RESUMO
In a genome-wide screen using DMH (differential methylation hybridization) we have identified a CpG island within the 5' region and untranslated first exon of the secretory granule neuroendocrine protein 1 gene (SGNE1/7B2) that showed hypermethylation in low- and high-grade astrocytomas compared to normal brain tissue. Pyrosequencing was performed to confirm the methylation status of this CpG island in 89 astrocytic gliomas of different malignancy grades and six glioma cell lines. Hypermethylation of SGNE1/7B2 was significantly more frequent in diffuse low-grade astrocytomas as well as secondary glioblastomas and anaplastic astrocytomas as compared to primary glioblastomas. mRNA expression analysis by real-time RT-PCR indicates that SGNE1/7B2 expression is downregulated in astrocytic gliomas compared to white matter samples. Treatment of glioma cells with the demethylating agent 5-aza-2'-deoxycytidine restores the transcription of SGNE1/7B2. Overexpression of SGNE1/7B2 in T98G, A172 and U373MG glioblastoma cells significantly suppressed focus formation and led to a significant increase in apoptotic cells as determined by flow cytometric analysis in T98G cells. In summary, we have identified SGNE1/7B2 as a novel target silenced by DNA methylation in astrocytic gliomas. The high incidence of this alteration and the significant effects of SGNE1/7B2 on the growth and apoptosis of glioblastoma cells provide a first proof for a functional implication of SGNE1/7B2 inactivation in the molecular pathology of gliomas.
Assuntos
Regiões 5' não Traduzidas , Astrocitoma/genética , Ilhas de CpG , Metilação de DNA , Proteína Secretora Neuroendócrina 7B2/genética , Proteína Secretora Neuroendócrina 7B2/metabolismo , Apoptose , Astrocitoma/metabolismo , Astrocitoma/patologia , Azacitidina/análogos & derivados , Azacitidina/farmacologia , Sequência de Bases , Neoplasias Encefálicas/genética , Neoplasias Encefálicas/metabolismo , Neoplasias Encefálicas/patologia , Linhagem Celular Tumoral , Decitabina , Epigênese Genética , Éxons , Regulação Neoplásica da Expressão Gênica , Humanos , Proteína Secretora Neuroendócrina 7B2/biossíntese , Regiões Promotoras Genéticas , Análise de Sequência de DNA , Transcrição Gênica/efeitos dos fármacosRESUMO
7B2 is a chaperone for the prohormone/proneuropeptide convertase PC2. Its mRNA is readily detectable in most neuronal and endocrine cells; the protein, in contrast, is often found at relatively low levels, suggesting that translation of the corresponding mRNA may be repressed. Because the 5' untranslated region (5'-UTR) of this mRNA is relatively long and burdened with multiple AUGs, it has been speculated that it contributes to this repression. In this report, the influence of this region was assessed using in vitro and ex vivo approaches. The results showed that, in a cell-free system, full-length 7B2 mRNA was a poor template for translation. Its translatability dramatically improved when its 5'-UTR was truncated or when it was replaced with the 5'-UTR of carboxypeptidase E mRNA. These observations were confirmed in transfected mouse insulinoma MIN6 cells and human embryonic kidney HEK293 cells. Acute exposure of MIN6 cells to high glucose increased endogenous 7B2 biosynthesis without affecting the levels of its mRNA, suggesting that translation repression of this mRNA can be relieved by physiological stimuli.
Assuntos
Regiões 5' não Traduzidas/fisiologia , Proteína Secretora Neuroendócrina 7B2/biossíntese , RNA Mensageiro/metabolismo , Animais , Carboxipeptidase H/metabolismo , Linhagem Celular Tumoral , Sistema Livre de Células/metabolismo , Regulação da Expressão Gênica , Células HEK293 , Humanos , Insulinoma/metabolismo , Camundongos , Proteína Secretora Neuroendócrina 7B2/metabolismo , Pró-Proteína Convertase 2/metabolismo , RNA Mensageiro/genética , Coelhos , Ratos , TransfecçãoRESUMO
Follicle-associated epithelium (FAE) covering Peyer's patches contains specialized epithelial M cells that take up ingested macromolecules and microorganisms from the lumen of the gut by transcytosis. Using high-density oligonucleotide microarrays, we analyzed the gene expression profiles of FAE and M cells in order to characterize their cellular phenotypes. The microarray data revealed that, among approximately 14,000 genes, 409 were expressed in FAE at twofold or higher levels compared to the intestinal epithelial cells (IECs) of the villi. These included genes involved in membrane traffic, host defense and transcriptional regulation, as well as uncharacterized genes. Real-time PCR and in situ hybridization analyses identified three molecules, ubiquitin D (Ub-D), tumor necrosis factor receptor superfamily 12a (TNFRsf12a), and transmembrane 4 superfamily 4 (Tm4sf4), which were predominantly distributed throughout FAE, but were expressed little, if at all, in IECs. By contrast, transcripts of secretory granule neuroendocrine protein 1 (Sgne-1) were scattered in FAE, and were co-localized with Ulex europaeus agglutinin-1 (UEA-1)-positive cells. This clearly suggests that expression of Sgne-1 in the gut is specific to M cells. Such a unique pattern of gene expression distinguishes FAE and M cells from IECs, and may reflect their cellular phenotype(s) associated with specific functional features.
