The Role Of RNA Helicase P68 In Wound Healing

As the largest organ of human body, skin serves as a protective barrier against the outside world, any damage of it should be rapidly and efficiently repaired. Wound healing is a complex and well-ordered process, which involved in the cell around wound margin proliferating, migrating and differentiating. Tissue injury causes vascular vessels disruption and extravasation. Inflammatory cells migrated to the wound site secreted many cytokines, which also play important roles in the wound healing regulation. A major goal of wound healing biology is to figure out how skin can be induced to reconstruct the damaged parts more perfectly. In histological level, the process has been well documented, while the genes involved in the wound healing process needs further study.The nuclear RNA helicase p68 is a prototypical member of DEAD box family of RNA helicase. p68 has been highly conserved in evolution and appears to be essential for normal cell growth. p68 plays a very important role in cell proliferation and early organ development and maturation. The protein is expressed in all dividing cells of different vertebrates. The protein shows clear cell cycle-related localization in the nucleus. Additionally, p68 has been detected to be overexpressed in nuclei of epithelial cells under hyperproliferative conditions of colorectal adenocarcinoma. Related to the role of p68 RNA helicase in cell proliferation, the expression of the protein was shown to be correlated with tumor progression and transformation. A previous report has indicated that p68 RNA helicase down-expressed after wound. While the characterization of p68 expression in skin wound healing process has not been fully demonstrated. This prompts us to extensively study the expression pattern of p68 RNA helicase in wound healing process and uncover its roles in the regulation of tissue repairing cell proliferation and migration.In this study we used various methods on morphology and molecular biology to study the important roles of p68 in wound healing. First, we detected the expression pattern of p68 in cutaneous tissues at different stages of postnatal development in rats by immunohistochemistry. Then, we detected the expression of p68 in the wounded tissues from both simple wounds and impaired-healing wounds induced by total body irradiation. We also detected the expression of p68 in clinical human specimens of hyperplastic scar and squamous cell carcinoma (SCC). The functional studies on the cell proliferation and migration were performed in culture systems by treatment with wound fluids or sequence-specific RNAi of p68.The main results and conclusions of this study are listed as follows:Results:1. The expression of p68 was higher in the new-born (day 1) rat skin and distributed extensively in the epidermis, hair follicles and dermis and its expression level decreased in the skin from rats at days 4 and days 10. After days 15, the p68 expression was not detectable. These results supported that p68 expression was correlated with the skin development.2. The expression of p68 significnatly increased in the skin tissues including epidermal cells and dermal cells after wounding. The protein expression reached the peak at 14 days post wounding, then decreased to the basal level with the complete of the healing process. It was observed that p68 mainly expressed in the nuclei of keratinocytes, dermal multipotent stem cells and vascular endothelial cells in wounded tissues. When combined with 6 Gy total body irradiation, the wound healing process was significantly inhibited and the expression of p68 was also inhibited in the wounded tissues.We spectulated that the expression of p68 was involved in the regulation of wound healing process.3. Compared with the normal human skin, the expression of p68 is overexpresed in hyperplastic scar and squamous cell carcinoma (SCC) tissues. In addition, the p68 protein expression was higher in the squamous cell carcinoma cases with metastasis than those in the primary cases without metastasis. p68 also showed similar expression pattern with PCNA in SCC cases. It was observed for the first time that the overexpression of p68 protein correlated with the development of hyperplastic scar and metastasis of SCC.4. By in vitro study, the expression level of p68 is much lower in the quiescent cells than that in the rapidly proliferating cells of three major repairing cell types, including keratinocytes, dermal multipotent cells and vascular endothelial cells. The acute wound fluid increased the cell proliferation and migration, and increased the expression of p68 in all the three cell types. After knock-down of p68 expression by RNAi, the proliferation and migration of the cells were inhibited; and the expression of PCNA was down-regulated, while the phosphorylated p53 was up-regulated. These strengthened the important role of p68 in the regulation of cellular proliferation and migration.