Genetic Analysis of MicroRNA Functions in Skin Stem Cells in Vivo

描述（由申请人提供）：哺乳动物皮肤及其附件作为身体的最外层屏障，保护内部器官免受环境危害，并将必需液体保持在体内。哺乳动物皮肤的稳态和完整性由存在于不同皮肤区室中的多个祖细胞和干细胞群体维持。特别是，位于隆突区域的多能毛囊干细胞（HFSC）在正常毛发周期和表皮伤口愈合期间维持毛囊（HF）。了解调节HFSCs自我更新和激活的分子机制是利用这些迷人的细胞进行再生医学的基本先决条件。在这些机制中，microRNA（miRNAs）是一类小的非编码RNA，其在哺乳动物不同细胞类型和组织中的基因调控中起重要作用。与有效激活或抑制基因表达的转录机制不同，miRNA在转录后水平调节mRNA的蛋白质产生，并且通常用于微调转录组的输出。尽管对单个靶点的调节有限，但miRNA广泛调节大量（60%）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途径在胚胎皮肤发育和成年HF谱系维持中的关键功能已得到充分认识。与此形成鲜明对比的是，没有报道确定成人皮肤中HFSC自我更新所需的任何单个miRNA。因此，在HFSC中miRNA介导的调节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，我们建议关注miR-205，HFSC中最高表达和功能重要的miRNA，并检查其在成体HFSC自我更新和迁移中的作用（目的1和2）。为了提供对这些功能的机制见解，我们将miRISC及其相关mRNA靶标的生物化学纯化应用于WT和miR-205敲除的HFSC，并以HFSC特异性方式鉴定所有miR-205相关靶标。该项目的长期目标是阐明单个miRNA的功能，并在HFSC中的稳态和应激条件（包括损伤反应和肿瘤发生）期间为这些分子提供机制见解。综上所述，本文提出的研究，如果成功，将显着提高我们对个体miRNAs在成人HFSC中功能的认识。在正常和应激条件下从这些研究中获得的知识将为操纵miRNAs并将这些干细胞用于再生医学铺平道路。

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.