Circular RNA (CircRNA) plays a potential role in bone formation. We aimed to study the circRNAs expression profiles and their functions in osteogenic differentiation of human bone marrow stromal cells (BMSCs). Firstly, we established osteogenic differentiation of BMSCs displaying increased mRNA expression of osteogenic differentiation marker (RUNX2, OPN, and OCN), increased ALP activity and protein expression, and increased mineralized nodules formation, as well as morphological alteration. Then, we employed high-throughput sequencing to analyze circRNA expression and found that 3440 and 3893 circRNAs in non-induced and induced groups, respectively. We further validated the 10 differentially expressed circRNAs with the most significant difference between induced and non-induced groups. Among these ten circRNAs, five of them with more than one miRNA binding site were used to construct a ceRNA network exhibiting 81 miRNAs and 182 target mRNAs. Furthermore, among these five circRNAs, we found only circ_0005564 significantly reduced the mRNA expression of RUNX2, OPN, and OCN. The circularity of circ_0005564 was verified. Our results showed that knockdown of circ_0005564 inhibited osteoblast differentiation in BMSCs. Taken together, our study demonstrates that circ_0005564 is a potential positive regulator of osteogenic differentiation of BMSCs.

Background Circular RNAs (circRNAs) are a new type of stable noncoding RNA and have been proven to play a crucial role in osteoporosis. This study explored the role and mechanism of hsa_circ_0001485 in osteogenic differentiation. Methods Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis and Gene Ontology (GO) enrichment analysis were performed according to the previous sequencing data in human bone marrow mesenchymal stem cells (BMSC) before and after the induction of osteogenic differentiation on the differentially expressed circRNAs, to screen out signaling pathways associated with osteogenic differentiation. The hFOB 1.19 cells were used to verify the function and mechanism of specific circRNAs in osteogenic differentiation. Additionally, small interfering fragments and overexpression plasmids were used to determine the role of specific circRNAs during osteogenic differentiation. Furthermore, pull-down experiments and mass spectrometry were performed to determine the proteins that bind to specific circRNAs. Results The KEGG and GO enrichment analyses showed that the TGFβ-BMP signaling pathway was related to the osteogenic differentiation process, and four circRNAs were associated with the pathway. The quantitative polymerase chain reaction analysis revealed that hsa_circ_0001485 expression was increased during the osteogenic differentiation process of BMSCs. Knockdown of hsa_circ_0001485 suppressed the activity of the alkaline phosphatase enzyme and the expression of RUNX2, osteopontin, and osteocalcin in the osteogenic hFOB 1.19 cells, whereas overexpression of hsa_circ_0001485 promoted their expression. Additionally, we found that hsa_circ_0001485 and BMPR2 targeted binding to activate the TGFβ-BMP signaling pathway and promoted osteogenic differentiation through mass spectrometry analysis. Conclusion This study demonstrates that hsa_circ_0001485 is highly expressed in the osteogenic hFOB 1.19 cells, which activate the TGFβ-BMP pathway through targeted binding of BMPR2, and plays a positive role in regulating osteogenic differentiation.