Circ_0003072 Mediates the Pro-osteogenic Differentiation Effect of Betulinic Acid on Human Periodontal Ligament Stem Cells.

BACKGROUND: Betulinic acid (BetA) exhibits a good pro-osteogenic differentiation effect on human periodontal ligament stem cells (hPDLSCs), making it a promising supplement for periodontal regeneration. Circular RNAs (circRNAs) have emerged as important regulators of cellular behaviour, and whether circRNAs are involved in the effects of BetA remains unknown. METHODS: Bioinformatics analysis was used to screen for dysregulated circRNAs involved in osteogenic differentiation based on public datasets. Osteogenic differentiation was evaluated using quantitative PCR detection of RUNX2, ALPL, COL1A1, and BGLAP levels, alkaline phosphatase staining, and Alizarin Red S staining of calcified nodules. The role of circ_0003072 in the BetA-regulated osteogenic differentiation of hPDLSCs was investigated, and its downstream microRNAs and mRNA were confirmed using RNA-sequencing, competing endogenous RNA network construction, gene ontology analysis, dual-luciferase reporter assays, and functional assays. RESULTS: circ_0003072 showed the highest fold-change among the 18 candidate circRNAs, and knockdown of circ_0003072 inhibited the pro-osteogenic differentiation effect of BetA on hPDLSCs. RNA-sequencing combined with gene ontology analysis identified 11 osteogenesis-relevant genes. Five genes that shared microRNAs between circ_0003072 and two candidate genes (chordin-like 1 [CHRDL1] and XIAP) were screened, and miR-488-3p showed the highest increase after silencing circ_0003072. Knockdown of circ_0003072 inhibited the pro-osteogenic differentiation effect of silencing miR-488-3p. miR-488-3p bound to circ_0003072 and CHRDL1. Furthermore, overexpression of CHRDL1 rescued the miR-488-3p-induced inhibition of osteogenic differentiation. CONCLUSION: BetA promoted the osteogenic differentiation of hPDLSCs by regulating the circ_0003072/miR-488-3p/CHRDL1 pathway, and circ_0003072 acted as a sponge for miR-488-3p, thus upregulating the level of CHRDL1.