Department of Spinal Surgery, Zhuzhou Central Hospital, Zhuzhou, Hunan, China.
Eur Rev Med Pharmacol Sci. 2018 Oct;22(20):6616-6624. doi: 10.26355/eurrev_201810_16136.
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.
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.
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.
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.
强直性脊柱炎（AS）是一种痉挛性和脊柱关节疾病，其特征为病理性骨化。生物信息学分析表明，微小 RNA-124（miR-124）与糖原合酶激酶-3β（GSK-3β）mRNA 的 3'-UTR 之间存在互补结合位点。我们旨在研究 miR-124 调节 GSK-3β 表达、Wnt/β-连环蛋白通路活性和脊柱韧带成纤维细胞成骨分化的作用。
收集 AS 患者和股骨颈骨折患者的韧带组织。采用实时定量 PCR（qRT-PCR）检测 miR-124 和 GSK-3β mRNA 的表达。采用 Western blot 检测 GSK-3β 和 β-连环蛋白的表达。分离韧带成纤维细胞并诱导分化为成骨细胞。茜素红 S 染色（ARS）鉴定成骨细胞分化。分化过程中检测 miR-124、GSK-3β、β-连环蛋白、成骨转录因子 2（Osterix）和 runt 相关转录因子 2（RUNX2）的表达。将细胞分为 agomiR-正常对照（NC）转染组和 agomir miR-124 转染组。检测碱性磷酸酶（ALP）活性和茜素红 S 染色。
与对照组相比，AS 患者韧带组织中 miR-124 和 β-连环蛋白表达增加，而 GSK-3β 水平降低。随着成骨细胞分化的增加，miR-124、β-连环蛋白、Osterix 和 RUNX2 的表达逐渐升高，而 GSK-3β 的表达逐渐降低。抗 miR-124 转染明显上调成骨细胞分化中 GSK-3β 的表达，明显降低 β-连环蛋白、Osterix 和 RUNX2 的表达，明显抑制成骨细胞分化。
AS 韧带组织中 miR-124 降低，GSK-3β 升高。下调 miR-124 表达增强 GSK-3β 表达，减弱 Wnt/β-连环蛋白通路活性，抑制韧带成纤维细胞向成骨细胞分化。
