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Published on: July 3, 2020
miR-29a-5p Targets SATB2 and Regulates the SIRT1/Smad3 Deacetylation Pathway to Inhibit Thoracic Ligamentum Flavum
Fabo Feng1, Haiyan Qiu2, Danjie Zhu1
1Department of Orthopedics, Zhejiang Provincial People's Hospital, People's Hospital of Hangzhou Medical College, Hangzhou, PR China.
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.
Insights
MicroRNA-29a-5p prevents thoracic ligamentum flavum ossification by targeting SATB2, inhibiting osteogenesis. This finding offers insights into TOLF pathogenesis and potential therapeutic targets.
Area of Science:
- Molecular biology
- Biochemistry
- Pathology
Background:
- Thoracic ossification of the ligamentum flavum (TOLF) causes spinal stenosis and myelopathy.
- MiR-29a-5p is downregulated in TOLF ligament cells, suggesting a potential role in disease pathogenesis.
Purpose of the Study:
- To investigate the role of miR-29a-5p and SATB2 in the pathological osteogenic process of TOLF.
- To elucidate the molecular mechanisms underlying TOLF.
Main Methods:
- Quantitative real-time PCR (qRT-PCR) to measure miR-29a-5p expression.
- Alkaline phosphatase activity assay and Alizarin red staining to assess osteogenesis.
- Western blotting and immunohistochemistry to evaluate protein expression (SATB2, SIRT1, Smad3).
- Dual luciferase reporter assays to confirm direct targeting of SATB2 by miR-29a-5p.
Main Results:
- MiR-29a-5p was downregulated, while SATB2 was upregulated in TOLF tissues.
- Overexpression of miR-29a-5p inhibited TOLF cell osteogenic differentiation and reduced SATB2, SIRT1, and Smad3 acetylation.
- Inhibition of miR-29a-5p enhanced osteogenic differentiation.
- miR-29a-5p directly targets and suppresses SATB2 expression.
- SATB2 knockdown partially reversed the inhibitory effect of miR-29a-5p on osteogenesis.
Conclusions:
- MiR-29a-5p plays a protective role against TOLF osteogenesis.
- The mechanism involves targeting SATB2, leading to suppression of the SIRT1/Smad3 deacetylation pathway.
- These findings highlight miR-29a-5p as a potential therapeutic target for TOLF.
