Disorders of bone homeostasis are the key factors leading to metabolic bone disease, such as senile osteoporosis, which is characterized by age-related bone loss. Bone marrow stromal cells (BMSCs) possess high osteogenic capacity which has been regarded as a practical approach to preventing bone loss. Previous studies have shown that the osteogenic differentiation ability of BMSCs is significantly decreased in senile osteoporosis. Recently, circular RNAs (circRNAs) have been regarded as critical regulators in controlling the osteogenic differentiation of BMSCs by sponging microRNAs (miRNAs). Our study aimed to discover new and critical osteogenesis-related circRNAs that can promote bone formation in senile osteoporosis.

We detected the dysregulated circRNAs of BMSCs upon osteogenic differentiation induction and identified the critical osteogenic circRNA (circ-3626). The relationship between circ-3626 and osteoporosis was further verified in clinical bone samples and aged mice by qPCR. Moreover, circ-3626 AAV was constructed to examine the osteogenic effect of circ-3626 on bone formation via using Micro-CT, double calcein labeling, and the three-point bending tests. Bioinformatics analysis, Luciferase report gene assays, FISH, RNA pull-down, qPCR, Western Blots, and alizarin red staining assay explore the effects and mechanisms of circ-3626 on osteogenic differentiation of BMSCs.

Circ-3626 was identified as a pivotal osteogenesis-related circRNA via RNA sequencing. The results of alizarin red staining, Western blots, and qPCR assays suggest that overexpressing circ-3626 dramatically accelerates the osteogenic capability of BMSCs. Furthermore, the bone repair capability of aging mice could be significantly improved by circ-3626 AAV treatment. Micro RNA miR-338-3p was identified as the downstream target of circ-3626. Overexpression of circ-3626 increases the expression of Runx2 by sponging miR-338-3p, thereby promoting the osteogenic differentiation of BMSCs by upregulating the expression of osteogenic genes. In addition, Western blots, and qPCR assays suggest circ-3626 AAV treatment promote the expression of Runx2 and osteogenic marker genes.

Thus, we demonstrate that circ-3626 plays a pivotal role in promoting bone formation through the miR-338-3p/Runx2 axis and may provide new strategies for preventing and treating the bone loss of senile osteoporosis.