Regulation of anti-endocrine resistance of breast cancer by a network of non-codi

描述（由申请人提供）：非编码rna网络对乳腺癌抗内分泌抵抗的调控非编码rna的发现增加了一层复杂性，以了解正常细胞中基因表达的精确调控是如何实现的，以及它们的失调如何导致包括癌症在内的病理状态。MicroRNAs （miRNAs或miRs）是这些非编码rna中定义最好的一类。MiRs被认为是通过以序列特异性的方式与目标信使rna结合并引起抑制而起作用的。miRs的异常表达与包括乳腺癌在内的几种恶性肿瘤的发病机制有关。用于研究miR-mRNA相互作用的常规技术包括表达谱分析（通过微阵列或高通量测序），miR-mRNA靶点的生物信息学预测，mir的过表达和敲除，随后测量转录物和蛋白质水平的变化，以及基于报告的mir的3'非翻译区结合分析。尽管这些方法提供了关于miR-mRNA相互作用的有价值的信息，但它们在直接证明体内特异性miR-mRNA相互作用方面存在不足。为了解决这些缺点，最近描述了一种高通量测序交联免疫沉淀（HITS-CLIP）的生化技术，该技术直接分离miR-mRNA相互作用组。该技术依赖于紫外线（UV）将miRs和mrna与argonaute蛋白交联的能力，然后通过免疫沉淀分离RNA-蛋白复合物，分离RNA并测序。我们已经成功地采用这种技术来定义乳腺癌细胞的miR-mRNA相互作用组。这项职业发展指导基金的申请旨在应用基于HITS-CLIP的系统生物学方法来定义miRs在乳腺癌抗内分泌治疗耐药性中的作用。由于其高效、低毒的特点，抗内分泌治疗成为所有雌激素受体阳性乳腺癌患者治疗的支柱。然而，初级和次级电阻仍然是一个问题。其他研究者的结果表明，多种miRs（包括miR-221）参与乳腺癌中雌激素受体的调节。在aim 1中，我们将描述雌激素对乳腺癌miR-mRNA相互作用组的特异性改变。在目标2中，我们将验证我们的假设，即特定的miRs网络有助于乳腺癌对抗内分泌治疗的反应性和/或耐药性。从前两个目标中定义的特定miR网络将在目标3的临床样本中进行验证。在博士的指导下，提出了培训和指导计划。索伯恩和伊莱亚斯将为申请人提供小rna生物学和乳腺癌方面的深入专业知识。科罗拉多大学提供了一个优秀的学术环境，结合了乳腺癌生物学、RNA和生物信息学方面的专业知识。这些研究的数据有望有助于更好地理解小rna在乳腺癌激素反应性和耐药中的作用，并最终开发出新的诊断和治疗方法。

DESCRIPTION (provided by applicant): Regulation of anti-endocrine resistance of breast cancer by a network of non-coding RNAs The discovery of regulatory non-coding RNAs has added a layer of complexity to understanding how precise regulation of gene expression is achieved in normal cells and how their dysregulation may contribute to pathological states including cancer. MicroRNAs (miRNAs or miRs) are the best defined class amongst these non-coding RNAs. MiRs are thought to function by binding to their target messenger RNAs in a sequence- specific fashion and cause repression. Abnormal expression of miRs has been linked to the pathogenesis of several malignancies including breast cancer. Conventional techniques employed to study miR-mRNA interactions include profiling of expression (by either microarray or high throughput sequencing), bio-informatic prediction of miR-mRNA targets, over-expression and knock-down of miRs followed by measurement of changes in transcript and protein level, and reporter-based analysis of 3' untranslated region-binding of miRs. Although these approaches have provided valuable information about miR-mRNA interactions, they fall short in directly demonstrating specific miR-mRNA interactions in vivo. To address these shortcomings, a biochemical technique of High Throughput Sequencing following Cross-Linked Immunoprecipitation (HITS-CLIP) was recently described, which directly isolates the miR-mRNA interactome. This technique relies on the ability of ultraviolet light (UV) to cross-link miRs and mRNAs to the argonaute proteins followed by the isolation of RNA- protein complexes by immune precipitation, isolation and sequencing of the RNA. We have successfully adapted this technique to define the miR-mRNA interactome of breast cancer cells. This application for a Mentored Career Development Grant seeks to apply a systems biology approach based on HITS-CLIP to define the role of miRs in resistance to anti-endocrine therapy in breast cancer. Given their high efficacy and low toxicity, anti-endocrine therapies form the backbone of treatments for all patients with estrogen receptor positive breast cancer. However, both primary and secondary resistance remains a problem. Results from other investigators have suggested that multiple miRs (including miR-221) are involved in estrogen receptor regulation in breast cancer. In aim 1, specific changes brought about by estrogen in the miR-mRNA interactome of breast cancer will be characterized. In aim 2, we will test our hypothesis that a specific network of miRs contributes to the responsiveness and / or resistance of breast cancer to anti-endocrine therapy. Specific miR network defined from the first two aims will be validated in clinical samples in aim 3. The training and mentoring program proposed under the guidance Drs. Thorburn and Elias will provide the applicant with an in depth expertise in the biology of small RNAs and breast cancer. University of Colorado provides an outstanding academic environment that combines expertise in breast cancer biology, RNA and bioinformatics. Data from these studies is hoped to contribute to a better understanding of the role of small RNAs in hormone responsiveness and resistance of breast cancer and ultimately to novel diagnostics and therapeutics.