RESUMO
The Dlk1-Gtl2 imprinting locus is located on mouse distal chromosome 12 and consists of multiple maternally expressed non-coding RNAs and several paternally expressed protein-coding genes. The imprinting of this locus plays a crucial role in embryonic development and postnatal growth. At least one cis-element, the intergenic differentially methylated region (IG-DMR) is required for expression of maternally expressed genes and repression of silenced paternally expressed genes. The mechanism by which the IG-DMR functions is largely unknown. However, it has been suggested that the unmethylated IG-DMR acts as a positive regulator activating expression of non-coding RNAs. Gtl2 is the first non-coding RNA gene downstream of the IG-DMR. Although its in vivo function in the mouse is largely unknown, its human ortholog MEG3 has been linked to tumor suppression in human tumor-derived cell lines. We generated a knockout mouse model, in which the first five exons and adjacent promoter region of the Gtl2 gene were deleted. Maternal deletion of Gtl2 resulted in perinatal death and skeletal muscle defects, indicating that Gtl2 plays an important role in embryonic development. The maternal deletion also completely abolished expression of downstream maternally expressed genes, activated expression of silenced paternally expressed genes and resulted in methylation of the IG-DMR. By contrast, the paternal inherited deletion did not have this effect. These data strongly indicate that activation of Gtl2 and its downstream maternal genes play an essential role in regulating Dlk1-Gtl2 imprinting, possibly by maintaining active status of the IG-DMR.
Assuntos
Deleção de Genes , Regulação da Expressão Gênica no Desenvolvimento , Proteínas/metabolismo , Alelos , Animais , Sequência de Bases , Proteínas de Ligação ao Cálcio , Metilação de DNA , Feminino , Inativação Gênica , Peptídeos e Proteínas de Sinalização Intercelular/genética , Peptídeos e Proteínas de Sinalização Intercelular/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Microscopia Eletrônica de Transmissão , Músculo Esquelético/embriologia , Músculo Esquelético/metabolismo , Músculo Esquelético/ultraestrutura , Placenta/metabolismo , Placentação , Gravidez , Proteínas/genética , RNA Longo não CodificanteRESUMO
DLK1-MEG3 is an imprinted locus consisting of multiple maternally expressed noncoding RNA genes and paternally expressed protein-coding genes. The expression of maternally expressed gene 3 (MEG3) is selectively lost in clinically nonfunctioning adenomas (NFAs) of gonadotroph origin; however, expression status of other genes at this locus in human pituitary adenomas has not previously been reported. Using quantitative real-time RT-PCR, we evaluated expression of 24 genes from the DLK1-MEG3 locus in 44 human pituitary adenomas (25 NFAs, 7 ACTH-secreting, 7 GH-secreting, and 5 PRL-secreting adenomas) and 10 normal pituitaries. The effects on cell proliferation of five miRNAs whose expression was lost in NFAs were investigated by flow cytometry analysis. We found that 18 genes, including 13 miRNAs at the DLK1-MEG3 locus, were significantly down-regulated in human NFAs. In ACTH-secreting and PRL-secreting adenomas, 12 and 7 genes were significantly down-regulated, respectively; no genes were significantly down-regulated in GH-secreting tumors. One of the five miRNAs tested induced cell cycle arrest at the G2/M phase in PDFS cells derived from a human NFA. Our data indicate that the DLK1-MEG3 locus is silenced in NFAs. The growth suppression by miRNAs in PDFS cells is consistent with the hypothesis that the DLK1-MEG3 locus plays a tumor suppressor role in human NFAs.
