Circznf236 Mediates Scaps Differentiation By Regulating Lgr4-Induced Autophagy

The study identified a novel circRNA, circ-ZNF236 and showed the significance of circ-ZNF236 in the odonto/osteogenic differentiation of SCAPs and its underlying regulatory mechanisms. The circular structure of circ-ZNF236 was identified via Sanger sequencing, amplification of convergent and divergent primers. The proliferation of SCAPs was detected by CCK-8, flow cytometry analysis and EdU incorporation assay. Western blotting, qRT-PCR, Alkaline phosphatase and Alizarin red staining were performed to explore the regulatory effect of circ-ZNF236/miR-218-5p/LGR4 axis in the odonto/osteogenic differentiation of SCAPs in vitro. Fluorescence in situ hybridization, as well as dual-luciferase reporting assays, revealed that circ-ZNF236 binds to miR-218-5p. Transmission electron microscopy and mRFP-GFP-LC3 lentivirus were performed to detect the activation of autophagy. Circ-ZNF236 was identified as a highly stable circRNA with a covalently closed loop structure, no detectable influence on cell proliferation, but positively regulated SCAPs odonto/osteogenic differentiation. Furthermore, circ-ZNF236 was confirmed as a sponge of miR-218-5p in SCAPs, while miR-218-5p targets LGR4 mRNA at its 3′-UTR. Subsequent rescue experiments revealed that circZNF236 regulates odonto/osteogenic differentiation by miR-218-5p/LGR4 in SCAPs. Circ-ZNF236 activated autophagy, and the activation of autophagy strengthened the committed differentiation capability of SCAPs.