RESUMEN
An amendment to this paper has been published and can be accessed via a link at the top of the paper.
RESUMEN
High-dose radiation is the main component of glioblastoma therapy. Unfortunately, radio-resistance is a common problem and a major contributor to tumor relapse. Understanding the molecular mechanisms driving response to radiation is critical for identifying regulatory routes that could be targeted to improve treatment response. We conducted an integrated analysis in the U251 and U343 glioblastoma cell lines to map early alterations in the expression of genes at three levels: transcription, splicing, and translation in response to ionizing radiation. Changes at the transcriptional level were the most prevalent response. Downregulated genes are strongly associated with cell cycle and DNA replication and linked to a coordinated module of expression. Alterations in this group are likely driven by decreased expression of the transcription factor FOXM1 and members of the E2F family. Genes involved in RNA regulatory mechanisms were affected at the mRNA, splicing, and translation levels, highlighting their importance in radiation-response. We identified a number of oncogenic factors, with an increased expression upon radiation exposure, including BCL6, RRM2B, IDO1, FTH1, APIP, and LRIG2 and lncRNAs NEAT1 and FTX. Several of these targets have been previously implicated in radio-resistance. Therefore, antagonizing their effects post-radiation could increase therapeutic efficacy. Our integrated analysis provides a comprehensive view of early response to radiation in glioblastoma. We identify new biological processes involved in altered expression of various oncogenic factors and suggest new target options to increase radiation sensitivity and prevent relapse.
Asunto(s)
Neoplasias Encefálicas/genética , Neoplasias Encefálicas/radioterapia , Regulación Neoplásica de la Expresión Génica/efectos de la radiación , Glioblastoma/genética , Glioblastoma/radioterapia , Empalme del ARN/genética , Transcripción Genética/genética , Neoplasias Encefálicas/patología , Ciclo Celular/genética , Línea Celular Tumoral , Replicación del ADN/genética , Proteína Forkhead Box M1/genética , Proteína Forkhead Box M1/metabolismo , Expresión Génica , Glioblastoma/patología , Humanos , Proteínas Oncogénicas/genética , Proteínas Oncogénicas/metabolismo , Biosíntesis de Proteínas/genética , Tolerancia a Radiación , Radiación Ionizante , Dosificación RadioterapéuticaRESUMEN
The conserved RNA-binding protein Musashi1 (MSI1) has been characterized as a stem cell marker, controlling the balance between self-renewal and differentiation and as a key oncogenic factor in numerous solid tumors, including glioblastoma. To explore the potential use of MSI1 targeting in therapy, we studied MSI1 in the context of radiation sensitivity. Knockdown of MSI1 led to a decrease in cell survival and an increase in DNA damage compared to control in cells treated with ionizing radiation. We subsequently examined mechanisms of double-strand break repair and found that loss of MSI1 reduces the frequency of nonhomologous end-joining. This phenomenon could be attributed to the decreased expression of DNA-protein kinase catalytic subunit, which we have previously identified as a target of MSI1. Collectively, our results suggest a role for MSI1 in double-strand break repair and that its inhibition may enhance the effect of radiotherapy.
