MicroRNA Based Pathway Discovery in Cellular Reprogramming

描述（由申请人提供）：对于在一个系统、多基因水平而不是一次一个基因水平上解剖重要生物过程的新颖变革方法的基本需求。由于生物系统中复杂的反馈和前馈机制以及冗余性，单基因方法往往存在缺陷。如果没有对特定生物过程中涉及的所有途径的更全面的了解，生物学家和临床医生将非常难以操纵这些过程来改善人类健康。该实验室的长期目标是使用小的非编码rna，即mirna，来提供与特定生物学结果相关的所有途径的更完整的图谱。这里的目标是使用mirna来解剖大多数（如果不是全部的话）促进成体体细胞向诱导多能干细胞去分化所需的途径。核心假设是，人们可以利用mirna的独特特征，它具有多个靶点和共同的生理结果，作为一种强大的手段来揭示蛋白质、途径、途径中的模块和细胞过程，这些过程是重编程诱导多能性的基础。这一假设来源于初步数据，这些数据显示了特异性mirna如何影响重编程，尽管这些mirna每个都有数百个靶点，但这些靶点可以被组织成途径和蛋白质网络，从而提供了对重编程机制的日益全面的了解。提出了以下具体目标：1)改进基于网络关联的miRNA靶点预测；2)使用预测来分析单个miRNA家族促进自我更新和多能性的所有途径；3)通过全基因组miRNA方法确定大多数（如果不是全部）调节重编程的途径。在Aim 1中，将结合分子实验和经验测试的关联过滤器来定义基于网络的参数，从而更准确、更全面地识别单个mirna的靶标。在目标2中，将分别测试ESCC mirna的分子和生物信息学鉴定目标对重编程、细胞周期和自我更新的影响，以及与这些目标相关的途径。在Aim 3中，将测试所有mirna对重编程的影响，它们的靶标根据积极和消极影响组织成网络，并通过实验测试由此产生的丰富网络。这为揭示生理过程的分子机制提供了新的范式，具有重要意义。虽然专注于重编程，但所描述的实验开发的工具和方法可用于帮助系统地剖析任何感兴趣的过程。这样的系统级知识将允许对一个过程进行更智能的操作，以达到更好地治疗疾病所需的预期结果。

DESCRIPTION (provided by applicant): There is a fundamental need for novel transformative approaches to dissecting important biological processes at a system, multi-gene, level rather than one gene at a time. Single gene approaches are often flawed by the complex feed-back and feed-forward mechanisms as well as redundancies involved in biological systems. Without more comprehensive knowledge of all the pathways involved in a particular biological process, it will be exceedingly difficult for biologists and clinicians to manipulate these processes to improve human health. The long-term goal of the lab is to use the small non-coding RNAs, miRNAs, to provide a more complete map of all the pathways involved in specific biological outcomes. The objective here is to use miRNAs to dissect most, if not all, the pathways required to promote the dedifferentiation of adult somatic cells to induced pluripotent stem cells. The central hypothesis is that one can use the unique features of miRNAs, which have multiple targets with common physiological outcomes, as a robust means to uncover proteins, pathways, modules within pathways, and cellular processes underlying the reprogramming to induced pluripotency. This hypothesis derives from preliminary data showing how specific miRNAs can influence reprogramming and that, while these miRNAs have hundreds of targets each, the targets can be organized into pathways and protein networks that provide an increasingly comprehensive knowledge of the mechanisms of reprogramming. The following specific aims are proposed: 1) Improve miRNA target predictions based on network associations, 2) Use predictions to dissect all pathways by which a single family of miRNAs promotes self-renewal and pluripotency, 3) Determine most, if not all pathways, that regulate reprogramming through a genome-wide miRNA approach. In Aim 1, a combination of molecular experiments and empirically tested association filters will be used to define network based parameters that more accurately and comprehensively identify targets of individual miRNAs. In Aim 2, molecularly and bioinformatically identified targets of the ESCC miRNAs will be individually tested for their influence on reprogramming, cell cycle, and self-renewal as will the pathways to which the targets are associated. In Aim 3, all miRNAs will be tested for their influence on reprogramming, their targets organized into networks based on positive versus negative influences, and resulting enriched networks tested experimentally. This proposal is highly significant as it provides novel paradigms for uncovering molecular mechanisms underlying physiological processes. While focused on reprogramming, the tools and approach developed by the described experiments could be used to help systematically dissect any process of interest. Such systems level knowledge will allow for more intelligent manipulation of a process to reach a desired outcome required for the better treatment of disease.