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 were measured via qRT-PCR. Alkaline phosphatase (ALP) 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.