Qi Yuesun, Lin Chunnan, Zhao Chengwei, Wu Ye
Department of Stomatology, Jinshan Hospital, Fudan University, Shanghai, People's Republic of China.
Department of Stomatology, Fengxian District Institute of Dental Diseases, Shanghai, People's Republic of China.
Int Dent J. 2025 Apr;75(2):1390-1399. doi: 10.1016/j.identj.2024.10.017. Epub 2024 Nov 20.
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.
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.
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.
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.
桦木酸（BetA）对人牙周膜干细胞（hPDLSCs）具有良好的促成骨分化作用，使其成为牙周组织再生的一种有前景的补充剂。环状RNA（circRNAs）已成为细胞行为的重要调节因子，而circRNAs是否参与BetA的作用尚不清楚。
基于公共数据集，利用生物信息学分析筛选参与成骨分化的差异表达circRNAs。通过定量PCR检测RUNX2、ALPL、COL1A1和BGLAP水平、碱性磷酸酶染色以及钙化结节的茜素红S染色来评估成骨分化。研究了circ_0003072在BetA调节hPDLSCs成骨分化中的作用，并通过RNA测序、竞争性内源RNA网络构建、基因本体分析、双荧光素酶报告基因检测和功能检测来确认其下游的微小RNA和信使核糖核酸。
在18个候选circRNAs中，circ_0003072的倍数变化最高，敲低circ_0003072可抑制BetA对hPDLSCs的促成骨分化作用。RNA测序结合基因本体分析鉴定出11个与成骨相关的基因。筛选出circ_0003072与两个候选基因（腱蛋白样1[CHRDL1]和X连锁凋亡抑制蛋白[XIAP]）之间共享微小RNA的5个基因，沉默circ_0003072后，miR-488-3p的增加最为显著。敲低circ_0003072可抑制沉默miR-488-3p后的促成骨分化作用。miR-488-3p与circ_0003072和CHRDL1结合。此外，CHRDL1的过表达挽救了miR-488-3p诱导的成骨分化抑制。
BetA通过调节circ_0003072/miR-488-3p/CHRDL1通路促进hPDLSCs的成骨分化，circ_0003072作为miR-488-3p的海绵，从而上调CHRDL1的水平。
