RESUMEN
Current therapies for most non-infectious diseases are directed at or affect functionality of the human translated genome, barely 2% of all genetic information. By contrast, the therapeutic potential of targeting the transcriptome, ~ 70% of the genome, remains largely unexplored. RNA therapeutics is an emerging field that widens the range of druggable targets and includes elements such as microRNA. Here, we sought to screen for microRNA with tumor-suppressive functions in neuroblastoma, an aggressive pediatric tumor of the sympathetic nervous system that requires the development of new therapies. We found miR-323a-5p and miR-342-5p to be capable of reducing cell proliferation in multiple neuroblastoma cell lines in vitro and in vivo, thereby providing a proof of concept for miRNA-based therapies for neuroblastoma. Furthermore, the combined inhibition of the direct identified targets such as CCND1, CHAF1A, INCENP and BCL-XL could reveal new vulnerabilities of high-risk neuroblastoma.
Asunto(s)
Regulación Neoplásica de la Expresión Génica , MicroARNs/genética , Neoplasias del Sistema Nervioso/genética , Neuroblastoma/genética , Animales , Línea Celular Tumoral , Proliferación Celular , Niño , Factor 1 de Ensamblaje de la Cromatina/genética , Factor 1 de Ensamblaje de la Cromatina/metabolismo , Proteínas Cromosómicas no Histona/genética , Proteínas Cromosómicas no Histona/metabolismo , Ciclina D1/genética , Ciclina D1/metabolismo , Femenino , Células HEK293 , Ensayos Analíticos de Alto Rendimiento , Humanos , Ratones , Ratones Desnudos , MicroARNs/metabolismo , Neoplasias del Sistema Nervioso/mortalidad , Neoplasias del Sistema Nervioso/patología , Neoplasias del Sistema Nervioso/terapia , Neuroblastoma/mortalidad , Neuroblastoma/patología , Neuroblastoma/terapia , Neuronas/metabolismo , Neuronas/patología , Unión Proteica , Transducción de Señal , Análisis de Supervivencia , Carga Tumoral , Ensayos Antitumor por Modelo de Xenoinjerto , Proteína bcl-X/genética , Proteína bcl-X/metabolismoRESUMEN
Systemic lupus erythematosus (SLE) is a complex disease targeting multiple organs as a result of overactivation of the type I interferon (IFN) system, a feature currently being targeted by multiple biologic therapies against IFN-α. We have identified an estrogen-regulated microRNA, miR-302d, whose expression is decreased in SLE patient monocytes and identify its target as interferon regulatory factor (IRF)-9, a critical component of the transcriptional complex that regulates expression of interferon-stimulated genes (ISGs). In keeping with the reduced expression of miR-302d in SLE patient monocytes, IRF9 levels were increased, as was expression of a number of ISGs including MX1 and OAS1. In vivo evaluation revealed that miR-302d protects against pristane-induced inflammation in mice by targeting IRF9 and hence ISG expression. Importantly, patients with enhanced disease activity have markedly reduced expression of miR-302d and enhanced IRF9 and ISG expression, with miR-302d negatively correlating with IFN score. Together these findings identify miR-302d as a key regulator of type I IFN driven gene expression via its ability to target IRF9 and regulate ISG expression, underscoring the importance of non-coding RNA in regulating the IFN pathway in SLE.
Asunto(s)
Regulación de la Expresión Génica , Subunidad gamma del Factor 3 de Genes Estimulados por el Interferón/genética , Lupus Eritematoso Sistémico/genética , MicroARNs/genética , Interferencia de ARN , Animales , Análisis por Conglomerados , Modelos Animales de Enfermedad , Estrógenos/farmacología , Perfilación de la Expresión Génica , Regulación de la Expresión Génica/efectos de los fármacos , Humanos , Interferón Tipo I/metabolismo , Lupus Eritematoso Sistémico/inmunología , Lupus Eritematoso Sistémico/metabolismo , Ratones , Monocitos/efectos de los fármacos , Monocitos/inmunología , Monocitos/metabolismo , Transducción de Señal/efectos de los fármacosRESUMEN
Defects within a self-assembled monolayer (SAM) of dodecanethiol on gold have been used as nucleation sites for the electrodeposition of mushroom shaped platinum nanoparticles (PtNPs). The top surfaces of these PtNPs were then decorated with a layer of silver creating a hemispherical - platinum : silver core : shell nanoparticle (Pt-AgNP). Thiolated probe strand miRNA was then immobilised onto the upper silver surface. These regioselectively modified particles were desorbed by applying a current jump to yield nanoparticles capable of hybridising to a complementary miRNA target with electrocatalysis occurring on the non-functionalized lower surface. A second electrode was functionalized with single stranded capture miRNA that has a sequence that is complementary to an miRNA, miR-132, associated with the childhood cancer, Neuroblastoma but leaves a section of the target available to bind the nucleic acid sequence on the core : shell Pt-AgNPs. Following hybridization of the target and capture strands the surface was exposed to the miRNA labelled electrocatalytic Pt-AgNPs. The concentration of the target was then determined by monitoring the current associated with the reduction of hydrogen peroxide in a solution of H2SO4. Calibration plots of the log[miRNA] vs. faradaic current were linear from 1 aM to 1 µM and aM concentrations could be detected without the need for chemical amplification of the target, e.g., using PCR or NASBA. The regioselectively modified particles were also immobilised within the interior of gold microcavity arrays via miRNA hybridisation and their Raman properties investigated.
