TARGETING MIR-29 FUNCTION IN CUTANEOUS WOUND REPAIR

在我们的社会中，无法愈合的皮肤溃疡的发生率和对改善皮肤再生的需求不断增加。为了开发有效的策略来改善皮肤修复，了解正常和受损愈合的潜在机制是至关重要的。在我的项目中，我试图进一步描述小核糖核酸(RNA)分子miR-29的功能，我认为它是皮肤顶层表皮的调节器。在受伤时，表皮细胞(主要是角质形成细胞)负责用一层新的皮肤覆盖伤口部位，以保护我们的身体免受感染进入和水分流失。只有在健康的皮肤上，角质形成细胞才能充分生长和迁移到伤口部位。了解这一过程在正常皮肤中是如何调节的，以及在伤口愈合受损的患者中角质形成细胞的再生是如何被解除调节的，这是非常重要的。这项研究的总体目标是促进角质形成细胞的正常生长，从而促进表皮的再生。然而，另一方面，角质形成细胞的过度生长会导致皮肤过度增厚，表皮破裂，水分丢失，以及严重的继发性感染。因此，找到调节和确保微调控制角质形成细胞正常生长的分子是很重要的。小RNA，如miR-29，已经被描述为正常皮肤生长的精确和生理调节因子。重要的是，由于短核酸的小尺寸和化学性质，miRs可以用于分子治疗。MIR-29已经在临床上用于改善皮肤硬化患者的皮肤状况(表现为皮肤异常增厚)，因此有可能迅速用于包括伤口在内的其他皮肤病的分子治疗。然而，这需要了解角质形成细胞内与miR-29相互作用的所有可能的分子，以及可能由表皮中的miR-29调控的所有细胞内和细胞间过程。我将使用从非治疗性手术(例如整形手术或包皮环切术)后残留的人类皮肤活检组织中分离出的人类角质形成细胞，在人造胶原基质上将角质形成细胞培养成与人类皮肤相当的细胞。这将允许对miR-29水平的遗传操作、生化下拉、纯化和分析功能性miR-29与其他分子的复合体。我将研究miR-29对角质形成细胞生长和迁移的影响，并将使用短的合成核酸分子来抑制miR-29。这将允许开发新的策略，在伤口愈合期间、大规模烧伤后和重建手术中实现皮肤的成功再生。我还将研究miR-29s在小鼠伤口愈合模型中的功能，以模拟体内再生的复杂性。该项目的长期目标是利用miR-29改善患有大型急性伤口、压疮和糖尿病溃疡的患者的皮肤再生。

The incidence of non-healing skin ulcers and the need of improved skin regeneration is continuously increasing in our society. For the development of efficient strategies to improve skin repair it is essential to understand the mechanisms underlying normal and impaired healing. For my project, I am seeking to further describe functions of small ribonucleic acids (RNA) molecules, miR-29s, which I identified as regulators of the top layer of the skin called the epidermis. Upon wounding, the cells of the epidermis (mostly keratinocytes) are responsible for covering the wound site with a new layer of skin that protects our body from infection entry and water loss. Keratinocytes can sufficiently grow and migrate into the wound site only in a healthy skin. It is very important to understand how this process is regulated in the normal skin, and then how this regeneration of keratinocytes is deregulated in patients with impaired wound healing. The overall goal of this research is to enhance normal growth of keratinocytes and thereby, regeneration of the epidermis. However, on the other hand, the excessive growth of keratinocytes results in a hyper-thickened skin, cracking of the epidermis, water loss, and severe secondary infections. Thus, it is important to find molecules that regulate and ensure a fine-tuned control over the normal growth of keratinocytes. Small RNAs, like miR-29s, have been already described to function as precise and physiologic regulators of normal skin growth. Importantly, because of the small size and chemical properties of short nucleic acids, miRs can be used for molecular therapy. miR-29s have already been tried in the clinic to improve skin condition in patients with cutaneous sclerosis (manifested by the abnormal skin thickening), and thus can potentially be rapidly used for molecular therapy of other skin diseases, including wounds. However, it requires knowing all possible molecules that interact with miR-29s inside keratinocytes, and all intra- and intercellular processes, which may be regulated by miR-29s in the epidermis. I will use human keratinocytes isolated from human skin biopsies remaining after non-therapeutic surgeries (e.g., plastic surgery or circumcisions) and grow keratinocytes into human skin equivalents on artificial collagen matrix. This will allow genetic manipulation of miR-29 levels, biochemical pull-down, purification, and analysis of the functional miR-29 complexes with other molecules. I will study the effect of miR-29 on growth and migration of keratinocytes and will use short synthetic nucleic acid molecules to inhibit miR-29. This will allow development of the new strategy to achieve a successful regeneration of the skin during wound healing, after massive burns, and in reconstructive surgeries. I will also study the function of miR-29s in murine wound healing model, necessary to mimic the complexity of the in vivo regeneration. The long-term goal of the project is to utilize miR-29 to improve skin regeneration in patients suffering from large acute wounds, bedsores, and diabetic ulcers.