Asunto(s)
Reparación del ADN/fisiología , Glioblastoma/patología , Proteínas del Tejido Nervioso/metabolismo , Polinucleótido 5'-Hidroxil-Quinasa/metabolismo , Proteínas de Unión al ARN/metabolismo , Tolerancia a Radiación/fisiología , Dominio Catalítico/fisiología , Línea Celular Tumoral , Ensayo Cometa , Roturas del ADN de Doble Cadena/efectos de la radiación , ADN Catalítico , Técnica del Anticuerpo Fluorescente , Humanos , Immunoblotting , Reacción en Cadena de la PolimerasaRESUMEN
BACKGROUND: Glioblastoma (GBM) is the most common and aggressive type of brain tumor. Currently, GBM has an extremely poor outcome and there is no effective treatment. In this context, genomic and transcriptomic analyses have become important tools to identify new avenues for therapies. RNA-binding proteins (RBPs) are master regulators of co- and post-transcriptional events; however, their role in GBM remains poorly understood. To further our knowledge of novel regulatory pathways that could contribute to gliomagenesis, we have conducted a systematic study of RBPs in GBM. RESULTS: By measuring expression levels of 1542 human RBPs in GBM samples and glioma stem cell samples, we identified 58 consistently upregulated RBPs. Survival analysis revealed that increased expression of 21 RBPs was also associated with a poor prognosis. To assess the functional impact of those RBPs, we modulated their expression in GBM cell lines and performed viability, proliferation, and apoptosis assays. Combined results revealed a prominent oncogenic candidate, SNRPB, which encodes core spliceosome machinery components. To reveal the impact of SNRPB on splicing and gene expression, we performed its knockdown in a GBM cell line followed by RNA sequencing. We found that the affected genes were involved in RNA processing, DNA repair, and chromatin remodeling. Additionally, genes and pathways already associated with gliomagenesis, as well as a set of general cancer genes, also presented with splicing and expression alterations. CONCLUSIONS: Our study provides new insights into how RBPs, and specifically SNRPB, regulate gene expression and directly impact GBM development.
Asunto(s)
Neoplasias Encefálicas/genética , Transformación Celular Neoplásica/genética , Regulación Neoplásica de la Expresión Génica , Genómica , Glioblastoma/genética , Empalme del ARN/genética , Proteínas de Unión al ARN/genética , Proteínas Nucleares snRNP/genética , Apoptosis/genética , Neoplasias Encefálicas/metabolismo , Neoplasias Encefálicas/mortalidad , Neoplasias Encefálicas/patología , Proliferación Celular , Supervivencia Celular/genética , Análisis por Conglomerados , Biología Computacional/métodos , Exones , Perfilación de la Expresión Génica , Técnicas de Silenciamiento del Gen , Genómica/métodos , Glioblastoma/metabolismo , Glioblastoma/mortalidad , Glioblastoma/patología , Humanos , Intrones , Anotación de Secuencia Molecular , Clasificación del Tumor , Pronóstico , Proteínas de Unión al ARN/metabolismo , Transducción de Señal , Transcriptoma , Proteínas Nucleares snRNP/metabolismoRESUMEN
Insulin-like growth factor 2 mRNA binding protein 3 (IGF2BP3) expression correlates with malignancy, but its role(s) in pathogenesis remains enigmatic. We interrogated the IGF2BP3-RNA interaction network in pancreatic ductal adenocarcinoma (PDAC) cells. Using a combination of genome-wide approaches, we have identified 164 direct mRNA targets of IGF2BP3. These transcripts encode proteins enriched for functions such as cell migration, proliferation, and adhesion. Loss of IGF2BP3 reduced PDAC cell invasiveness and remodeled focal adhesion junctions. Individual nucleotide resolution crosslinking immunoprecipitation (iCLIP) revealed significant overlap of IGF2BP3 and microRNA (miRNA) binding sites. IGF2BP3 promotes association of the RNA-induced silencing complex (RISC) with specific transcripts. Our results show that IGF2BP3 influences a malignancy-associated RNA regulon by modulating miRNA-mRNA interactions.
