Molecular mechanism of Circ_PRKDC regulating macrophage polarization in wound healing.

Abstract

PURPOSE: This study explores the molecular mechanism of Circ_PRKDC regulating macrophage polarization in wound healing. METHODS: A mouse skin trauma model was established, and Circ_PRKDC expression was upregulated by subcutaneous injection of lentivirus-packaged Circ_PRKDC overexpression vector. Wound healing and skin pathological changes were observed; iNOS and CD206 expressions were detected by immunofluorescence; M1 and M2 macrophage markers were detected by ELISA. Macrophages were cultured in vitro, and the transition of M1 to M2 under different treatments was detected by flow cytometry. Macrophages were co-cultured with endothelial cells. Wound healing assay and tubular formation assay were performed to detect cell healing and angiogenesis. Circ_PRKDC, FUS, and PRDX4 expressions in tissues or cells were detected by qRT-PCR and Western blot. The binding between Circ_PRKDC and FUS, as well as FUS and PRDX4 mRNA was analyzed by RIP and RNA pull down. RESULTS: Overexpression of Circ_PRKDC inhibits wound healing in mice and alters the expression profiles of markers related to macrophage activation status in the skin. Overexpression of Circ_PRKDC in vitro promotes M1 polarization, while inhibition of Circ_PRKDC promotes M2 polarization. Overexpression of Circ_PRKDC in macrophages co-cultured with endothelial cells slows down wound healing in skin cells and inhibits angiogenesis. Mechanistically, Circ_PRKDC binds to FUS, thus promoting PRDX4 mRNA degradation and inhibiting PRDX4 protein expression; FUS binds to PRDX4 mRNA to stabilize its expression. CONCLUSION: Circ_PRKDC promotes PRDX4 mRNA degradation by binding to FUS, thus reducing the expression of PRDX4, and suppress the phenotypic transition of macrophages from M1 to M2 during wound healing, ultimately inhibiting wound healing. This effect may be realized by disrupting the binding between FUS and PRDX4 mRNA.