Asunto(s)
Nanopartículas del Metal , MicroARNs/análisis , Platino (Metal) , PlataRESUMEN
Disturbance of homeostasis within the endoplasmic reticulum (ER) lumen leads to the accumulation of unfolded and misfolded proteins. This results in the activation of an evolutionary conserved stress response termed ER stress that, if unresolved, induces apoptosis. Previously the Bcl-2 homology domain 3-Only Protein Puma was identified as a mediator of ER stress-induced apoptosis in neurons. In the search of alternative contributors to ER stress-induced apoptosis, a downregulation of the anti-apoptotic Bcl-2 family protein Mcl-1 was noted during ER stress in both mouse cortical neurons and human SH-SY5Y neuroblastoma cells. Downregulation of Mcl-1 was associated with an upregulation of microRNA-29a (miR-29a) expression, and subsequent experiments showed that miR-29a targeted the 3'-untranslated region of the anti-apoptotic Bcl-2 family protein, Mcl-1. Inhibition of miR-29a expression using sequence-specific antagomirs or the overexpression of Mcl-1 decreased cell death following tunicamycin treatment, while gene silencing of Mcl-1 increased cell death. miR-29a did not alter the signalling branches of the ER stress response, rather its expression was controlled by the ER stress-induced transcription factor activating-transcription-factor-4 (ATF4). The current data demonstrate that the ATF4-mediated upregulation of miR-29a enhances the sensitivity of neurons to ER stress-induced apoptosis.
Asunto(s)
Apoptosis , Estrés del Retículo Endoplásmico , MicroARNs/genética , Neuronas/metabolismo , Regulación hacia Arriba , Factor de Transcripción Activador 4/genética , Factor de Transcripción Activador 4/metabolismo , Animales , Proteínas Reguladoras de la Apoptosis/genética , Proteínas Reguladoras de la Apoptosis/metabolismo , Células Cultivadas , Células HeLa , Humanos , Ratones , Ratones Endogámicos C57BL , Proteína 1 de la Secuencia de Leucemia de Células Mieloides/genética , Proteína 1 de la Secuencia de Leucemia de Células Mieloides/metabolismo , Proteínas Supresoras de Tumor/genética , Proteínas Supresoras de Tumor/metabolismoRESUMEN
Temporal lobe epilepsy is associated with large-scale, wide-ranging changes in gene expression in the hippocampus. Epigenetic changes to DNA are attractive mechanisms to explain the sustained hyperexcitability of chronic epilepsy. Here, through methylation analysis of all annotated C-phosphate-G islands and promoter regions in the human genome, we report a pilot study of the methylation profiles of temporal lobe epilepsy with or without hippocampal sclerosis. Furthermore, by comparative analysis of expression and promoter methylation, we identify methylation sensitive non-coding RNA in human temporal lobe epilepsy. A total of 146 protein-coding genes exhibited altered DNA methylation in temporal lobe epilepsy hippocampus (n = 9) when compared to control (n = 5), with 81.5% of the promoters of these genes displaying hypermethylation. Unique methylation profiles were evident in temporal lobe epilepsy with or without hippocampal sclerosis, in addition to a common methylation profile regardless of pathology grade. Gene ontology terms associated with development, neuron remodelling and neuron maturation were over-represented in the methylation profile of Watson Grade 1 samples (mild hippocampal sclerosis). In addition to genes associated with neuronal, neurotransmitter/synaptic transmission and cell death functions, differential hypermethylation of genes associated with transcriptional regulation was evident in temporal lobe epilepsy, but overall few genes previously associated with epilepsy were among the differentially methylated. Finally, a panel of 13, methylation-sensitive microRNA were identified in temporal lobe epilepsy including MIR27A, miR-193a-5p (MIR193A) and miR-876-3p (MIR876), and the differential methylation of long non-coding RNA documented for the first time. The present study therefore reports select, genome-wide DNA methylation changes in human temporal lobe epilepsy that may contribute to the molecular architecture of the epileptic brain.