Asunto(s)
Proteínas de Unión al ARN/metabolismo , Complejo Silenciador Inducido por ARN/metabolismo , Adenocarcinoma/genética , Adenocarcinoma/patología , Proteínas Argonautas/genética , Proteínas Argonautas/metabolismo , Carcinoma Ductal Pancreático/genética , Carcinoma Ductal Pancreático/patología , Línea Celular Tumoral , Adhesiones Focales/metabolismo , Regulación Neoplásica de la Expresión Génica , Células HeLa , Humanos , MicroARNs/genética , MicroARNs/metabolismo , Invasividad Neoplásica , Polimorfismo de Nucleótido Simple/genética , ARN Mensajero/genética , ARN Mensajero/metabolismo , ARN Neoplásico/metabolismo , Proteínas de Unión al ARN/genéticaRESUMEN
OBJECTIVE: This article describes current trends in cognitive assessment use for adults with mild stroke by home health practitioners in the United States. METHOD: Participants were 56 home health occupational therapists and occupational therapy assistants. A self-administered survey about use of cognitive assessment tools in home health care and influences on their use was conducted. RESULTS: Ninety-six percent of participants assessed cognition in adults with mild stroke. Nonstandardized assessments were the most widely used method for evaluating cognition in the home health setting. Influences such as specialized training in specific assessments, confidence, and productivity requirements may have affected the practitioners' cognitive assessment decisions in home health care. CONCLUSION: Although cognitive assessments are widely used in home health care, occupational therapy practitioners are selecting nonstandardized assessments most frequently to assess cognition.
Asunto(s)
Actividades Cotidianas , Trastornos del Conocimiento/diagnóstico , Servicios de Atención de Salud a Domicilio , Terapia Ocupacional/tendencias , Rehabilitación de Accidente Cerebrovascular , Adulto , Anciano , Cognición , Femenino , Humanos , Masculino , Escala del Estado Mental , Persona de Mediana Edad , Pruebas Neuropsicológicas , Pautas de la Práctica en Medicina , Encuestas y Cuestionarios , Análisis y Desempeño de TareasRESUMEN
hnRNPs are polyvalent RNA binding proteins that have been implicated in a range of regulatory roles including splicing, mRNA decay, translation, and miRNA metabolism. A variety of genome wide studies have taken advantage of methods like CLIP and RIP to identify the targets and binding sites of RNA binding proteins. However, due to the complex nature of RNA-binding proteins, these studies are incomplete without assays that characterize the impact of RBP binding on mRNA target expression. Here we used a suite of high-throughput approaches (RIP-Seq, iCLIP, RNA-Seq and shotgun proteomics) to provide a comprehensive view of hnRNP H1s ensemble of targets and its role in splicing, mRNA decay, and translation. The combination of RIP-Seq and iCLIP allowed us to identify a set of 1,086 high confidence target transcripts. Binding site motif analysis of these targets suggests the TGGG tetramer as a prevalent component of hnRNP H1 binding motif, with particular enrichment around intronic hnRNP H1 sites. Our analysis of the target transcripts and binding sites indicates that hnRNP H1s involvement in splicing is 2-fold: it directly affects a substantial number of splicing events, but also regulates the expression of major components of the splicing machinery and other RBPs with known roles in splicing regulation. The identified mRNA targets displayed function enrichment in MAPK signaling and ubiquitin mediated proteolysis, which might be main routes by which hnRNP H1 promotes tumorigenesis.
Asunto(s)
Ribonucleoproteína Heterogénea-Nuclear Grupo F-H/genética , Secuenciación de Nucleótidos de Alto Rendimiento , Sitios de Unión , Células HeLa , Ribonucleoproteína Heterogénea-Nuclear Grupo F-H/metabolismo , Ribonucleoproteína Heterogénea-Nuclear Grupo F-H/fisiología , Humanos , Empalme del ARNRESUMEN
The conserved RNA-binding protein Musashi1 (MSI1) has emerged as a key oncogenic factor in numerous solid tumors, including glioblastoma. However, its mechanism of action has not yet been established comprehensively. To identify its target genes comprehensively and determine the main routes by which it influences glioblastoma phenotypes, we conducted individual-nucleotide resolution cross-linking and immunoprecipitation (iCLIP) experiments. We confirmed that MSI1 has a preference for UAG sequences contained in a particular structural context, especially in 3' untranslated regions. Although numerous binding sites were also identified in intronic sequences, our RNA transcriptome sequencing analysis does not favor the idea that MSI1 is a major regulator of splicing in glioblastoma cells. MSI1 target mRNAs encode proteins that function in multiple pathways of cell proliferation and cell adhesion. Since these associations indicate potentially new roles for MSI1, we investigated its impact on glioblastoma cell adhesion, morphology, migration, and invasion. These processes are known to underpin the spread and relapse of glioblastoma, in contrast to other tumors where metastasis is the main driver of recurrence and progression.
