Genetic Analysis of MicroRNA Functions in Skin Stem Cells in Vivo

描述（由申请人提供）：哺乳动物的皮肤及其附属物是身体最外层的屏障，保护内部器官免受环境危害，并保持体内必需的液体。哺乳动物皮肤的稳态和完整性是由居住在不同皮肤区室的多个祖细胞和干细胞群维持的。特别是，位于凸起区域的多能毛囊干细胞（HFSCs）在正常的毛发周期和表皮伤口愈合期间维持毛囊（HFs）。了解调节HFSCs自我更新和激活的分子机制是开发这些迷人细胞用于再生医学的基本前提。在这些机制中，microRNAs （miRNAs）是一类小的非编码rna，在哺乳动物不同细胞类型和组织的基因调控中发挥重要作用。与有效激活或抑制基因表达的转录机制不同，miRNAs在转录后水平调节mrna的蛋白质产生，通常用于微调转录组的输出。尽管对单个靶标的调节有限，但mirna广泛调节大量（60%）的基因，并在广泛的生物过程中发挥重要作用。在哺乳动物皮肤中，整个miRNA通路在胚胎皮肤发育和成年HF谱系维持中的关键功能已经得到了很好的认识。与此形成鲜明对比的是，目前还没有报道确定成人皮肤中HFSCs自我更新所需的任何单个miRNA。因此，mirna介导的HFSCs调控的潜在机制在很大程度上仍然未知。为了解决这些重要问题，我们建议将重点放在miR-205上，这是HFSCs中表达最高、功能最重要的miRNA，并研究其在成人HFSCs自我更新和迁移中的作用（目的1和2）。为了提供这些功能的机制见解，我们将对miRISC及其相关mRNA靶点进行生化纯化，用于WT和miR-205敲除的HFSC，并以HFSC特异性的方式识别所有miR-205相关靶点。该项目的长期目标是阐明单个mirna的功能，并提供这些分子在稳态和应激条件下的机制，包括HFSCs的损伤反应和肿瘤发生。综上所述，本文提出的研究如果成功，将显著增强我们对成人HFSCs中单个mirna功能的了解。从这些正常和应激条件下的研究中获得的知识将为操纵mirna和利用这些干细胞进行再生医学铺平道路。

DESCRIPTION (provided by applicant): Mammalian skin and its appendages function as the outermost barrier of the body to protect inner organs from environmental hazards and keep essential fluids within the body. Homeostasis and integrity of mammalian skin are maintained by multiple progenitor and stem cell populations residing in distinct skin compartments. In particular, multipotent hair follicle stem cells (HFSCs) located in the bulge region maintain hair follicles (HFs) during normal hair cycle and epidermal wound healing. Understanding molecular mechanisms that regulate self-renewal and activation of HFSCs is a fundamental prerequisite to exploit these fascinating cells for regenerative medicine. Among the mechanisms, microRNAs (miRNAs) are a class of small, noncoding RNAs that take essential roles in mammalian gene regulation in diverse cell types and tissues. Unlike transcriptional mechanisms that potently activate or suppress gene expression, miRNAs regulate protein production of mRNAs at post-transcriptional levels and usually function to fine tune the output of the transcriptome. Despite modest regulation of individual targets, miRNAs broadly modulate a large number (60%) of genes and play important roles in a wide range of biological processes. In mammalian skin, the critical functions of the entire miRNA pathway in both embryonic skin development and maintenance of adult HF lineages have been well appreciated. In sharp contrast, there is no report identifying any individual miRNA that is required for the self-renewal of the HFSCs in adult skin. Consequently, the underlying mechanism of miRNA-mediated regulation in the HFSCs remains largely unknown. To address these important issues, we propose to focus on miR-205, the most highly expressed and functionally important miRNA in the HFSCs, and examine its roles in self- renewal and migration of adult HFSCs (Aim 1 and 2). To provide mechanistic insights for these functions, we will apply biochemical purification of miRISC and its associated mRNA targets to the WT and miR-205 knockout HFSCs and identify all miR-205-associated targets in an HFSC- specific manner. The long-term goal of this project is to elucidate individual miRNAs' functions and provide mechanistic insights to these molecules during homeostasis and stressed conditions including injury response and tumorigenesis in the HFSCs. Taken together, studies proposed here, if successful, will significantly enhance our knowledge about individual miRNAs' functions in adult HFSCs. The knowledge gained from these studies under normal and stressed conditions will pave the way to manipulate miRNAs and utilize these stem cells for regenerative medicine.