PROJECT SUMMARY/ABSTRACT The oral mucosa is constantly exposed to a bacteria-rich environment coupled with continual physical trauma. For patients, chronic oral wounds aren't merely an inconvenience. They can impair nutrition intake and pose a substantial risk of debilitating infections, marking a significant oral health challenge. Defining factors involved in oral wound healing would allow the identification of novel therapeutic targets to improve tissue repair. Oral wound healing has long been considered a model of optimal wound resolution characterized by rapid and scarless wound healing. However, the intrinsic genetic and epigenetic controlling mechanisms that make oral mucosa highly adaptable to accelerated healing upon injury remain unclear. An important aspect of healing is re- epithelialization, which entails both proliferation and migration of keratinocytes at the periphery of the wound. Understanding the role and function of keratinocytes in re-epithelialization will benefit patients with chronic wounds. We recently identified a previously unnoticed landscape of non-coding RNA in saliva and found piwi-interacting RNA (piRNA) is surprisingly abundant in saliva. Of particular interest are three distinctive oral-piRNAs that are uniquely present in saliva and oral keratinocytes and appear to contribute to the oral wound re-epithelialization phenotype. The preliminary data involving inhibition of oral piRNAs concomitant with a failure of wounds to heal in a timely fashion suggests that these small RNAs may play a significant role in mediating wound healing. The overarching hypothesis of this proposal is that oral-piRNAs are intrinsic factors in human oral keratinocytes, that modulate the rate of wound closure. Two specific aims are in place to further characterize the wound healing phenotype and elucidate the cellular factors responsible for oral-piRNA-mediated phenotypes. Aim 1 is to investigate the role of saliva piRNAs on oral wound re-epithelialization using 3D ex vivo and in vivo oral wound healing models. Aim 2 is to elucidate the mechanism by which piRNAs regulate oral wound healing. This proposal aims to unravel how these novel oral-piRNAs accelerate human oral keratinocyte proliferation and migration, which may improve wound healing therapeutic developments. The long-term goals of this proposed study are to enhance our understanding of the biological roles of piRNA relevant to human oral health and diseases and leverage these findings to improve wound healing outcomes for patients.