Dicalcium silicate promotes osteogenic differentiation and bone defect healing via circ_1983–miR-6931–Gas7 interaction

Background: Dicalcium cilicate (C2S) is a potent biomaterial for bone regeneration application. Circular RNA (circRNAs) plays crucial role in osteogenic differentiation of mesenchymal stem cells (MSCs) and bone defect healing. In this study, we aim to elucidate the differential expression of circ_RNAs and mRNAs in C2S-treated MSCs, the role of circ_1983 in C2S-mediated bone regeneration, and its mechanism.Methods: The effect of C2S on osteogenic differentiation of mice bone marrow-derived MSCs (BMSCs) and rat cranial bone defect healing were analyzed. Differential expression of circ-RNAs and mRNAs in C2S-treated BMSCs were profiled by RNA-sequencing. The interaction between circ_1983 and miR-6931 was confirmed by pull-down and dual luciferase reporter assays. ceRNA function of circ_1983 via sponging miR-6931 and targeting Gas7 mRNA expression in BMSCs was analyzed by miRanda (http://www.microrna.org/microrna/). Role of circ_1983 in C2S-induced osteogenic differentiation of BMSCs was analyzed by shRNA-mediated knockdown of circ_1983.Results: C2S enhanced osteogenic differentiation of BMSCs and bone defect healing. CircRNAs (total 1384) and mRNAs (total 1725) were differentially upregulated in C2S-treated BMSCs. Upregulated circRNAs and ceRNA-interaction networks were associated with osteogenesis-related signaling pathways, i.e. MAPK, PI3K-Akt. RNA-sequencing and qRT-PCR results confirmed upregulation of circ_1983 in C2S-tretaed BMSCs. Circ_1983 showed ceRNA effect by sponging miR-6931 and overexpressing Gas mRNA that enhanced Runx2 expression and promoted osteogenic differentiation of BMSCs. Knockdown of circ_1983 inhibited the C2S-induced osteogenic differentiation of BMSCs.Interpretation: C2S-induced circ_1983 upregulation in BMSCs sponges miR-6931 and targets Gas7 gene to enhance Runx2 expression thereby promotes osteogenic differentiation and bone regeneration.