RESUMO
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.
Assuntos
Neoplasias Cerebelares/genética , Neoplasias Cerebelares/patologia , Redes Reguladoras de Genes/genética , Genes Neoplásicos/genética , Meduloblastoma/genética , Meduloblastoma/patologia , Proteínas do Tecido Nervoso/metabolismo , Proteínas de Ligação a RNA/metabolismo , Animais , Sequência de Bases , Linhagem Celular Tumoral , Proliferação de Células , Regulação Neoplásica da Expressão Gênica , Inativação Gênica , Genoma Humano/genética , Células HEK293 , Humanos , Imunoprecipitação , Masculino , Camundongos , Camundongos Nus , Dados de Sequência Molecular , Proteínas do Tecido Nervoso/genética , Análise de Sequência com Séries de Oligonucleotídeos , Prognóstico , Biossíntese de Proteínas/genética , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Proteínas de Ligação a RNA/genética , Indução de Remissão , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Musashi1 (Msi1) is an evolutionarily conserved RNA-binding protein (RBP) that has profound implications in cellular processes such as stem cell maintenance, nervous system development, and tumorigenesis. Msi1 is highly expressed in many cancers, including glioblastoma, whereas in normal tissues, its expression is restricted to stem cells. Unfortunately, the factors that modulate Msi1 expression and trigger high levels in tumors are largely unknown. The Msi1 mRNA has a long 3' untranslated region (UTR) containing several AU- and U-rich sequences. This type of sequence motif is often targeted by HuR, another important RBP known to be highly expressed in tumor tissue such as glioblastoma and to regulate a variety of cancer-related genes. In this report, we show an interaction between HuR and the Msi1 3'-UTR, resulting in a positive regulation of Msi1 expression. We show that HuR increased MSI1 mRNA stability and promoted its translation. We also present evidence that expression of HuR and Msi1 correlate positively in clinical glioblastoma samples. Finally, we show that inhibition of cell proliferation, increased apoptosis, and changes in cell-cycle profile as a result of silencing HuR are partially rescued when Msi1 is ectopically expressed. In summary, our results suggest that HuR is an important regulator of Msi1 in glioblastoma and that this regulation has important biological consequences during gliomagenesis.
Assuntos
Neoplasias Encefálicas/genética , Proteínas ELAV/fisiologia , Glioblastoma/genética , Proteínas do Tecido Nervoso/genética , Biossíntese de Proteínas/genética , Estabilidade de RNA/genética , Proteínas de Ligação a RNA/genética , Regiões 3' não Traduzidas/genética , Neoplasias Encefálicas/metabolismo , Neoplasias Encefálicas/patologia , Proteínas ELAV/antagonistas & inibidores , Proteínas ELAV/genética , Proteínas ELAV/metabolismo , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Glioblastoma/metabolismo , Glioblastoma/patologia , Células HeLa , Humanos , Proteínas do Tecido Nervoso/metabolismo , Oncogenes/genética , Biossíntese de Proteínas/efeitos dos fármacos , Estabilidade de RNA/efeitos dos fármacos , RNA Interferente Pequeno/farmacologia , Proteínas de Ligação a RNA/antagonistas & inibidores , Proteínas de Ligação a RNA/metabolismo , Células Tumorais CultivadasRESUMO
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.
Assuntos
Regiões 3' não Traduzidas/metabolismo , Regulação Neoplásica da Expressão Gênica , Proteínas de Neoplasias/biossíntese , Neoplasias/metabolismo , Proteínas do Tecido Nervoso/biossíntese , RNA Neoplásico/metabolismo , Proteínas de Ligação a RNA/biossíntese , Regiões 3' não Traduzidas/genética , Apoptose/genética , Ciclo Celular/genética , Diferenciação Celular/genética , Linhagem Celular , Humanos , Proteínas de Neoplasias/genética , Neoplasias/genética , Proteínas do Tecido Nervoso/genética , Proteômica/métodos , RNA Neoplásico/genética , Proteínas de Ligação a RNA/genéticaRESUMO
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.