Asunto(s)
Adhesión Celular/genética , Glioblastoma/genética , Glioblastoma/patología , Invasividad Neoplásica/genética , Proteínas del Tejido Nervioso/genética , Proteínas de Unión al ARN/genética , Regiones no Traducidas 3'/genética , Empalme Alternativo/genética , Secuencia de Bases , Sitios de Unión/genética , Línea Celular Tumoral , Movimiento Celular/genética , Proliferación Celular/genética , Supervivencia Celular/genética , Humanos , Invasividad Neoplásica/patología , Proteínas del Tejido Nervioso/biosíntesis , Interferencia de ARN , ARN Interferente Pequeño , Proteínas de Unión al ARN/biosíntesis , Análisis de Secuencia de ARNRESUMEN
miR-137 plays critical roles in the nervous system and tumor development; an increase in its expression is required for neuronal differentiation while its reduction is implicated in gliomagenesis. To evaluate the potential of miR-137 in glioblastoma therapy, we conducted genome-wide target mapping in glioblastoma cells by measuring the level of association between PABP and mRNAs in cells transfected with miR-137 mimics vs. controls via RIPSeq. Impact on mRNA levels was also measured by RNASeq. By combining the results of both experimental approaches, 1468 genes were found to be negatively impacted by miR-137--among them, 595 (40%) contain miR-137 predicted sites. The most relevant targets include oncogenic proteins and key players in neurogenesis like c-KIT, YBX1, AKT2, CDC42, CDK6 and TGFß2. Interestingly, we observed that several identified miR-137 targets are also predicted to be regulated by miR-124, miR-128 and miR-7, which are equally implicated in neuronal differentiation and gliomagenesis. We suggest that the concomitant increase of these four miRNAs in neuronal stem cells or their repression in tumor cells could produce a robust regulatory effect with major consequences to neuronal differentiation and tumorigenesis.
Asunto(s)
Diferenciación Celular/genética , Transformación Celular Neoplásica/genética , Predisposición Genética a la Enfermedad/genética , MicroARNs/genética , Neuronas/metabolismo , Apoptosis/genética , Western Blotting , Neoplasias Encefálicas/genética , Neoplasias Encefálicas/patología , Movimiento Celular/genética , Proliferación Celular , Transformación Celular Neoplásica/patología , Perfilación de la Expresión Génica , Regulación Neoplásica de la Expresión Génica , Redes Reguladoras de Genes , Estudio de Asociación del Genoma Completo , Glioblastoma/genética , Glioblastoma/patología , Humanos , Modelos Genéticos , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/metabolismo , Neurogénesis/genética , Neuronas/patología , Análisis de Secuencia por Matrices de Oligonucleótidos , Reacción en Cadena de la Polimerasa de Transcriptasa InversaRESUMEN
MOTIVATION: Post-transcriptional and co-transcriptional regulation is a crucial link between genotype and phenotype. The central players are the RNA-binding proteins, and experimental technologies [such as cross-linking with immunoprecipitation- (CLIP-) and RIP-seq] for probing their activities have advanced rapidly over the course of the past decade. Statistically robust, flexible computational methods for binding site identification from high-throughput immunoprecipitation assays are largely lacking however. RESULTS: We introduce a method for site identification which provides four key advantages over previous methods: (i) it can be applied on all variations of CLIP and RIP-seq technologies, (ii) it accurately models the underlying read-count distributions, (iii) it allows external covariates, such as transcript abundance (which we demonstrate is highly correlated with read count) to inform the site identification process and (iv) it allows for direct comparison of site usage across cell types or conditions. AVAILABILITY AND IMPLEMENTATION: We have implemented our method in a software tool called Piranha. Source code and binaries, licensed under the GNU General Public License (version 3) are freely available for download from http://smithlab.usc.edu. CONTACT: andrewds@usc.edu SUPPLEMENTARY INFORMATION: Supplementary data available at Bioinformatics online.
