LAUSR

Ossification of the ligamentum flavum (OLF) is a disease of heterotopic ossification in spinal ligaments. The key of the OLF pathogenesis is the differentiation of fibroblasts into osteoblasts. In this study, we explored the role of miR‐615‐3p in the osteogenic differentiation of human LF cells. The expression of miR‐615‐3p was detected during the osteogenic differentiation of hFOB1.19 human osteoblasts, human BMSCs, and human LF cells. The qPCR results showed that miR‐615‐3p was being decreased during the osteogenic differentiation of these cell lineages. Then, both gain‐ and loss‐function experiments, respectively performed by single‐strand miR‐615‐3p mimic and antagomir, revealed that miR‐615‐3p negatively regulated the osteogenesis of hLF cells, manifested by a lighter staining degree with Alizarin Red and a decreased level of osteogenic marker genes, including alkaline phosphatase (ALP), RUNX2, osterix (ostx), osteocalcin (OCN), and osteopontin (OPN). Subsequently, our data on bioinformatic analysis, 3′‐UTR luciferase activity assay, and protein level detection indicated that miR‐615‐3p directly targeted and suppressed the expression of FOXO1 and GDF5. Furthermore, knockdown of either FOXO1 or GDF5 could inhibit the osteogenic differentiation of hLF cells, which displayed a similar effect with the miR‐615‐3p mimic. In conclusion, miR‐615‐3p negatively regulates the osteogenic differentiation of hLF cells through post‐transcriptionally suppressing osteogenic regulators GDF5 and FOXO1. It can be regarded as a potential target for human OLF therapy.