MicroRNA Based Pathway Discovery in Cellular Reprogramming

描述(由申请人提供)：基本需要新的变革性方法，在一个系统、多基因、水平而不是一次一个基因的水平上解剖重要的生物过程。单基因方法常常因为复杂的反馈和前馈机制以及生物系统中的冗余而存在缺陷。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困难地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，提供涉及特定生物结果的所有途径的更完整的地图。这里的目标是使用miRNAs来剖析促进成年体细胞去分化为诱导的多能干细胞所需的大部分(如果不是全部)途径。中心假设是，人们可以利用miRNAs的独特功能，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发现蛋白质、通路、通路中的模块以及重新编程为诱导多能性的潜在细胞过程。这一假说源于初步数据，该数据显示了特定的miRNAs如何影响重编程，虽然这些miRNAs每个有数百个靶子，但这些靶子可以组织成路径和蛋白质网络，从而为重编程的机制提供越来越全面的知识。提出了以下具体目标：1)改进基于网络关联的miRNA靶标预测，2)使用预测来剖析单个miRNAs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方法确定调节重新编程的大部分(如果不是全部)途径。在目标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，以更准确和全面地识别单个miRNAs的靶标。在目标2中，将分别测试ESCC miRNAs的分子和生物信息识别的靶标对重新编程、细胞周期和自我更新的影响，以及与这些靶标相关的途径。在目标3中，将测试所有miRNAs对重新编程的影响，根据积极影响和消极影响将其目标组织成网络，并对由此产生的丰富网络进行实验测试。这一提议具有非常重要的意义，因为它为揭示生理过程背后的分子机制提供了新的范式。虽然侧重于重新编程，但所述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。这种系统水平的知识将允许对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。 公共卫生相关性：这项拟议的研究与公共健康相关，因为它开发了解剖生物过程中涉及的多个分子路径的新方法，这将使更全面和智能的方法在治疗从退行性疾病到癌症的任何疾病中操作细胞。此外，最初的重点是诱导多能性，它有可能产生替代组织，并为研究人类疾病提供新的强大工具。因此，这项研究与NIH促进基本创造性发现的使命有关，这些发现提高了国家保护和改善人类健康的能力。

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. PUBLIC HEALTH RELEVANCE: The proposed research is relevant to public health because it develops novel means of dissecting the multiple molecular pathways involved in a biological process, which will enable more comprehensive and intelligent means of manipulating cells in the treatment of anything from degenerative disease to cancer. Furthermore, the initial focus is on induced pluripotency, which has the potential to produce replacement tissues as well as provide new and powerful tools to study human disease. Therefore the research is relevant to NIH's mission to foster fundamental creative discoveries that increase the Nation's capacity to protect and improve human health.