Asunto(s)
Análisis de Secuencia de ARN/métodos , Programas Informáticos , Secuencia de Bases , Sitios de Unión , Biología Computacional/métodos , Células HEK293 , Células HeLa , Secuenciación de Nucleótidos de Alto Rendimiento/métodos , Humanos , ARN/genética , Proteínas de Unión al ARN/genéticaRESUMEN
Musashi1 (Msi1) is a highly conserved RNA-binding protein that is required during the development of the nervous system. Msi1 has been characterized as a stem cell marker, controlling the balance between self-renewal and differentiation, and has also been implicated in tumorigenesis, being highly expressed in multiple tumor types. We analyzed Msi1 expression in a large cohort of medulloblastoma samples and found that Msi1 is highly expressed in tumor tissue compared with normal cerebellum. Notably, high Msi1 expression levels proved to be a sign of poor prognosis. Msi1 expression was determined to be particularly high in molecular subgroups 3 and 4 of medulloblastoma. We determined that Msi1 is required for tumorigenesis because inhibition of Msi1 expression by small-interfering RNAs reduced the growth of Daoy medulloblastoma cells in xenografts. To characterize the participation of Msi1 in medulloblastoma, we conducted different high-throughput analyses. Ribonucleoprotein immunoprecipitation followed by microarray analysis (RIP-chip) was used to identify mRNA species preferentially associated with Msi1 protein in Daoy cells. We also used cluster analysis to identify genes with similar or opposite expression patterns to Msi1 in our medulloblastoma cohort. A network study identified RAC1, CTGF, SDCBP, SRC, PRL, and SHC1 as major nodes of an Msi1-associated network. Our results suggest that Msi1 functions as a regulator of multiple processes in medulloblastoma formation and could become an important therapeutic target.
Asunto(s)
Neoplasias Cerebelosas/genética , Neoplasias Cerebelosas/patología , Redes Reguladoras de Genes/genética , Genes Relacionados con las Neoplasias/genética , Meduloblastoma/genética , Meduloblastoma/patología , Proteínas del Tejido Nervioso/metabolismo , Proteínas de Unión al ARN/metabolismo , Animales , Secuencia de Bases , Línea Celular Tumoral , Proliferación Celular , Regulación Neoplásica de la Expresión Génica , Silenciador del Gen , Genoma Humano/genética , Células HEK293 , Humanos , Inmunoprecipitación , Masculino , Ratones , Ratones Desnudos , Datos de Secuencia Molecular , Proteínas del Tejido Nervioso/genética , Análisis de Secuencia por Matrices de Oligonucleótidos , Pronóstico , Biosíntesis de Proteínas/genética , ARN Mensajero/genética , ARN Mensajero/metabolismo , Proteínas de Unión al ARN/genética , Inducción de Remisión , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Translation regulation plays important roles in both normal physiological conditions and diseases states. This regulation requires cis-regulatory elements located mostly in 5' and 3' UTRs and trans-regulatory factors (e.g., RNA binding proteins (RBPs)) which recognize specific RNA features and interact with the translation machinery to modulate its activity. In this paper, we discuss important aspects of 5' UTR-mediated regulation by providing an overview of the characteristics and the function of the main elements present in this region, like uORF (upstream open reading frame), secondary structures, and RBPs binding motifs and different mechanisms of translation regulation and the impact they have on gene expression and human health when deregulated.