Asunto(s)
Metilación de ADN/genética , Epigénesis Genética , Epilepsia del Lóbulo Temporal/patología , Hipocampo/patología , Adolescente , Adulto , Biología Computacional , Islas de CpG/fisiología , Epilepsia del Lóbulo Temporal/genética , Femenino , Regulación de la Expresión Génica , Hipocampo/metabolismo , Humanos , Inmunoprecipitación , Masculino , MicroARNs/metabolismo , Microdisección , Persona de Mediana Edad , Proyectos Piloto , Regiones Promotoras Genéticas , Esclerosis , Adulto JovenRESUMEN
MicroRNAs (miRNAs) are small non-coding RNA molecules that regulate gene expression at a post-transcriptional level. An miRNA may target many messenger RNA (mRNA) transcripts, and each transcript may be targeted by multiple miRNAs. Our understanding of miRNA regulation is evolving to consider modules of miRNAs that regulate groups of functionally related mRNAs. Here we expand the model of miRNA functional modules and use it to guide the integration of miRNA and mRNA expression and target prediction data. We present evidence of cooperativity between miRNA classes within this integrated miRNA-mRNA association matrix. We then apply bicluster analysis to uncover miRNA functional modules within this integrated data set and develop a novel application to visualize and query these results. We show that this wholly unsupervised approach can discover a network of miRNA-mRNA modules that are enriched for both biological processes and miRNA classes. We apply this method to investigate the interplay of miRNAs and mRNAs in integrated data sets derived from neuroblastoma and human immune cells. This study is the first to apply the technique of biclustering to model functional modules within an integrated miRNA-mRNA association matrix. Results provide evidence of an extensive modular miRNA functional network and enable characterization of miRNA function and dysregulation in disease.
Asunto(s)
MicroARNs/metabolismo , Modelos Genéticos , ARN Mensajero/metabolismo , Análisis por Conglomerados , Gráficos por Computador , Perfilación de la Expresión Génica , Regulación de la Expresión Génica , Humanos , Sistema Inmunológico/metabolismo , MicroARNs/clasificación , Neuroblastoma/genética , Neuroblastoma/metabolismo , Programas InformáticosRESUMEN
The acquisition of multidrug resistance is a major impediment to the successful treatment of neuroblastoma, a clinically heterogeneous cancer accounting for â¼15% of all pediatric cancer deaths. The MYCN transcription factor, whose gene is amplified in â¼30% of high-risk neuroblastoma cases, influences drug resistance by regulating a cadre of genes, including those involved with drug efflux, however, other high-risk subtypes of neuroblastoma lacking MYCN amplification, such as those with chromosome 11q deletions, also acquire multidrug resistance. To elucidate additional mechanisms involved with drug resistance in non-MYCN amplified tumour cells, an SK-N-AS subline (SK-N-AsCis24) that is significantly resistant to cisplatin and cross resistant to etoposide was developed through a pulse-selection process. High resolution aCGH analysis of SK-N-AsCis24 revealed a focal gain on chromosome 5 containing the coding sequence for the neural apoptosis inhibitory protein (NAIP). Significant overexpression of NAIP mRNA and protein was documented, while experimental modulation of NAIP levels in both SK-N-AsCis24 and in parental SK-N-AS cells confirmed that NAIP was responsible for the drug resistant phenotype by apoptosis inhibition. Furthermore, a decrease in the NAIP targeting microRNA, miR-520f, was also demonstrated to be partially responsible for increased NAIP levels in SK-N-AsCis24. Interestingly, miR-520f levels were determined to be significantly lower in postchemotherapy treatment tumours relative to matched prechemotherapy samples, consistent with a role for this miRNA in the acquisition of drug resistance in vivo, potentially through decreased NAIP targeting. Our findings provide biological novel insight into neuroblastoma drug-resistance and have implications for future therapeutic research.
Asunto(s)
Resistencia a Antineoplásicos/genética , MicroARNs/genética , Neuroblastoma/genética , Proteína Inhibidora de la Apoptosis Neuronal/genética , Antineoplásicos/farmacología , Antineoplásicos/uso terapéutico , Línea Celular Tumoral , Cisplatino/farmacología , Hibridación Genómica Comparativa , Expresión Génica , Regulación Neoplásica de la Expresión Génica , Genómica , Humanos , Neuroblastoma/tratamiento farmacológico , Fenotipo , Interferencia de ARNRESUMEN
While a polymorphism located within the promoter region of the MDM2 proto-oncogene, SNP309 (T > G), has previously been associated with increased risk and aggressiveness of neuroblastoma and other tumor entities, a protective effect has also been reported in certain other cancers. In this study, we evaluated the association of MDM2 SNP309 with outcome in 496 patients with neuroblastoma and its effect on MDM2 expression. No significant difference in overall or event-free survival was observed among patients with neuroblastoma with or without MDM2 SNP309. The presence of SNP309 does not affect MDM2 expression in neuroblastoma.
