Prospective Identification of Translational Regulators in EMT

EMT 中翻译调节因子的前瞻性鉴定

基本信息

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
项目类别：
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&apos 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

项目摘要

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尚未充分探索的基因调控机制，在许多情况下，它对细胞生理学的几乎各个方面都有贡献。给定 mRNA 转录物的非翻译区 (UTR) 赋予对 microRNA 和/或结合蛋白的反应性，这可能决定转录物的命运，其中已证实基因表达的转录后调节。发挥重要作用的是上皮间质转化（EMT），据认为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 会响应 EMT。使用细胞因子 TGF-ß 处理，使用密度梯度分离和下一代测序，我们检查了两种细胞中 MCF10a 细胞的多核糖体富集和消耗。在我们的模型系统中，我们鉴定了 54 个在间质状态下优先翻译的基因产物（比总 mRNA 富集 4 倍或更多），其中 SNAI1 是多种 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，均不清楚。拟议的研究旨在评估。在我们的系统中前瞻性鉴定新型翻译调节因子的模板的可行性我们的假设有两个方面：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，它们的优先翻译受到调节。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 敲低来确定是否存在这种相互作用。所鉴定的基因产物对于该过程的三个不同模型中的 EMT 是必要且充分的。同时，我们将采用一种新颖的、可扩展的、基于转座子的报告平台来定义这些 mRNA 转录本中的哪些 3'UTR 赋予翻译控制。最后，我们将直接评估许多这些转录物共有的顺式调控元件是否构成这种控制的基础，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反式调节因子。顺式元件，根据这些反式调节因子可能将转录后基因调节整合到我们的模型系统中识别的EMT程序中的新调节因子，无论是直接效应子还是调节它们的反式因子，都是潜在的目标。此外，旨在预防早期癌症转移的治疗干预将产生一个可用于其他基于细胞的肿瘤发生模型的通用模板。