The Circular RNA Landscape of Periodontal Ligament Stem Cells During Osteogenesis

Background: The present study aims to investigate the distinct expression pattern of circular RNAs (circRNAs) in periodontal ligament stem cells (PDLSCs) during osteogenesis.

Methods: PDLSCs were isolated and cultured in osteogenic medium. Total RNA was extracted from cells at day 0 (D0), day 3 (D3), day 7 (D7), and day 14 (D14) and submitted to RNA-sequencing to detect expression profiles of circRNAs, messenger RNAs (mRNAs), and microRNAs (miRNAs). Real-time quantitative reverse-transcription polymerase chain reaction (qRT-PCR) was performed to validate expression of circRNAs and miRNAs. Differential expression analysis and gene ontology analysis were performed. A circRNA-miRNA-mRNA network was constructed to reveal the potential regulatory role of circRNAs.

Results: A total of 12,693 circRNA transcripts were detected, and circRNAs displayed stage-specific expression. Expression of four well-known circRNAs was validated by qRT-PCR. In total, 118 circRNAs were differentially expressed at D3, 128 circRNAs were differentially expressed at D7, and 139 circRNAs were differentially expressed at D14 compared with D0. Host genes of differentially expressed circRNAs were enriched in cytoplasmic or membrane-bound vesicles and extracellular matrix, indicating their potential roles in modulating biogenesis of extracellular vesicles. Moreover, mRNAs that were potentially regulated by circRNAs were enriched in bone-formation-associated processes, including extracellular matrix organization, cell differentiation, and bone morphogenetic protein signaling pathway.

Conclusion: Expression profiles of circRNAs were significantly altered during osteogenic differentiation of PDLSCs, providing a clue for future studies on the role of circRNAs in osteoblast differentiation.