Project Summary More than 20% of oral squamous cell carcinoma (OSCC) patients present with bone invasion and require maxillary/mandibular continuity restoration after tumor excision. Postoperative adjuvant radiotherapy or chemoradiotherapy is also needed for advanced OSCC tumors. However, despite standard-of-care treatments, the 5-year survival rate for advanced-stage OSCC is less than 50%. Notably, adverse effects of radiotherapy can impair bone formation and delay graft osseointegration after reconstructive surgery. Patients receiving adjuvant radiotherapy have an approximately 20% long-term failure rate because of nonunion of bone grafts, hardware infection, and ultimately flap malunion and loss. Thus, a safer and more effective therapeutic with strong osteoinduction and anti-OSCC capacities are needed to improve reconstructive surgery outcomes of OSCC treatments. MicroRNAs (miRs) are small non-coding RNAs that play crucial roles in craniofacial bone metabolism and OSCC initiation and progression via epigenetically regulating cellular biological processes. miRs have emerged as a potential tool to improve OSCC treatment and reconstructive bone regeneration. Our previously funded studies have demonstrated that miR-200c targets many proinflammatory cytokines and osteoclastogenic mediators and effectively attenuates inflammation and bone resorption. Local application of plasmid DNA (pDNA) encoding miR-200c also significantly promotes osteogenic differentiation of hBMSC and bone regeneration by activating Wnt and BMP activities. pDNA miR-200c incorporated into 3D-printed scaffolds or delivered by CaCO3-based nanoparticles effectively enhance bone regeneration at calvarial and maxillary defects. In addition, our preliminary studies have identified that radiation in OSCC patients and rat mandibles suppress miR-218 expression, a miRNA similar to miR-200c that potently suppresses OSCC and osteoclastogenesis and promotes bone regeneration. Therefore, we hypothesize that pDNA encoding miR- 200c and miR-218 delivered by CaCO3 will promote bone regeneration and bone graft integration in irradiated mandibular bone defects by improving osteogenesis and mitigating inflammation and osteoclastogenesis. We propose to determine the functions of miR-200c in promoting mandibular bone regeneration under radiation and preventing bone graft resorption (Aim 1) and the additional/synergistic function of miR-218 in miR-200c- mediated bone regeneration (Aim 2). We will also elucidate the molecular mechanism(s) and cellular effects underlying anti-OSCC radiation and osteoinductive miRNAs (Aim 3). After accomplishing the project, we expect to lay the groundwork for developing a novel miRNA-based therapeutic that will enhance osteoinduction and suppress bone resorption for advanced OSCC patients.