RESUMEN
The ubiquitously expressed RNA-binding protein Hu antigen R (HuR) or ELAVL1 is implicated in a variety of biological processes as well as being linked with a number of diseases, including cancer. Despite a great deal of prior investigation into HuR, there is still much to learn about its function. We take an important step in this direction by conducting cross-linking and immunoprecipitation and RNA sequencing experiments followed by an extensive computational analysis to determine the characteristics of the HuR binding site and impact on the transcriptome. We reveal that HuR targets predominantly uracil-rich single-stranded stretches of varying size, with a strong conservation of structure and sequence composition. Despite the fact that HuR sites are observed in intronic regions, our data do not support a role for HuR in regulating splicing. HuR sites in 3'-UTRs overlap extensively with predicted microRNA target sites, suggesting interplay between the functions of HuR and microRNAs. Network analysis showed that identified targets containing HuR binding sites in the 3' UTR are highly interconnected.
Asunto(s)
Regiones no Traducidas 3'/fisiología , Proteínas ELAV/metabolismo , MicroARNs/metabolismo , Elementos de Respuesta/fisiología , Proteínas ELAV/genética , Genómica/métodos , Células HeLa , Humanos , MicroARNs/genéticaRESUMEN
Musashi1 (Msi1) is an evolutionarily conserved RNA-binding protein that has been implicated in processes like stem cell fate, nervous system development, and tumorigenesis via its activities as a specific regulator of translation. While Msi1 is barely detected in normal adult tissue, it has been observed to be highly expressed in numerous tumor types (e.g. breast, colon, medulloblastoma, glioblastoma, and et cetera). Unfortunately, the molecular cues that are responsible for Msi1 upregulation in cancer cells are largely unknown. Tumor suppressor microRNAs (miRNAs) are known for targeting genes with oncogenic properties like Msi1 and for being either downregulated or deleted in tumor tissue. We observed that Msi1 long 3'UTR region is potentially targeted by several tumor suppressor miRNAs (miR-34a, -101, -128, -137, and -138). Western blotting of endogenous Msi1 protein as well as luciferase assays confirmed Msi1 regulation by these tumor suppressor miRNAs. Furthermore, we observed when examining different cellular states that these miRNAs and Msi1 have opposite expression profiles. Cell proliferation inhibition induced by the tumor suppressor miRNAs was partially rescued by Msi1 transgenic expression. We conclude that tumor suppressor miRNAs are direct and influential regulators of Msi1, affecting its expression pattern during tumorigenesis of malignant nervous system tumors.
Asunto(s)
Transformación Celular Neoplásica , MicroARNs/genética , MicroARNs/metabolismo , Neoplasias/genética , Proteínas del Tejido Nervioso/genética , Proteínas de Unión al ARN/genética , Regiones no Traducidas 3' , Secuencia de Bases , Línea Celular Tumoral , Proliferación Celular , Genes Supresores de Tumor , Células HeLa , Humanos , Neoplasias/metabolismo , Células Madre Neoplásicas , Proteínas del Tejido Nervioso/metabolismo , Interferencia de ARN , ARN Interferente Pequeño , Proteínas de Unión al ARN/metabolismo , Alineación de Secuencia , Transducción de Señal/genética , Regulación hacia ArribaRESUMEN
Transcription, mRNA decay, translation and protein degradation are essential processes during eukaryotic gene expression, but their relative global contributions to steady-state protein concentrations in multi-cellular eukaryotes are largely unknown. Using measurements of absolute protein and mRNA abundances in cellular lysate from the human Daoy medulloblastoma cell line, we quantitatively evaluate the impact of mRNA concentration and sequence features implicated in translation and protein degradation on protein expression. Sequence features related to translation and protein degradation have an impact similar to that of mRNA abundance, and their combined contribution explains two-thirds of protein abundance variation. mRNA sequence lengths, amino-acid properties, upstream open reading frames and secondary structures in the 5' untranslated region (UTR) were the strongest individual correlates of protein concentrations. In a combined model, characteristics of the coding region and the 3'UTR explained a larger proportion of protein abundance variation than characteristics of the 5'UTR. The absolute protein and mRNA concentration measurements for >1000 human genes described here represent one of the largest datasets currently available, and reveal both general trends and specific examples of post-transcriptional regulation.