Asunto(s)
Neuroblastoma/genética , Neuroblastoma/mortalidad , Polimorfismo de Nucleótido Simple , Regiones Promotoras Genéticas , Proteínas Proto-Oncogénicas c-mdm2/genética , Supervivencia sin Enfermedad , Predisposición Genética a la Enfermedad , Genotipo , Humanos , Estimación de Kaplan-Meier , Pronóstico , Regiones Promotoras Genéticas/genética , Proto-Oncogenes MasRESUMEN
Prolonged seizures (status epilepticus) produce pathophysiological changes in the hippocampus that are associated with large-scale, wide-ranging changes in gene expression. Epileptic tolerance is an endogenous program of cell protection that can be activated in the brain by previous exposure to a non-harmful seizure episode before status epilepticus. A major transcriptional feature of tolerance is gene downregulation. Here, through methylation analysis of 34,143 discrete loci representing all annotated CpG islands and promoter regions in the mouse genome, we report the genome-wide DNA methylation changes in the hippocampus after status epilepticus and epileptic tolerance in adult mice. A total of 321 genes showed altered DNA methylation after status epilepticus alone or status epilepticus that followed seizure preconditioning, with >90% of the promoters of these genes undergoing hypomethylation. These profiles included genes not previously associated with epilepsy, such as the polycomb gene Phc2. Differential methylation events generally occurred throughout the genome without bias for a particular chromosomal region, with the exception of a small region of chromosome 4, which was significantly overrepresented with genes hypomethylated after status epilepticus. Surprisingly, only few genes displayed differential hypermethylation in epileptic tolerance. Nevertheless, gene ontology analysis emphasized the majority of differential methylation events between the groups occurred in genes associated with nuclear functions, such as DNA binding and transcriptional regulation. The present study reports select, genome-wide DNA methylation changes after status epilepticus and in epileptic tolerance, which may contribute to regulating the gene expression environment of the seizure-damaged hippocampus.
Asunto(s)
Región CA3 Hipocampal/metabolismo , Metilación de ADN/genética , Estado Epiléptico/genética , Estado Epiléptico/metabolismo , Animales , Regulación hacia Abajo/genética , Estudio de Asociación del Genoma Completo/métodos , Masculino , Ratones , Ratones Endogámicos C57BL , Estado Epiléptico/prevención & controlRESUMEN
BACKGROUND: Neuroblastoma is responsible for 15% of all childhood cancer deaths. Despite advances in treatment and disease management, the overall 5-year survival rates remain poor in high-risk disease (25-40%). MiR-497 was previously identified by our laboratory as a member of a miRNA expression signature, predictive of neuroblastoma patient survival and has been reported as a tumor suppressor in a variety of other cancers. WEE1, a tyrosine kinase regulator of the cell cycle and predicted target of miR-497, has emerged as an oncogene in several cancer types and therefore represents an attractive potential target for novel therapy approaches in high-risk neuroblastoma. Our aim was to investigate the potential tumor suppressive role of miR-497 in high-risk neuroblastoma. METHODS: Expression levels of miR-497 and WEE1 in tissues and cells were determined using RT-PCR. The effect of miR-497 and siWEE1 on cell viability was evaluated using MTS assays, apoptosis levels were determined using FACS analysis of Annexin V/PI stained cells, and target protein expression was determined using western blot. Luciferase reporter plasmids were constructed to confirm direct targeting. Results were reported as mean±S.E.M and differences were tested for significance using 2-tailed Students t-test. RESULTS: We determined that miR-497 expression was significantly lower in high-risk MYCN amplified (MNA) tumors and that low miR-497 expression was associated with worse EFS and OS in our cohort. Over-expression of miR-497 reduced cell viability and increased apoptosis in MNA cells. We identified WEE1 as a novel target for miR-497 in neuroblastoma. Furthermore, our analysis showed that high WEE1 levels are significantly associated with poor EFS and OS in neuroblastoma and that siRNA knockdown of WEE1 in MNA cell lines results in significant levels of apoptosis, supporting an oncogenic role of WEE1 in neuroblastoma. Cisplatin (CDDP) treatment of both miR-497 over-expressing cells and WEE1 inhibited cells, resulted in a significant increase in apoptosis in MNA cells, describing a synergistic effect and therefore a potential therapeutic for high-risk neuroblastoma. CONCLUSION: Our study's results are consistent with miR-497 being a candidate tumor suppressor in neuroblastoma, through the direct targeting of WEE1. These findings re-enforce the proposal of WEE1 as a therapeutic target in neuroblastoma.
