TARGETING MIR-29 FUNCTION IN CUTANEOUS WOUND REPAIR

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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.