Assuntos
Adenoma/genética , Inativação Gênica , Loci Gênicos/genética , Impressão Genômica/genética , Peptídeos e Proteínas de Sinalização Intercelular/genética , Proteínas de Membrana/genética , Neoplasias Hipofisárias/genética , Proteínas/genética , Adenoma/patologia , Adulto , Idoso , Idoso de 80 Anos ou mais , Proteínas de Ligação ao Cálcio , Pontos de Checagem do Ciclo Celular/genética , Linhagem Celular Tumoral , Pai , Feminino , Regulação Neoplásica da Expressão Gênica , Humanos , Imuno-Histoquímica , Peptídeos e Proteínas de Sinalização Intercelular/metabolismo , Masculino , Proteínas de Membrana/metabolismo , MicroRNAs/genética , MicroRNAs/metabolismo , Pessoa de Meia-Idade , Mães , Neoplasias Hipofisárias/patologia , Proteínas/metabolismo , RNA Longo não CodificanteRESUMO
Meningiomas are common tumors, representing 15% to 25% of all central nervous system tumors. NF2 gene inactivation on chromosome 22 has been shown as an early event in tumorigenesis; however, few factors underlying tumor growth and progression have been identified. The chromosomal abnormalities of 14q32 are often associated with meningioma pathogenesis and progression; therefore, it has been proposed that an as yet unidentified tumor suppressor is present at this locus. Maternally expressed gene 3 (MEG3) is an imprinted gene located at 14q32 which encodes a noncoding RNA with an antiproliferative function. We found that MEG3 mRNA is highly expressed in normal arachnoidal cells. However, MEG3 is not expressed in the majority of human meningiomas or the human meningioma cell lines IOMM-Lee and CH157-MN. There is a strong association between loss of MEG3 expression and tumor grade. Allelic loss at the MEG3 locus is also observed in meningiomas, with increasing prevalence in higher grade tumors. In addition, there is an increase in CpG methylation within the promoter and the imprinting control region of MEG3 gene in meningiomas. Functionally, MEG3 suppresses DNA synthesis in both IOMM-Lee and CH157-MN cells by approximately 60% in bromodeoxyuridine incorporation assays. Colony-forming efficiency assays show that MEG3 inhibits colony formation in CH157-MN cells by approximately 80%. Furthermore, MEG3 stimulates p53-mediated transactivation in these cell lines. Therefore, these data are consistent with the hypothesis that MEG3, which encodes a noncoding RNA, may be a tumor suppressor gene at chromosome 14q32 involved in meningioma progression via a novel mechanism.
Assuntos
Transformação Celular Neoplásica/genética , Meningioma/genética , Proteínas/genética , Aracnoide-Máter/metabolismo , Aracnoide-Máter/patologia , Processos de Crescimento Celular/genética , Linhagem Celular Tumoral , Transformação Celular Neoplásica/metabolismo , Transformação Celular Neoplásica/patologia , Metilação de DNA , DNA Complementar/genética , Dosagem de Genes , Genes Supressores de Tumor , Impressão Genômica , Humanos , Meningioma/metabolismo , Meningioma/patologia , Regiões Promotoras Genéticas , Proteínas/metabolismo , RNA Longo não Codificante , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Ativação Transcricional , Proteína Supressora de Tumor p53/biossíntese , Proteína Supressora de Tumor p53/genéticaRESUMO
Maternally expressed gene 3 (MEG3) is a noncoding RNA highly expressed in the normal human brain and pituitary. Expression of MEG3 is lost in gonadotroph-derived clinically nonfunctioning pituitary adenomas. Meg3 knockout mice were generated to identify targets and potential functions of this gene in embryonic development and tumorigenesis. Gene expression profiles were compared in the brains of Meg3-null embryos and wild-type littermate controls using microarray analysis. Microarray data were analyzed with GeneSifter, which uses Kyoto Encyclopedia of Genes and Genomes pathways and Gene Ontology classifications to identify signaling cascades and functional categories of interest within the dataset. Differences were found in signaling pathways and ontologies related to angiogenesis between wild-type and knockout embryos. Quantitative RT-PCR and immunohistological staining showed increased expression of some Vascular Endothelial Growth Factor pathway genes and increased cortical microvessel density in the Meg3-null embryos. In conclusion, Meg3 may play an important role in control of vascularization in the brain and may function as a tumor suppressor in part by inhibiting angiogenesis.