Asunto(s)
Proteínas de Ciclo Celular/genética , Amplificación de Genes , MicroARNs/metabolismo , Neuroblastoma/metabolismo , Proteínas Nucleares/genética , Proteínas Oncogénicas/genética , Proteínas Tirosina Quinasas/genética , Regiones no Traducidas 3' , Antineoplásicos/farmacología , Apoptosis/efectos de los fármacos , Proteínas de Ciclo Celular/metabolismo , Línea Celular Tumoral , Supervivencia Celular , Cisplatino/farmacología , Expresión Génica , Regulación Neoplásica de la Expresión Génica , Técnicas de Silenciamiento del Gen , Humanos , Lactante , Estimación de Kaplan-Meier , MicroARNs/genética , MicroARNs/fisiología , Análisis Multivariante , Proteína Proto-Oncogénica N-Myc , Neuroblastoma/mortalidad , Proteínas Nucleares/metabolismo , Modelos de Riesgos Proporcionales , Proteínas Tirosina Quinasas/metabolismo , ARN Interferente Pequeño/genéticaRESUMEN
BACKGROUND: Neuroblastoma (NB) tumours are commonly divided into three cytogenetic subgroups. However, by unsupervised principal components analysis of gene expression profiles we recently identified four distinct subgroups, r1-r4. In the current study we characterized these different subgroups in more detail, with a specific focus on the fourth divergent tumour subgroup (r4). METHODS: Expression microarray data from four international studies corresponding to 148 neuroblastic tumour cases were subject to division into four expression subgroups using a previously described 6-gene signature. Differentially expressed genes between groups were identified using Significance Analysis of Microarray (SAM). Next, gene expression network modelling was performed to map signalling pathways and cellular processes representing each subgroup. Findings were validated at the protein level by immunohistochemistry and immunoblot analyses. RESULTS: We identified several significantly up-regulated genes in the r4 subgroup of which the tyrosine kinase receptor ERBB3 was most prominent (fold change: 132-240). By gene set enrichment analysis (GSEA) the constructed gene network of ERBB3 (n = 38 network partners) was significantly enriched in the r4 subgroup in all four independent data sets. ERBB3 was also positively correlated to the ErbB family members EGFR and ERBB2 in all data sets, and a concurrent overexpression was seen in the r4 subgroup. Further studies of histopathology categories using a fifth data set of 110 neuroblastic tumours, showed a striking similarity between the expression profile of r4 to ganglioneuroblastoma (GNB) and ganglioneuroma (GN) tumours. In contrast, the NB histopathological subtype was dominated by mitotic regulating genes, characterizing unfavourable NB subgroups in particular. The high ErbB3 expression in GN tumour types was verified at the protein level, and showed mainly expression in the mature ganglion cells. CONCLUSIONS: Conclusively, this study demonstrates the importance of performing unsupervised clustering and subtype discovery of data sets prior to analyses to avoid a mixture of tumour subtypes, which may otherwise give distorted results and lead to incorrect conclusions. The current study identifies ERBB3 as a clear-cut marker of a GNB/GN-like expression profile, and we suggest a 7-gene expression signature (including ERBB3) as a complement to histopathology analysis of neuroblastic tumours. Further studies of ErbB3 and other ErbB family members and their role in neuroblastic differentiation and pathogenesis are warranted.
Asunto(s)
Biomarcadores de Tumor/metabolismo , Ganglioneuroblastoma/metabolismo , Ganglioneuroma/metabolismo , Neoplasias del Sistema Nervioso Periférico/metabolismo , Receptor ErbB-3/metabolismo , Biomarcadores de Tumor/genética , Regulación Neoplásica de la Expresión Génica , Ontología de Genes , Redes Reguladoras de Genes , Humanos , Análisis de Secuencia por Matrices de Oligonucleótidos , Receptor ErbB-3/genética , Transcriptoma , Regulación hacia ArribaRESUMEN
Neuroblastoma is the most common extracranial solid tumor of childhood, and accounts for â¼15% of all childhood cancer deaths. The histone demethylase, lysine-specific demethylase 1 (KDM1A, previously known as LSD1), is strongly expressed in neuroblastomas, and overexpression correlates with poor patient prognosis. Inducing differentiation in neuroblastoma cells has previously been shown to down regulate KDM1A, and siRNA-mediated KDM1A knockdown inhibited neuroblastoma cell viability. The microRNA, miR-137, has been reported to be downregulated in several human cancers, and KDM1A mRNA was reported as a putative target of miR-137 in colon cancer. We hypothesized that miR-137 might have a tumor-suppressive role in neuroblastoma mediated via downregulation of KDM1A. Indeed, low levels of miR-137 expression in primary neuroblastomas correlated with poor patient prognosis. Re-expressing miR-137 in neuroblastoma cell lines increased apoptosis and decreased cell viability and proliferation. KDM1A mRNA was repressed by miR-137 in neuroblastoma cells, and was validated as a direct target of miR-137 using reporter assays in SHEP and HEK293 cells. Furthermore, siRNA-mediated KDM1A knockdown phenocopied the miR-137 re-expression phenotype in neuroblastoma cells. We conclude that miR-137 directly targets KDM1A mRNA in neuroblastoma cells, and activates cell properties consistent with tumor suppression. Therapeutic strategies to re-express miR-137 in neuroblastomas could be useful to reduce tumor aggressiveness.
