The Role of miRNAs in Glioma Stem Cell and Glioma Biology

在肿瘤中过度表达的microRNAs可能会降低靶向抑癌基因的表达水平，而下调的microRNAs可能会抑制与肿瘤过程有关的致癌基因(OncomiRS)。此外，miRNAs经常位于杂合性缺失区域、扩增区域或公共断裂区，它们已被发现在包括GBM在内的几种肿瘤中调节肿瘤相关基因的表达。到目前为止，已经发表了几项研究，使用不同的检测方法分析了正常大脑和脑肿瘤中的miRNA表达谱。对小鼠和人脑miRNA的分析表明，miR-9、-101、-124和-132等基因有明显的表达。已发现miRNAs-10b和-21在星形细胞肿瘤中表达上调，前者可能作为癌基因减少恶性细胞的凋亡，而miRNA-124和-137表达下调，参与促进脑肿瘤起始细胞(BTIC)的神经元分化和GBM细胞周期停滞。然而，关于miRNAs在星形细胞脑瘤或BTIC中的表达水平和靶基因调控的参与程度还知之甚少。为了更好地了解miRNAs在GBM调控中的作用，我们已经开始使用基于定量逆转录聚合酶链式反应(qRT-PCR)的分析方法，在原代GBM和BTIC以及神经干细胞和已建立的处于增殖和分化条件下的胶质瘤细胞系中生成并比较365个miRNAs的全球表达谱，以确定特定的miRNAs变化。这项分析揭示了在肿瘤以及BTIC与非肿瘤样本中miRNAs表达的统计学意义上的下调。此外，这种表达谱证实了BTIC是在miRNA水平上比已建立的胶质瘤细胞系更好的研究星形细胞脑瘤的模型，正如我们先前基于它们的基因组/基因表达谱所描述的那样。我们还在确定我们研究中包括的miRNAs的表达与我们的肿瘤/BTISC样本的特定CNA或拷贝数变化(缺失/扩增区域)之间的相关性。CNA/表达水平不一致的miRNAs表明可能存在表观遗传调控，这可能使它们成为有趣的治疗靶点。这份初步简介有两种用途。一方面，miRNA分析将扩展到GMDI脑肿瘤的收集，为我们已经从它们那里获得的大量数据增加了非常有价值的信息。另一方面，在我们的BTIC体外模型和体内小鼠模型中，正在进行特定miRNAs及其特定靶向基因参与胶质瘤发生、细胞增殖或侵袭的生物学验证。

MicroRNAs that are over expressed in tumors might diminish the level of expression of targeted tumor suppressor genes whereas microRNAs down regulated might repress oncogenic genes contributing to the neoplasic process (oncomirs). Also, miRNAs are frequently located in regions of loss of heterozygosity, regions of amplification, or common breakpoint regions and they have been identified to regulate the expression of tumor-associated genes in several tumors including GBM. Several studies have been published to date analyzing miRNA expression profiles in normal brain and brain tumors using different detection methods. Analysis of murine and human brain miRNA indicated distinctive expression of miR-9,-101,-124 and -132 among others. miRNAs -10b and -21 have been found upregulated in astrocytic tumors appearing the former to work as an oncogene decreasing apoptosis in the malignant cells, whereas miRNA-124 and -137 were down regulated and involved in promotion of neuronal differentiation of brain tumor initiating cells (BTIC) and GBM cell cycle arrest. However, little is know about the expression levels and involvement by target genes regulation of miRNAs in astrocytic brain tumors or BTIC. To better understand the role of miRNAs in the regulation of GBM, we have started generating and comparing the global profile of expression of 365 miRNAs using quantitative reverse transcriptase polymerase chain reaction (qRT-PCR) based assays in primary GBM and BTIC derived from them, as well as neuronal stem cells and established glioma cell lines in proliferating and differentiating conditions, to identify specific miRNAs alterations. This analysis is revealing statistically significant downregulation miRNAs in tumors as well as in BTIC vs. non-tumor samples. Furthermore, this expression profile is corroborating BTIC as better models at miRNA level for the study of astrocytic brain tumors than established glioma cell lines, as we have previously described based on their genomic/gene expression profiles. We are also determining the correlation between expression of the miRNAs included in our study and the particular CNA or copy number alterations (deleted/amplified regions) of our tumor/ BTISC samples. miRNAs with non concordant CNA/expression level indicate a possible epigenetic regulation, that could point them as interesting therapeutics targets. This preliminary profile is being used in two ways. On one hand, miRNA analysis will be extended to GMDI collection of brain tumors adding highly valuable information to our already extensive data from them. On the other hand, biological validation of the involvement in gliomagenesis, cell proliferation or invasion of particular miRNAs and their specific targeted genes is being performed in our BTIC in vitro model as well as in vivo mouse models.