The Role of miRNAs in Glioma Stem Cell and Glioma Biology

在肿瘤中过度表达的MicroRNAs可能会降低靶向肿瘤抑制基因的表达水平，而MicroRNAs的下调可能会抑制促进肿瘤过程的致癌基因（oncomirs）。此外，mirna经常位于杂合性缺失区域、扩增区域或共同断点区域，并且已确定它们在包括GBM在内的几种肿瘤中调节肿瘤相关基因的表达。迄今为止，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miRNA的表达谱。对小鼠和人脑miRNA的分析表明，miR-9、-101、-124和-132等的表达存在差异。研究发现，mirna -10b和-21在星形细胞肿瘤中表达上调，前者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细胞周期阻滞。然而，对于mirna在星形细胞性脑肿瘤或BTIC中的表达水平和靶基因调控作用知之甚少。为了更好地了解mirna在GBM调控中的作用，我们已经开始使用基于定量逆转录酶聚合酶链反应（qRT-PCR）的方法，在原发性GBM和由此衍生的BTIC，以及神经干细胞和已建立的胶质瘤细胞系中生成和比较365种mirna的全球表达谱，以确定特定的mirna改变。该分析揭示了肿瘤以及BTIC与非肿瘤样本中具有统计学意义的下调mirna。此外，这种表达谱证实了BTIC在miRNA水平上比已建立的胶质瘤细胞系更适合星形细胞性脑肿瘤的研究，正如我们之前基于它们的基因组/基因表达谱所描述的那样。我们也正在确定我们研究中包含的mirna的表达与肿瘤/ BTISC样本的特定CNA或拷贝数改变（删除/扩增区域）之间的相关性。CNA/表达水平不一致的mirna可能存在表观遗传调控，可能成为治疗靶点。这个初步的概要文件有两种用途。一方面，miRNA分析将扩展到GMDI收集的脑肿瘤，为我们已经广泛的数据增加非常有价值的信息。另一方面，在BTIC体外模型和体内小鼠模型中，我们正在对特定mirna及其特定靶向基因参与胶质瘤形成、细胞增殖或侵袭进行生物学验证。

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.