MicroRNA-221 Regulates Hypertrophy of Ligamentum Flavum in Lumbar Spinal Stenosis by Targeting TIMP-2.

STUDY DESIGN: A study of lumbar ligamentum flavum (LF).

OBJECTIVE: The aim of this study was to identify LF hypertrophy related microRNAs (miRNAs) expression profile and to investigate the role of miRNAs in the development of LF hypertrophy in lumbar spinal stenosis (LSS).

SUMMARY OF BACKGROUND DATA: Although histologic and biologic literature on LF hypertrophy is available, the pathomechanism is still unknown. Accumulating evidence suggests that microRNAs (miRNAs) participate in many physiologic processes, including cell proliferation, differentiation, and fibrosis, but the role of specific miRNAs involved in LF hypertrophy remains elusive.

METHODS: An initial screening of LF tissues miRNA expression by miRNA microarray was performed using samples from 10 patients and 10 controls, respectively. Subsequently, differential expression was validated using qRT-PCR. Then, functional analysis of the miRNAs in regulating collagens I and III expression was carried out. Western blotting and luciferase reporter assay were also used to detect the target gene. In addition, the thickness of the LF at the level of the facet joint was measured on axial T1-weighted magnetic resonance images.

RESULTS: We identified 18 miRNAs that were differentially expressed in patients compared with controls. Following qRT-PCR confirmation, miR-221 was significantly lower in LF tissues of patients than controls. The LF was significantly thicker in patients than that in controls. Bioinformatics target prediction identified tissue inhibitors of matrix metalloproteinase (TIMP)-2 as a putative target of miR-221. Furthermore, luciferase reporter assays demonstrated that miR-221 directly targets TIMP-2 and affects the protein expression of TIMP-2 in fibroblasts isolated from LF. Of note, miR-221 mimic reduced mRNA and protein expression of collagens I and collagen III in fibroblasts isolated from LF.

CONCLUSION: The downregulation of miR-221 might contribute to LF hypertrophy by promoting collagens I and III expression via the induction of TIMP-2. Our study also underscores the potential of miR-221 as a novel therapeutic target in LSS.

LEVEL OF EVIDENCE: 3.