Ossification of the ligamentum flavum (OLF) is a disorder of heterotopic ossification of spinal ligaments and is the main cause of thoracic spinal canal stenosis. Previous studies suggested that miR-132-3p negatively regulates osteoblast differentiation. However, whether miR-132-3p is involved in the process of OLF has not been investigated. In this study, we investigated the effect of miR-132-3p and its target genes forkhead box O1 (FOXO1), growth differentiation factor 5 (GDF5) and SRY-box 6 (SOX6) on the osteogenic differentiation of ligamentum flavum (LF) cells. We demonstrated that miR-132-3p was down-regulated during the osteogenic differentiation of LF cells and negatively regulated the osteoblast differentiation. Further, miR-132-3p targeted FOXO1, GDF5 and SOX6 and down-regulated the protein expression of these genes. Meanwhile, FOXO1, GDF5 and SOX6 were up-regulated after osteogenic differentiation and the down-regulation of endogenous FOXO1, GDF5 or SOX6 suppressed the osteogenic differentiation of LF cells. In addition, we also found FOXO1, GDF5 and SOX6 expression in the ossification front of OLF samples. Overall, these results suggest that miR-132-3p inhibits the osteogenic differentiation of LF cells by targeting FOXO1, GDF5 and SOX6.

Ossification of the ligamentum flavum (OLF) is a pathology almost only reported in East Asian countries. The leading cause of OLF is thoracic spinal canal stenosis and myelopathy. In this study, the role of miR‐199b‐5p and jagged 1 (JAG1) in primary ligamentum flavum cell osteogenesis was examined. MiR‐199b‐5p was found to be down‐regulated during osteogenic differentiation in ligamentum flavum cells, while miR‐199b‐5p overexpression inhibited osteogenic differentiation. In addition, JAG1 was found to be up‐regulated during osteogenic differentiation in ligamentum flavum cells, while JAG1 knockdown via RNA interference caused an inhibition of Notch signalling and osteogenic differentiation. Moreover, target prediction analysis and dual luciferase reporter assays supported the notion that JAG1 was a direct target of miR‐199b‐5p, with miR‐199b‐5p found to down‐regulate both JAG1 and Notch. Further, JAG1 knockdown was demonstrated to block the effect of miR‐199b‐5p inhibition. These findings imply that miR‐199b‐5p performs an inhibitory role in osteogenic differentiation in ligamentum flavum cells by potentially targeting JAG1 and influencing the Notch signalling pathway.

Thoracic ossification of the ligamentum flavum (TOLF)is a common cause of thoracic spinal canal stenosis and has been reported almost exclusively in East Asian countries. In this study, we established a relationship between bone morphogenic protein 2 (BMP-2) and TOLF. We divided patients into two groups according to severity of ossification and identified susceptible loci through exome sequencing. We identified 39 novel likely pathogenic variants in 29 genes in the transforming growth factor-beta (TGF-β) superfamily or TGF-β/BMPs signaling pathway, including two missense variants in BMP-2 (NM_001200.3) exon region, c.460C>G:p.(R154G) and c.584G>T:p.(R195M). Further Sanger sequencing and genotyping suggested the variants were only found in patients with long regional OLF. Bioinformatic assays predicted the two BMP-2 variants to cause significant alterations to gene and protein expression. Functional assays showed upregulation of BMP-2 expression, increased osteogenic marker expression, and enhanced osteogenic differentiation. Collectively, these results suggest a genetic contribution to the pathogenesis of TOLF, particularly in patients with long segment disease, and that nucleotide substitutions associated with increased BMP-2 expression may be involved in TOLF pathogenesis.

Thoracic ossification of the ligamentum flavum (TOLF) is characterized by ectopic bone formation in the ligamentum flavum and is considered to be a leading cause of thoracic spinal canal stenosis and myelopathy. However, the underlying etiology is not well understood. An iTRAQ proteomics was used to reveal the involvement of inflammation factors in TOLF. TNF-α is a pro-inflammatory cytokine implicated in the pathogenesis of many human diseases. Protein profiling analysis showed that the protein level of TNF-α increased in the ossified ligamentum flavum of TOLF, which was confirmed by western blot. The effects of TNF-α on primary ligamentum flavum cells was examined. Cell proliferation assay demonstrated that primary cells from the ossified ligamentum flavum of TOLF grew faster than the control. Flow cytometry assay indicated that the proportions of cells in S phase of cell cycle of primary cells increased after TNF-α stimulation. To address the effect of TNF-α on gene expression, primary cells were derived from ligamentum flavum of TOLF patients. Culture cells were stimulated by TNF-α. RNA was isolated and analyzed by quantitative RT-PCR. G1/S-specific proteins cyclin D1 and c-Myc were upregulated after TNF-α stimulation. On the other hand, osteoblast differentiation related genes such as Bmp2 and Osterix (Osx) were upregulated in the presence of TNF-α. TNF-α activated Osx expression in a dose-dependent manner. Interestingly, a specific mitogen-activated protein kinase ERK inhibitor U0126, but not JNK kinase inhibitor SP600125, abrogated TNF-α activation of Osx expression. This suggests that TNF-α activates Osx expression through the mitogen-activated protein kinase ERK pathway. Taken together, we provide the evidence to support that TNF-α involves in TOLF probably through regulating cell proliferation via cyclin D1 and c-Myc, and promoting osteoblast differentiation via Osx.