RESUMO
OBJECTIVE: Glioma is one of the most common and invasive brain tumors worldwide. Long non-coding RNAs (LncRNAs) play an important role in the development of glioma. However, the regulatory mechanism of LncRNAs in glioma has not been fully elucidated. This study aimed to explore the interaction of lncRNA maternally expressed gene 3 (MEG3) and aberrant histone modification in glioma. MATERIALS AND METHODS: The expression levels of MEG3 and miR-21-3p in glioma cells were measured by quantitative polymerase chain reaction (qPCR). EZH2 (enhancer of zeste homolog 2) and H3K27me3 expression in glioma cells were detected by Western Blot (WB). The binding site of the promoter of MEG3 by H3K27me3 was confirmed by ChIP-Real-time PCR. The direct target of MEG3 and miR-21-3p in glioma cells was measured by a luciferase reporter assay. Cell proliferation was detected by Cell Counting Kit-8 (CCK8), and cell invasion and migration were measured by Transwell assays. RESULTS: EZH2 and miR-21-3p were upregulated and MEG3 was downregulated in glioma cells. Silencing of EZH2 inhibited cell proliferation, migration, and invasion in U87 and U251 cells. Meanwhile, the expression of H3K27me3 could be significantly inhibited by EZH2 interference. H3K27me3 protein can bind to MEG3 promoter directly. EZH2 inhibition and MEG3 down-expression in U87 cells reversed the effects of silencing of EZH2 on glioma cell growth and metastasis. However, EZH2 inhibition and MEG3 overexpression in U251 cells restricted cell proliferation, migration, and invasion. Furthermore, miR-21-3p was verified to interact with MEG3 by direct binding. Inhibition of MEG3 promoted U87 cell growth and metastasis, which was further strengthened following the co-transfection of si-MEG3 and miR-21-3p. Overexpressed MEG3 inhibited U251 cell growth and metastasis and a complete reversal of the results observed in the co-transfection of LV-MEG3 and miR-21-3p. CONCLUSIONS: EZH2 was highly expressed in glioma cells and EZH2-mediated H3K27me3 enrichment on the MEG3 promoter regulated the growth and metastasis of glioma cells by targeting miR-21-3p. It potentially provided a new therapeutic marker targeting glioma.
Assuntos
Proteína Potenciadora do Homólogo 2 de Zeste/metabolismo , Glioma/metabolismo , MicroRNAs/metabolismo , RNA Longo não Codificante/metabolismo , Proliferação de Células , Células Cultivadas , Proteína Potenciadora do Homólogo 2 de Zeste/genética , Glioma/patologia , Histonas/metabolismo , Humanos , MicroRNAs/genética , Regiões Promotoras Genéticas/genética , RNA Longo não Codificante/genéticaRESUMO
Using a combination of hybridization of PAC to a cDNA library and RACE technique, we isolated a novel cDNA, designated as C17orf25 (Chromosome 17 open reading frame 25, previously named it HC71A), from the deletion region on chromosome 17p13.3. The cDNA encodes a protein of 313 amino acids with a calculated molecular mass of 34.8 kDa. C17orf25 is divided into 10 exons and 9 introns, spanning 23 kb of genomic DNA. Northern blot analysis showed that the mRNA expression of C17orf25 was decreased in hepatocellular carcinoma samples as compared to adjacent noncancerous liver tissues from the same patients. The transfection of C17orf25 into the hepatocellular carcinoma cell SMMC7721 and overexpression could inhibit the cell growth. The above results indicate that C17orf25 is a novel human gene, and the cloning and preliminary characterization of C17orf25 is a prerequisite for further functional analysis of this novel gene in human hepatocellular carcinoma.
Assuntos
Carcinoma Hepatocelular/genética , Deleção Cromossômica , Cromossomos Humanos Par 17/genética , Neoplasias Hepáticas/genética , Proteínas/genética , Sequência de Aminoácidos , Animais , Sequência de Bases , Clonagem Molecular , Éxons , Biblioteca Gênica , Humanos , Íntrons , Perda de Heterozigosidade , Dados de Sequência Molecular , Proteínas de Neoplasias , Fases de Leitura Aberta/genética , Proteínas/química , Proteínas/metabolismo , Alinhamento de Sequência , Células Tumorais CultivadasRESUMO
We have identified LASS2, a previously unknown human homologue of the yeast longevity assurance gene LAG1. The LASS2 transcript is highly expressed in liver and kidney, which is very different from the expression of the previously identified human LAG1 homologue LAG1Hs-1. Radiation hybrid mapping studies indicated that LASS2 is located on chromosome 1q11. Yeast two-hybrid screening and glutathione S-transferase pull-down assays showed that the LASS2 protein interacts with several membrane-associated receptors or transporters. Furthermore, LASS2 protein was able to inhibit the colony formation of human hepatoma cells in vitro, which suggests that this gene may be involved in the regulation of cell growth.
Assuntos
Genes/genética , Proteínas de Membrana/genética , Proteínas de Saccharomyces cerevisiae , Sequência de Aminoácidos , Sequência de Bases , Northern Blotting , Carcinoma Hepatocelular/genética , Carcinoma Hepatocelular/patologia , Mapeamento Cromossômico , Cromossomos Humanos Par 1/genética , Clonagem Molecular , DNA Complementar/química , DNA Complementar/genética , DNA Complementar/isolamento & purificação , Éxons , Feminino , Proteínas Fúngicas/genética , Glutationa Transferase/genética , Glutationa Transferase/metabolismo , Humanos , Íntrons , Proteínas de Membrana/metabolismo , Dados de Sequência Molecular , Ligação Proteica , Proteínas/genética , Proteínas/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Mapeamento de Híbridos Radioativos , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Saccharomyces cerevisiae/genética , Alinhamento de Sequência , Análise de Sequência de DNA , Homologia de Sequência de Aminoácidos , Esfingosina N-Aciltransferase , Distribuição Tecidual , Transfecção , Células Tumorais Cultivadas , Ensaio Tumoral de Célula-Tronco , Proteínas Supressoras de Tumor , Técnicas do Sistema de Duplo-HíbridoRESUMO
The distribution of a novel calcium-binding protein with a molecular mass of 18 kDa (CBP-18) in the rat brain was studied by means of biochemical methods and immunohistochemistry on cryostat-sectioned tissue and compared with staining patterns of parvalbumin on adjacent sections. The biochemical analysis revealed high levels of CPB-18 in cortex and cerebellum, low levels in the lungs, and undetectable levels in all other tissues tested. Immunohistochemically, the polyclonal rabbit-derived antibody for CPB-18 showed selective affinity with periglomerular cells and dendrites in the olfactory bulb. Distinct immunostaining of scattered cells and their proximal dendrites was found in the anterior olfactory nuclei and in the perirhinal and entorhinal cortex. Strong staining of neuropil with recognizable but diffusely outlined cells was observed in the retrosplenial cortex, central amygdala, hippocampal rudiment, septum, area preoptica, hypothalamus, colliculus superior, and parabrachial nuclei. The cerebellum showed strong neuropil staining of both the molecular and the granule cell layer. Less intense neuropil staining and a few scattered cells were found in the neocortex, the remaining basal forebrain, and in the entire brainstem. Immunoreactivity was barely detectable or missing in the striatum, the hippocampus, the thalamus, and in the colliculus inferior. Thus, CPB-18 shows a unique staining pattern in the CNS, different from all other Ca(2+)-binding proteins studied so far.
