to study their association with vitiligo susceptibility. METHODS: Nine single nucleotide polymorphisms (SNPs) in the MYG1 locus were investigated by SNPlex assay and/or sequencing in vitiligo patients (n = 124) and controls (n = 325). MYG1 expression in skin biopsies was detected by quantitative-real time PCR (Q-RT-PCR) and polymorphisms were further analysed using luciferase and YFP reporters in the cell culture. RESULTS: Control subjects with -119G promoter allele (rs1465073) exhibited significantly higher MYG1 mRNA levels than controls with -119C allele (P = 0.01). Higher activity of -119G promoter was confirmed by luciferase assay. Single marker association analysis showed that the -119G allele was more frequent in vitiligo patients (47.1%) compared to controls (39.3%, P < 0.05, OR 1.37, 95%CI 1.02-1.85). Analysis based on the stage of progression of the vitiligo revealed that the increased frequency of -119G allele occurred prevalently in the group of patients with active vitiligo (n = 86) compared to the control group (48.2% versus 39.3%, P < 0.05; OR 1.44, 95%CI 1.02-2.03). Additionally, we showed that glutamine in the fourth position (in Arg4Gln polymorphism) completely eliminated mitochondrial entrance of YFP-tagged Myg1 protein in cell culture. The analysis of available EST, cDNA and genomic DNA sequences revealed that Myg1 4Gln allele is remarkably present in human populations but is never detected in homozygous state according to the HapMap database. CONCLUSIONS: Our study demonstrated that both MYG1 promoter polymorphism -119C/G and Arg4Gln polymorphism in the mitochondrial signal of Myg1 have a functional impact on the regulation of the MYG1 gene and promoter polymorphism (-119C/G) is related with suspectibility for actively progressing vitiligo.
Asunto(s)
Predisposición Genética a la Enfermedad , Mitocondrias/metabolismo , Polimorfismo Genético , Regiones Promotoras Genéticas , Proteínas/genética , Vitíligo/genética , Adolescente , Adulto , Anciano , Anciano de 80 o más Años , Sustitución de Aminoácidos , ADN/sangre , ADN/genética , ADN/aislamiento & purificación , Exonucleasas , Femenino , Humanos , Masculino , Persona de Mediana Edad , Polimorfismo de Nucleótido Simple , Proteínas/metabolismo , ARN Mensajero/genética , Valores de Referencia , Pigmentación de la Piel/genética , Adulto JovenRESUMEN
BACKGROUND INFORMATION: MYG1 [Melanocyte proliferating gene 1, also known as Gamm1 (NM_021640)] is a recently described gene of unknown function. MYG1 orthologues are found in simple as well as complex eukaryotes. According to sequence homology, MYG1 is considered to have a metal-dependent protein hydrolase (UPF0160) domain. The purpose of the present study was to determine the expression and subcellular localization of MYG1 protein and to identify physiological processes connected to MYG1 function. RESULTS: Human and mouse MYG1 is ubiquitously expressed, with the highest level in the testis. Analysis of mouse embryos moreover revealed a uniform Myg1 expression at E (embryonic day) 8.5, but at E11.75 expression becomes restricted predominantly to the developing brain and eye, limb buds and tail region. MYG1 exhibits a mitochondrial targeting signal in the N-terminal region and a Pat7-type nuclear localization signal in the region between amino acids 33-39 and localizes to these compartments. No active shuttling of MYG1 between the nucleus and the mitochondria was detected and the distribution of MYG1 was not dependent on the phase of the cell cycle. Immunoprecipitation of C-terminally FLAG-tagged MYG1 from HeLa cells did not identify any co-precipitated proteins. siRNA (short interfering RNA)-mediated knockdown of MYG1 mRNA was mainly followed by changes in the level of transcripts encoding factors involved in developmental tissue patterning and growth as well as immune-related processes. CONCLUSIONS: Taken together, we infer that MYG1 is a ubiquitous nucleo-mitochondrial protein, with differential pattern and level of expression during embryonic development. MYG1 expression in normal adult tissues is stable and our data suggest MYG1 involvement in early developmental processes and also in adult stress/illness conditions.