Asunto(s)
Genes Supresores de Tumor , Histona Demetilasas/genética , MicroARNs/fisiología , Neuroblastoma/genética , Línea Celular Tumoral , Supervivencia Celular , Regulación hacia Abajo , Histona Demetilasas/fisiología , Humanos , MicroARNs/análisisRESUMEN
BACKGROUND: Ultra-conserved regions (UCRs) are segments of the genome (≥ 200 bp) that exhibit 100% DNA sequence conservation between human, mouse and rat. Transcribed UCRs (T-UCRs) have been shown to be differentially expressed in cancers versus normal tissue, indicating a possible role in carcinogenesis. All-trans-retinoic acid (ATRA) causes some neuroblastoma (NB) cell lines to undergo differentiation and leads to a significant decrease in the oncogenic transcription factor MYCN. Here, we examine the impact of ATRA treatment on T-UCR expression and investigate the biological significance of these changes. METHODS: We designed a custom tiling microarray to profile the expression of 481 T-UCRs in sense and anti-sense orientation (962 potential transcripts) in untreated and ATRA-treated neuroblastoma cell lines (SH-SY5Y, SK-N-BE, LAN-5). Following identification of significantly differentially expressed T-UCRs, we carried out siRNA knockdown and gene expression microarray analysis to investigate putative functional roles for selected T-UCRs. RESULTS: Following ATRA-induced differentiation, 32 T-UCRs were differentially expressed (16 up-regulated, 16 down-regulated) across all three cell lines. Further insight into the possible role of T-UC.300A, an independent transcript whose expression is down-regulated following ATRA was achieved by siRNA knockdown, resulting in the decreased viability and invasiveness of ATRA-responsive cell lines. Gene expression microarray analysis following knockdown of T-UC.300A revealed a number of genes whose expression was altered by changing T-UC.300A levels and that might play a role in the increased proliferation and invasion of NB cells prior to ATRA-treatment. CONCLUSIONS: Our results indicate that significant numbers of T-UCRs have altered expression levels in response to ATRA. While the precise roles that T-UCRs might play in cancer or in normal development are largely unknown and an important area for future study, our findings strongly indicate that the function of non-coding RNA T-UC.300A is connected with proliferation, invasion and the inhibition of differentiation of neuroblastoma cell lines prior to ATRA treatment.
Asunto(s)
Antineoplásicos/farmacología , Neuroblastoma/genética , Neuroblastoma/patología , ARN no Traducido/genética , Tretinoina/farmacología , Línea Celular Tumoral , Análisis por Conglomerados , Secuencia Conservada , Perfilación de la Expresión Génica , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Técnicas de Silenciamiento del Gen , Humanos , Clasificación del Tumor , Interferencia de ARN , ARN no Traducido/química , ARN no Traducido/metabolismo , Reproducibilidad de los Resultados , Transcripción GenéticaRESUMEN
Neuroblastoma is a highly heterogeneous tumor accounting for 15 % of all pediatric cancer deaths. Clinical behavior ranges from the spontaneous regression of localized, asymptomatic tumors, as well as metastasized tumors in infants, to rapid progression and resistance to therapy. Genomic amplification of the MYCN oncogene has been used to predict outcome in neuroblastoma for over 30 years, however, recent methodological advances including miRNA and mRNA profiling, comparative genomic hybridization (array-CGH), and whole-genome sequencing have enabled the detailed analysis of the neuroblastoma genome, leading to the identification of new prognostic markers and better patient stratification. In this review, we will describe the main genetic factors responsible for these diverse clinical phenotypes in neuroblastoma, the chronology of their discovery, and the impact on patient prognosis.
Asunto(s)
Epigenómica/métodos , Neuroblastoma/genética , Niño , Deleción Cromosómica , Mapeo Cromosómico/métodos , HumanosRESUMEN
Many neuroblastoma cell lines can be induced to differentiate into a mature neuronal cell type with retinoic acid and other compounds, providing an important model system for elucidating signalling pathways involved in this highly complex process. Recently, it has become apparent that miRNAs, which act as regulators of gene expression at a post-transcriptional level, are differentially expressed in differentiating cells and play important roles governing many aspects of this process. This includes the down-regulation of DNA methyltransferases that cause the de-methylation and transcriptional activation of numerous protein coding gene sequences. The purpose of this article is to review involvement of miRNAs and DNA methylation alterations in the process of neuroblastoma cell differentiation. A thorough understanding of miRNA and genetic pathways regulating neuroblastoma cell differentiation potentially could lead to targeted therapies for this disease.
