MiR-124 Regulates Osteoblast Differentiation Through GSK-3β in Ankylosing Spondylitis

Objective: Ankylosing spondylitis (AS) is a spastic and spinal joint disease with the characteristic of pathological ossification. Bioinformatics analysis demonstrated that there is a complementary binding site between microRNA-124 (miR-124) and the 3'-UTR of glycogen synthase kinase-3β (GSK-3β) mRNA. We aimed to investigate the role of miR-124 in regulating GSK-3β expression, Wnt/β-catenin pathway activity, and osteoblast differentiation of spinal ligament fibroblasts.

Patients And Methods: The ligament tissues of AS and the femoral neck fracture patients were collected. MiR-124 and GSK-3β mRNA expressions were detected by using quantitative Real-time PCR (qRT-PCR). GSK-3β and β-catenin protein expressions were detected by using Western blot. Ligament fibroblasts were isolated and induced to differentiate into osteoblasts. Alizarin red S staining (ARS) was used to identify osteoblast differentiation. Expressions of miR-124, GSK-3β, β-catenin, Osterix, and runt-related transcription factor 2 (RUNX2) were detected during differentiation. The cells were divided into two groups as agomiR-normal control (NC) transfection group and agomir miR-124 transfection group. Alkaline phosphatase (ALP) activity and Alizarin Red S staining were detected.

Results: MiR-124 and β-catenin expressions in the ligament of AS patients increased, while GSK-3β level reduced compared with control. MiR-124, β-catenin, Osterix, and RUNX2 expressions gradually elevated, whereas GSK-3β level gradually declined following increased osteoblasts differentiation. Antagomir miR-124 transfection significantly up-regulated the expression of GSK-3β in osteoblast differentiation, significantly decreased the expression of β-catenin, Osterix, and RUNX2, and significantly inhibited osteoblast differentiation.

Conclusions: MiR-124 decreased and GSK-3β elevated in AS ligament tissue. Down-regulation of miR-124 expression enhanced GSK-3β expression, weakened Wnt/β-catenin pathway activity, and inhibited the differentiation of ligament fibroblasts into osteoblasts.