miR-29a-5p Targets SATB2 and Regulates the SIRT1/Smad3 Deacetylation Pathway to Inhibit Thoracic Ligamentum Flavum Cell Osteogenesis

Study Design. Experimental analysis of the thoracic ligamentum flavum cell osteogenic differentiation process. Objective. This study aimed to explore the role of miR-29a-5p and special AT-rich sequence-binding protein 2 (SATB2) in a pathological osteogenic process. Summary of Background Data. Thoracic ossification of the ligamentum flavum (TOLF) is an uncommon disease wherein ligaments within the spine undergo progressive ossification, resulting in stenosis of the spinal canal and myelopathy. MiR-29a-5p was found to be downregulated in ligament cells from ossified ligament tissue in a previous study. However, whether miR-29a-5p is involved in the process of TOLF has not been investigated. Methods. The expression of miR-29a-5p in ligament tissues or in the context of TOLF osteogenic cell differentiation was measured via qRT-PCR. Alkaline phosphatase activity assay and Alizarin red staining were used to analyze cellular osteogenesis. The protein-level expression of SATB2, SIRT1, and Smad3 were measured via immunohistochemistry or western blotting. Dual luciferase reporter assays and western blotting were used to confirm that miR-29a targets SATB2. Results. SATB2 was found to be upregulated and miR-29a-5p was downregulated in TOLF tissue. We additionally observed decreased miR-29a-5p expression during the process of TOLF osteogenic cell differentiation, and there was a marked reduction in the expression of key mediators of osteogenesis when miR-29a-5p was overexpressed. Consistent with this, when miR-29a-5p was inhibited this led to enhanced osteogenic cell differentiation of these cells. We further found miR-29a-5p to directly target and suppress the expression of SATB2. Knock-down of SATB2 was sufficient to reduce the ability of miR-29a-5p to inhibit osteogenesis, and this also led to decreased SIRT1 expression and Smad3 acetylation. Conclusion. Together our findings indicate that miR-29a-5p is able to prevent thoracic ligamentum flavum cell osteogenesis at least in part via targeting SATB2 and thereby suppressing the SIRT1/Smad3 deacetylation pathway. Level of Evidence: N/A