Asunto(s)
Antineoplásicos/farmacología , Diferenciación Celular/genética , Metilación de ADN/genética , MicroARNs/genética , Neuroblastoma/genética , Tretinoina/farmacología , Animales , Diferenciación Celular/efectos de los fármacos , Metilación de ADN/efectos de los fármacos , Humanos , MicroARNs/efectos de los fármacos , Neuroblastoma/patologíaRESUMEN
[This corrects the article DOI: 10.1371/journal.pone.0078428.].
RESUMEN
Transforming growth factor-ß (TGF-ß) signaling regulates many diverse cellular activities through both canonical (SMAD-dependent) and non-canonical branches, which includes the mitogen-activated protein kinase (MAPK), Rho-like guanosine triphosphatase and phosphatidylinositol-3-kinase/AKT pathways. Here, we demonstrate that miR-335 directly targets and downregulates genes in the TGF-ß non-canonical pathways, including the Rho-associated coiled-coil containing protein (ROCK1) and MAPK1, resulting in reduced phosphorylation of downstream pathway members. Specifically, inhibition of ROCK1 and MAPK1 reduces phosphorylation levels of the motor protein myosin light chain (MLC) leading to a significant inhibition of the invasive and migratory potential of neuroblastoma cells. Additionally, miR-335 targets the leucine-rich alpha-2-glycoprotein 1 (LRG1) messenger RNA, which similarly results in a significant reduction in the phosphorylation status of MLC and a decrease in neuroblastoma cell migration and invasion. Thus, we link LRG1 to the migratory machinery of the cell, altering its activity presumably by exerting its effect within the non-canonical TGF-ß pathway. Moreover, we demonstrate that the MYCN transcription factor, whose coding sequence is highly amplified in a particularly clinically aggressive neuroblastoma tumor subtype, directly binds to a region immediately upstream of the miR-335 transcriptional start site, resulting in transcriptional repression. We conclude that MYCN contributes to neuroblastoma cell migration and invasion, by directly downregulating miR-335, resulting in the upregulation of the TGF-ß signaling pathway members ROCK1, MAPK1 and putative member LRG1, which positively promote this process. Our results provide novel insight into the direct regulation of TGF-ß non-canonical signaling by miR-335, which in turn is downregulated by MYCN.
Asunto(s)
MicroARNs/genética , MicroARNs/metabolismo , Neuroblastoma/genética , Neuroblastoma/metabolismo , Factor de Crecimiento Transformador beta/genética , Factor de Crecimiento Transformador beta/metabolismo , Línea Celular Tumoral , Movimiento Celular/genética , Progresión de la Enfermedad , Regulación hacia Abajo , Glicoproteínas/antagonistas & inhibidores , Glicoproteínas/genética , Glicoproteínas/metabolismo , Humanos , Proteína Quinasa 1 Activada por Mitógenos/antagonistas & inhibidores , Proteína Quinasa 1 Activada por Mitógenos/genética , Proteína Quinasa 1 Activada por Mitógenos/metabolismo , Cadenas Ligeras de Miosina/genética , Cadenas Ligeras de Miosina/metabolismo , Proteína Proto-Oncogénica N-Myc , Invasividad Neoplásica , Neuroblastoma/patología , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Proteínas Oncogénicas/genética , Proteínas Oncogénicas/metabolismo , Fosforilación , Procesamiento Proteico-Postraduccional , Transducción de Señal , Factores de Transcripción/genética , Factores de Transcripción/metabolismo , Regulación hacia Arriba , Quinasas Asociadas a rho/antagonistas & inhibidores , Quinasas Asociadas a rho/genética , Quinasas Asociadas a rho/metabolismoRESUMEN
BACKGROUND: Protein tyrosine phosphatase receptor delta (PTPRD) is a member of a large family of protein tyrosine phosphatases which negatively regulate tyrosine phosphorylation. Neuroblastoma is a major childhood cancer arising from precursor cells of the sympathetic nervous system which is known to acquire deletions and alterations in the expression patterns of PTPRD, indicating a potential tumor suppressor function for this gene. The molecular mechanism, however, by which PTPRD renders a tumor suppressor effect in neuroblastoma is unknown. RESULTS: As a molecular mechanism, we demonstrate that PTPRD interacts with aurora kinase A (AURKA), an oncogenic protein that is over-expressed in multiple forms of cancer, including neuroblastoma. Ectopic up-regulation of PTPRD in neuroblastoma dephosphorylates tyrosine residues in AURKA resulting in a destabilization of this protein culminating in interfering with one of AURKA's primary functions in neuroblastoma, the stabilization of MYCN protein, the gene of which is amplified in approximately 25 to 30% of high risk neuroblastoma. CONCLUSIONS: PTPRD has a tumor suppressor function in neuroblastoma through AURKA dephosphorylation and destabilization and a downstream destabilization of MYCN protein, representing a novel mechanism for the function of PTPRD in neuroblastoma.
