MicroRNA Based Pathway Discovery in Cellular Reprogramming

描述（由申请人提供）：根本上需要新的变革性方法来在系统、多基因水平上剖析重要的生物过程，而不是一次一个基因。单基因方法往往是有缺陷的复杂的反馈和前馈机制，以及冗余参与生物系统。如果没有对特定生物过程中所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（miRNAs）来提供涉及特定生物学结果的所有途径的更完整的图谱。本文的目的是利用miRNAs来剖析促进成体细胞去分化为诱导多能干细胞所需的大多数（如果不是全部的话）途径。核心假设是，人们可以使用具有共同生理结果的多个靶点的miRNA的独特特征，作为一种强大的手段来揭示蛋白质、途径、途径内的模块以及重编程诱导多能性的细胞过程。这一假设来自于初步数据，这些数据显示特定的miRNAs如何影响重编程，虽然这些miRNAs每个都有数百个靶标，但这些靶标可以组织成通路和蛋白质网络，从而提供对重编程机制越来越全面的了解。提出了以下具体目标：1）基于网络关联改进miRNA靶点预测，2）使用预测来剖析单个miRNA家族促进自我更新和多能性的所有途径，3）确定通过全基因组miRNA方法调节重编程的大多数（如果不是全部）途径。在目标1中，将使用分子实验和经验测试的关联过滤器的组合来定义基于网络的参数，以更准确和全面地识别单个miRNA的靶标。在目标2中，将单独测试ESCC miRNA的分子和生物信息学鉴定的靶标对重编程、细胞周期和自我更新的影响，以及靶标相关的途径。在目标3中，将测试所有miRNA对重编程的影响，基于正面与负面影响将其靶标组织成网络，并通过实验测试所得的富集网络。这一建议是非常重要的，因为它提供了新的范例，揭示生理过程的分子机制。虽然专注于重新编程，但所描述的实验开发的工具和方法可以用来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过程进行更智能的操纵，以达到更好地治疗疾病所需的期望结果。

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.