Asunto(s)
Regulación de la Expresión Génica , Proteínas/análisis , Proteínas/genética , ARN Mensajero/análisis , ARN Mensajero/genética , Línea Celular Tumoral , Bases de Datos de Proteínas , Perfilación de la Expresión Génica , Humanos , Proteínas/metabolismo , ARN Mensajero/metabolismoRESUMEN
Musashi1 (Msi1) is a highly conserved RNA-binding protein with pivotal functions in stem cell maintenance, nervous system development, and tumorigenesis. Despite its importance, only three direct mRNA targets have been characterized so far: m-numb, CDKN1A, and c-mos. Msi1 has been shown to affect their translation by binding to short elements located in the 3'-untranslated region. To better understand Msi1 functions, we initially performed an RIP-Chip analysis in HEK293T cells; this method consists of isolation of specific RNA-protein complexes followed by identification of the RNA component via microarrays. A group of 64 mRNAs was found to be enriched in the Msi1-associated population compared with controls. These genes belong to two main functional categories pertinent to tumorigenesis: 1) cell cycle, cell proliferation, cell differentiation, and apoptosis and 2) protein modification (including ubiquitination and ubiquitin cycle). To corroborate our findings, we examined the impact of Msi1 expression on both mRNA (transcriptomic) and protein (proteomic) expression levels. Genes whose mRNA levels were affected by Msi1 expression have a Gene Ontology distribution similar to RIP-Chip results, reinforcing Msi1 participation in cancer-related processes. The proteomics study revealed that Msi1 can have either positive or negative effects on gene expression of its direct targets. In summary, our results indicate that Msi1 affects a network of genes and could function as a master regulator during development and tumor formation.
Asunto(s)
Regiones no Traducidas 3'/metabolismo , Regulación Neoplásica de la Expresión Génica , Proteínas de Neoplasias/biosíntesis , Neoplasias/metabolismo , Proteínas del Tejido Nervioso/biosíntesis , ARN Neoplásico/metabolismo , Proteínas de Unión al ARN/biosíntesis , Regiones no Traducidas 3'/genética , Apoptosis/genética , Ciclo Celular/genética , Diferenciación Celular/genética , Línea Celular , Humanos , Proteínas de Neoplasias/genética , Neoplasias/genética , Proteínas del Tejido Nervioso/genética , Proteómica/métodos , ARN Neoplásico/genética , Proteínas de Unión al ARN/genéticaRESUMEN
BACKGROUND: Musashi1 (Msi1) is an RNA binding protein with a central role during nervous system development and stem cell maintenance. High levels of Msi1 have been reported in several malignancies including brain tumors thereby associating Msi1 and cancer. METHODS: We used the human medulloblastoma cell line Daoy as model system in this study to knock down the expression of Msi1 and determine the effects upon soft agar growth and neurophere formation. Quantitative RT-PCR was conducted to evaluate the expression of cell proliferation, differentiation and survival genes in Msi1 depleted Daoy cells. RESULTS: We observed that MSI1 expression was elevated in Daoy cells cultured as neurospheres compared to those grown as monolayer. These data indicated that Msi1 might be involved in regulating proliferation in cancer cells. Here we show that shRNA mediated Msi1 depletion in Daoy cells notably impaired their ability to form colonies in soft agar and to grow as neurospheres in culture. Moreover, differential expression of a group of Notch, Hedgehog and Wnt pathway related genes including MYCN, FOS, NOTCH2, SMO, CDKN1A, CCND2, CCND1, and DKK1, was also found in the Msi1 knockdown, demonstrating that Msi1 modulated the expression of a subset of cell proliferation, differentiation and survival genes in Daoy. CONCLUSION: Our data suggested that Msi1 may promote cancer cell proliferation and survival as its loss seems to have a detrimental effect in the maintenance of medulloblastoma cancer cells. In this regard, Msi1 might be a positive regulator of tumor progression and a potential target for therapy.