Asunto(s)
Neuroblastoma/genética , Proteínas Oncogénicas/genética , Proteínas Serina-Treonina Quinasas/genética , Proteínas Tirosina Fosfatasas Clase 2 Similares a Receptores/genética , Proteínas Supresoras de Tumor/genética , Apoptosis/genética , Aurora Quinasa A , Aurora Quinasas , Línea Celular Tumoral , Estabilidad de Enzimas , Expresión Génica , Perfilación de la Expresión Génica , Humanos , Neuroblastoma/mortalidad , Proteínas Oncogénicas/metabolismo , Fosforilación , Unión Proteica , Proteínas Serina-Treonina Quinasas/metabolismo , Proteínas Tirosina Fosfatasas Clase 2 Similares a Receptores/metabolismo , Proteínas Supresoras de Tumor/metabolismo , Tirosina/metabolismoRESUMEN
Neuroblastoma is an aggressive embryonal tumor that accounts for â¼15% of childhood cancer deaths. Hitherto, despite the availability of comprehensive genomic data on DNA copy number changes in neuroblastoma, relatively little is known about the genes driving neuroblastoma tumorigenesis. In this study, high resolution array comparative genome hybridization (CGH) was performed on 188 primary neuroblastoma tumors and 33 neuroblastoma cell lines to search for previously undetected recurrent DNA copy number gains and losses. A new recurrent distal chromosome 1q deletion (del(1)(q42.2qter)) was detected in seven cases. Further analysis of available array CGH datasets revealed 13 additional similar distal 1q deletions. The majority of all detected 1q deletions was found in high risk 11q deleted tumors without MYCN amplification (Fisher exact test p = 5.61 × 10(-5) ). Using ultra-high resolution (â¼115 bp resolution) custom arrays covering the breakpoints on 1q for 11 samples, clustering of nine breakpoints was observed within a 12.5-kb region, of which eight were found in a 7-kb copy number variable region, whereas the remaining two breakpoints were colocated 1.4-Mb proximal. The commonly deleted region contains one miRNA (hsa-mir-1537), four transcribed ultra conserved region elements (uc.43-uc.46) and 130 protein coding genes including at least two bona fide tumor suppressor genes, EGLN1 (or PHD2) and FH. This finding further contributes to the delineation of the genomic profile of aggressive neuroblastoma, offers perspectives for the identification of genes contributing to the disease phenotype and may be relevant in the light of assessment of response to new molecular treatments.
Asunto(s)
Deleción Cromosómica , Cromosomas Humanos Par 11 , Dosificación de Gen , Neuroblastoma/genética , Proteínas Nucleares/genética , Proteínas Oncogénicas/genética , Línea Celular Tumoral , Hibridación Genómica Comparativa , Fumarato Hidratasa/genética , Humanos , Prolina Dioxigenasas del Factor Inducible por Hipoxia , Proteína Proto-Oncogénica N-Myc , Procolágeno-Prolina Dioxigenasa/genéticaRESUMEN
When an otherwise harmful insult to the brain is preceded by a brief, noninjurious stimulus, the brain becomes tolerant, and the resulting damage is reduced. Epileptic tolerance develops when brief seizures precede an episode of prolonged seizures (status epilepticus). MicroRNAs (miRNAs) are small, noncoding RNAs that function as post-transcriptional regulators of gene expression. We investigated how prior seizure preconditioning affects the miRNA response to status epilepticus evoked by intra-amygdalar kainic acid in mice. The miRNA was extracted from the ipsilateral CA3 subfield 24 hours after focal-onset status epilepticus in animals that had previously received either seizure preconditioning (tolerance) or no preconditioning (injury), and mature miRNA levels were measured using TaqMan low-density arrays. Expression of 21 miRNAs was increased, relative to control, after status epilepticus alone, and expression of 12 miRNAs was decreased. Increased miR-132 levels were matched with increased binding to Argonaute-2, a constituent of the RNA-induced silencing complex. In tolerant animals, expression responses of >40% of the injury-group-detected miRNAs differed, being either unchanged relative to control or down-regulated, and this included miR-132. In vivo microinjection of locked nucleic acid-modified oligonucleotides (antagomirs) against miR-132 depleted hippocampal miR-132 levels and reduced seizure-induced neuronal death. Thus, our data strongly suggest that miRNAs are important regulators of seizure-induced neuronal death.