Prospective Identification of Translational Regulators in EMT

EMT 中翻译调节因子的前瞻性鉴定

• 批准号: 9068884
• 负责人: Joel R Neilson
• 金额: $ 17.24万
• 依托单位: BAYLOR COLLEGE OF MEDICINE
• 依托单位国家: 美国
• 财政年份: 2015
• 资助国家: 美国
• 起止时间: 2015-05-15 至 2018-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9068884
• 关键词: 3' Untranslated Regions; Adherens Junction; Affinity Chromatography; Automobile Driving; Behavior; Binding; Binding Proteins; Biochemical; Biological Assay; Biological Models; Breast Epithelial Cells; Cell Line; Cell model; Cell physiology; Cells; Density Gradient Centrifugation; Development; Diagnostic Neoplasm Staging; E-Cadherin; Elements; Embryo; Engineering; Epithelial; Family; Flow Cytometry; Gene Expression; Gene Expression Regulation; Genes; Genetic Translation; Genomic approach; Goals; Health; Human; Immunoprecipitation; Investigation; MCF10A cells; Maintenance; Malignant Epithelial Cell; Malignant Neoplasms; Measures; Mediating; Mesenchymal; Messenger RNA; Methods; MicroRNAs; Modeling; Molecular; Monitor; Mutate; Nature; Neoplasm Metastasis; Phase; Phenotype; Physiological Processes; Play; Poly G; Post-Transcriptional Regulation; Primary Neoplasm; Process; Proteins; Proteomics; RNA Interference; RNA-Binding Proteins; Regulation; Regulatory Element; Reporter; Repression; Research; Role; Shapes; Signal Pathway; Snails; Stimulus; System; Testing; Therapeutic Intervention; Transcript; Transcriptional Regulation; Transforming Growth Factor beta; Translating; Translational Regulation; Translations; Untranslated Regions; Work; Xenograft Model; base; cancer cell; cell motility; cell type; comparative; crosslink; cytokine; design; epithelial to mesenchymal transition; functional genomics; gene product; in vivo; knock-down; mRNA Stability; molecular marker; next generation sequencing; novel; overexpression; overexpression analysis; prevent; programs; prospective; protein expression; targeted treatment; transcription factor; transcriptomics; treatment response; tumor; tumorigenesis; vector

 DESCRIPTION (provided by applicant): Post-transcriptional control of mRNA localization, stability, and translation is a fundamentally important, yet largely underexplored, mechanism of gene regulation that contributes to virtually every aspect of cellular physiology. In many cases, elements within the 3' untranslated region (UTR) of a given mRNA transcript confer responsiveness to microRNAs and/or binding proteins, which may dictate the fate of the transcript. One physiological process in which post-transcriptional regulation of gene expression has been demonstrated to play an important role is the epithelial to mesenchymal transition (EMT). EMT is thought to initiate tumor metastasis by altering the shape, invasiveness, and motility of carcinoma cells within the primary tumor. The breast epithelial cell line MCF10a undergoes EMT in response to treatment with the cytokine TGF-ß. Using density gradient fractionation and Next Generation Sequencing, we have examined polysomal enrichment and depletion in MCF10a cells in both the epithelial and mesenchymal states. Within our model system, we have identified 54 gene products that are preferentially translated (4-fold or more enrichment over total mRNA) in the mesenchymal state. Among these gene products is SNAI1, a known driver of EMT in several experimental systems. The nature of the mechanism driving this polysomal enrichment, and whether the remainder of the observed gene products contribute to the initiation or maintenance of the mesenchymal state, are both unclear. The proposed research is designed to assess the feasibility of a template for prospective identification of nove translational regulators within our system. Our hypothesis is two-fold: firstly, that these gene products functionally contribute to the initiation or maintenance of the mesenchymal state, and secondly, that their preferential translation is modulated by the interaction of a common factor with the 3' UTRs of these transcripts. To test these hypotheses, we will employ paired functional genomic approaches that are mutually informative but non-interdependent. Via overexpression and RNAi knockdown, we will determine whether the identified gene products are necessary and sufficient for EMT in three distinct models of this process. In parallel, we will employ a novel, scalable, transposon-based reporter platform to define for which of these mRNA transcripts the 3' UTR confers the translational control. Whether a cis-regulatory element common to many of these transcripts underlies this control will also be directly assessed. Finally, we will use biochemical and molecular methods to prospectively identify and characterize putative trans-regulators binding these putative cis-elements, under the rationale that these trans-regulators may integrate post-transcriptional gene regulation in the EMT programs. Novel regulators of EMT identified in our model system, both direct effectors and the trans-factors that regulate them, are potential targets for therapeutic intervention aimed at preventing metastasis of early-stage cancers. Further, successful implementation of our approach will result in a generalized template that can be leveraged in other cell-based models of tumorigenesis.

 描述（由申请人提供）：mRNA 定位、稳定性和翻译的转录后控制是一种极其重要但在很大程度上尚未得到充分探索的基因调控机制，它对细胞生理学的几乎各个方面都有贡献。在许多情况下，给定 mRNA 转录物 3' 非翻译区 (UTR) 内的元件赋予对 microRNA 和/或结合蛋白的响应性，这可能决定转录物的命运。基因表达的转录后调控已被证明发挥重要作用的一种生理过程是上皮间质转化（EMT）。 EMT被认为是通过改变原发肿瘤内癌细胞的形状、侵袭性和运动性来引发肿瘤转移。乳腺上皮细胞系 MCF10a 响应细胞因子 TGF-ß 的治疗而经历 EMT。使用密度梯度分离和下一代测序，我们检查了上皮状态和间充质状态的 MCF10a 细胞中多核糖体的富集和消耗。在我们的模型系统中，我们已经鉴定了 54 个基因产物，这些产物在间充质状态下优先翻译（比总 mRNA 富集 4 倍或更多）。这些基因产物中有 SNAI1，它是多个实验系统中 EMT 的已知驱动因素。驱动这种多核糖体富集的机制的性质，以及观察到的基因产物的其余部分是否有助于间充质状态的启动或维持，都尚不清楚。拟议的研究旨在评估我们系统中前瞻性识别新型翻译调节因子的模板的可行性。我们的假设有两个：首先，这些基因产物在功能上有助于间充质状态的启动或维持，其次，它们的优先翻译是通过共同因子与这些转录本的 3' UTR 的相互作用来调节的。为了检验这些假设，我们将采用相互提供信息但不相互依赖的配对功能基因组方法。通过过表达和 RNAi 敲除，我们将确定所识别的基因产物对于 EMT 过程的三个不同模型是否是必要和充分的。与此同时，我们将采用一个新颖的、可扩展的、基于转座子的报告平台来定义这些 mRNA 转录本的 3' UTR 赋予翻译控制。还将直接评估许多这些转录物共有的顺式调控元件是否是这种控制的基础。最后，我们将使用生化和分子方法前瞻性地识别和表征结合这些假定的顺式元件的假定的反式调节因子，理由是这些反式调节因子可能将转录后基因调控整合到 EMT 程序中。我们的模型系统中发现的新的 EMT 调节因子，包括直接效应因子和调节它们的反式因子，都是旨在预防早期癌症转移的治疗干预的潜在目标。此外，我们的方法的成功实施将产生一个通用模板，可用于其他基于细胞的肿瘤发生模型。