Genetic Analysis of MicroRNA Functions in Skin Stem Cells In Vivo

项目摘要： 哺乳动物的皮肤及其附属物是身体最外层的屏障， 保护内脏免受环境危害，并保持体内的基本液体。 哺乳动物皮肤的稳态和完整性由多个祖细胞和干细胞维持 存在于不同皮肤隔室中的细胞群 表皮和毛囊中的隆突干细胞。在上皮细胞中，细胞粘附、迁移和 增殖是由许多机制控制的基本性质。各主要 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中的基因调控。尽管有适度的监管， 作为单个靶点，miRNAs广泛调节大量（60%）基因，并发挥重要作用。 在广泛的生物过程中发挥作用。在哺乳动物的皮肤中， 胚胎皮肤发育和成人HF维持中的完整miRNA途径 血统得到了很好的赞赏。相比之下，单个miRNAs的知识， 目标和功能仍然很少。重要的是，与其他调节因子类似， 调节细胞迁移和增殖尚未在完整皮肤的背景下进行研究， 活的动物为了解决这些重要问题，我们开发了直接 捕获miRNA及其靶向mRNA片段，并对细胞迁移和增殖进行成像 以及活体动物完整皮肤中的细胞骨架动力学。使用这些最先进的工具 与我们的小鼠模型一起，我们发现miR-205，在小鼠中表达最高的miRNA， 上皮干细胞，通过靶向粘附连接的组分促进细胞迁移， 表皮和毛囊中的肌动蛋白细胞骨架和机械感应基因。在这 我们将进一步研究miR-205调节的细胞迁移如何改变平衡 表皮增殖和表皮分化之间的关系（目的1）; miR-205- 诱导的细胞迁移触发年轻和老年小鼠的毛囊生长，以及如何增强 毛囊生长影响毛囊干细胞（目标2）;并探讨如何失去Piezo 1， 机械激活的离子通道和一个新的miR-205靶点，控制头发的静止 卵泡干/祖细胞（Aim 3）。总之，这里提出的研究，如果成功，将 显著提高了我们对单个miRNAs介导机制的认识， 在活体动物中控制细胞迁移和增殖。从这些研究中获得的知识 在正常和应激条件下， 上皮干细胞用于再生医学。

Project Summary: 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 such as basal cells in the interfollicular epidermis and bulge stem cells in hair follicles. In epithelial cells, cell adhesion, migration and proliferation are fundamental properties that are controlled by many mechanisms. Among key regulators, microRNAs (miRNAs) are a class of small, noncoding RNAs that take essential roles in mammalian gene regulation in diverse cell types and tissues. 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 contrast, the knowledge of individual miRNAs for their targets and function remains scarce. Importantly, similar to other regulators, how miRNAs regulate cell migration and proliferation has not been examined in the context of intact skin in live animals. To address these important issues, we have developed techniques to directly capture miRNA and their targeted mRNA fragments and to image cell migration and proliferation as well as cytoskeleton dynamics in intact skin of live animals. Using these state-of-art tools together with our mouse models, we find that miR-205, the most highly expressed miRNA in epithelial stem cells, promotes cell migration by targeting components of adherens junctions, actin cytoskeleton and mechanosensing genes in both epidermis and hair follicles. In this project, we will further examine how miR-205-regulated cell migration alters the balance between epidermal proliferation and differentiation in the epidermis (Aim 1); how miR-205- induced cell migration triggers hair follicle growth in young and aged mice and how enhanced hair follicle growth affects hair follicle stem cells (Aim 2); and probe how the loss of Piezo1, a mechanically activated ion channel and a new miR-205 target, governs the quiescence of hair follicle stem/progenitor cells (Aim 3). Taken together, studies proposed here, if successful, will significantly enhance our knowledge about mechanisms mediated by individual miRNAs that govern cell migration and proliferation in live animals. The knowledge gained from these studies under normal and stressed conditions will pave the way to manipulate miRNAs and utilize epithelial stem cells for regenerative medicine.