Developing miRNA diagnostic methods and identifying tumor regulatory networks

描述（由申请人提供）：本申请涉及广泛的挑战领域（08）基因组学和特定的挑战主题08-CA-103：癌症中的微小RNA。微小RNA（miRNA）是通过降低完全或部分序列互补的靶mRNA的稳定性和/或翻译来调节基因表达的小的非编码RNA（Eulalio等人，2008; Friedman等人，2009年）。已经在哺乳动物中鉴定了数百个miRNA基因家族（Landfieldet al.，2007），其中许多在特定组织中表达，并且已发现在肿瘤中失调（综述于Ventura和Jacks，2009）。miRNA谱已经基于其起源细胞与特定肿瘤相关，并且在某些情况下与预后相关（Lu et al.，2005; He等人，2005; Yanaihara等人，2006年）。由于miRNA改变多个基因的表达，从而可以调节疾病途径中的多个调节步骤，因此它们代表了令人感兴趣的药物靶标;小鼠中的反义寡核苷酸靶向实验（Krutzfeldt等人，2005）和非人灵长类动物（Elmen等人，2008）已经证明了操纵miRNA水平的可行性。与此同时，阐明miRNA靶网络可能为定义导致癌症的调控过程提供重要见解。该提案的总体目标是推进小RNA分析和小RNA组织学方法，用于大量患者样本的成本效益表征。为了解释在miRNA表达模式中观察到的改变，我们将应用我们最近开发的用于直接测序miRNA靶向mRNA网络的强大生物化学方法到相关肿瘤细胞培养模型中。最后，这些最新的发展将在乳腺癌研究中实施，分析177个组织学和临床特征的肿瘤样本。首先，基于RNA深度测序的方法将发展到一个阶段，在这个阶段，它们适合于用有限的材料来定义大量临床样本的miRNA表达模式和突变状态。其次，检测到miRNA表达模式的改变后，将进行miRNA原位杂交，为此，我们最近开发了新的小RNA固定技术，并克服了阻碍组织切片中miRNA组织学检测的最大障碍。第三，为了深入了解miRNA失调的生物学，我们将应用我们最近开发的miRNA结合Argonaute蛋白复合物的光反应性尿苷增强交联和免疫沉淀（PURE-CLIP）方法来鉴定肿瘤细胞转录组中的miRNA靶位点。在最后一个目标中，我们将与荷兰阿姆斯特丹学术医学中心的Marc货车de Vijver合作，应用我们的方法评估miRNA分析在大量三阴性（HER 2基因扩增、雌激素受体（ER）和孕激素受体（PR）阴性）和其他类型乳腺肿瘤中的价值。标本采集包括80个三阴性、51个HER 2阳性、26个ER阳性、11个正常和20个导管原位癌（DCIS）样本。这些标本已经在组织学上以及mRNA转录表达水平上进行了表征，并且可以获得患者的临床特征和结局。我们将确定miRNA的绝对含量、相对miRNA组成或谱、miRNA突变状态和miRNA表达模式，并评估与已确定的临床参数的相关性。我们还将定义肿瘤细胞中的miRNA靶RNA调控网络，以便能够解释乳腺癌中miRNA失调的后果。肿瘤miRNA诊断方法的开发及miRNA调控网络的鉴定 公共卫生相关性：miRNAs是近年来发现的一种非编码小分子RNA，参与调控包括肿瘤在内的多种发育和生理过程。该提案旨在开发新的小RNA克隆方案以满足临床研究的要求，增强用于miRNA细胞定位的原位杂交方法，同时使这些方法适用于诊断实验室的自动化平台，并通过调整我们最近开发的用于鉴定miRNA mRNA靶点的方法来阐明癌细胞系中的miRNA调控网络。

DESCRIPTION (provided by applicant): This application addresses Broad Challenge Area (08) Genomics and Specific Challenge Topic 08-CA-103: Micro-RNAs in Cancer. MicroRNAs (miRNAs) are small non-coding RNAs that regulate gene expression by reducing stability and/or translation of fully or partially sequence-complementary target mRNAs (Eulalio et al., 2008; Friedman et al., 2009). Several hundred miRNA gene families have been identified in mammals (Landgraf et al., 2007), many of which are expressed in specific tissues and have been found to be dysregulated in tumors (reviewed in Ventura and Jacks, 2009). miRNA profiles have been correlated to particular tumors, based on their cell of origin, and in some cases to prognosis (Lu et al., 2005; He et al., 2005; Yanaihara et al., 2006). Because miRNAs alter the expression of multiple genes and thereby may be tuning multiple regulatory steps in disease pathways, they represent interesting drug targets; antisense oligonucleotide targeting experiments in mice (Krutzfeldt et al., 2005) and non-human primates (Elmen et al., 2008) have demonstrated the feasibility of manipulating miRNA levels. At the same time, elucidating miRNA target networks may shed important insights for defining regulatory processes contributing to cancer. The overall aim of this proposal is to advance small RNA profiling and small RNA histological methods for cost-effective characterization of large numbers of patient samples. To interpret the alterations observed in miRNA expression patterns, we will apply our recently developed powerful biochemical approach for direct sequencing of miRNA-targeted mRNA networks to relevant tumor cell culture models. Finally, these recent developments will be implemented in breast cancer research analyzing 177 histologically and clinically characterized tumor samples. First, RNA-deep-sequencing-based methods will be to a stage where they are suitable to define miRNA expression patterns and mutational status for large numbers of clinical samples with limited material. Second, alterations detected in miRNA expression patterns will be followed by miRNA in situ hybridization, for which we recently developed new small RNA fixation techniques and overcame the biggest obstacle hindering miRNA histological detection in tissue sections. Third, to gain insights into the biology of miRNA deregulation, we will apply our recently developed Photoreactive-Uridine-Enhanced Crosslinking and Immunoprecipitation (PURE- CLIP) method of miRNA-bound Argonaute protein complexes to identify the miRNA target sites in transcriptomes of tumor cells. In the last aim, we will apply our methods to assess the value of miRNA analysis in a large collection of triple negative (HER2 gene amplification, Estrogen Receptor (ER), and Progesterone Receptor (PR) negative) and other types of breast tumors in collaboration with Marc van de Vijver at the Academic Medical Center in Amsterdam, The Netherlands. The specimen collection includes 80 triple negative, 51 HER2 positive, 26 ER positive, 11 normal and 20 ductal carcinoma in situ (DCIS) samples. These specimens are already characterized histologically, as well as at the mRNA transcript expression level, and patient clinical characteristics and outcome are available. We will determine absolute miRNA content, relative miRNA composition or profile, miRNA mutational status, and miRNA expression patterns and assess correlations with already determined clinical parameters. We will also define the miRNA-target RNA regulatory networks in tumor cells to be able to interpret consequences of miRNA deregulation in breast cancer. Developing miRNA diagnostic methods and identifying miRNA regulatory networks in tumors PUBLIC HEALTH RELEVANCE: miRNAs are recently identified small non-coding RNAs that regulate many developmental and physiological processes including cancer. This proposal aims to develop new small RNA cloning protocols to meet requirements in clinical research, enhance in situ hybridization methods for cellular localization of miRNAs and at the same time adapt these methods for use in automated platforms of diagnostic laboratories, and clarify miRNA regulatory networks in cancer cell lines, by adapting our recently developed method for identification of miRNA mRNA targets.