Department of Spinal Surgery, Ningbo No.6 Hospital, 1059 East Zhongshan Road, Yinzhou District, Ningbo, 315040, Zhejiang, China.
Department of Medical Image, Ningbo No.6 Hospital, 1059 East Zhongshan Road, Yinzhou District, Ningbo, 315040, Zhejiang, China.
J Orthop Surg Res. 2023 Jun 2;18(1):402. doi: 10.1186/s13018-023-03850-1.
Ankylosing spondylitis (AS) is a chronic inflammatory arthritis. Upregulation of microRNA (miR)-92b-3p is associated with enhanced osteoblastic differentiation. The current study sought to investigate the functional mechanism of miR-92b-3p in osteogenic differentiation of AS fibroblasts.
First, fibroblasts were isolated from AS and non-AS patients and cultured. Next, cell morphology was observed, cell proliferation was assessed and the vimentin expression pattern was determined. Alkaline phosphatase (ALP) activity and levels of osteogenic markers RUNX2, OPN, OSX, and COL I were additionally measured, followed by determination of miR-92b-3p and TOB1 levels. The binding site of miR-92b-3p and TOB1 was predicted, and their target relationship was validated. Lastly, miR-92b-3p inhibitor, si-TOB1, and the BMP/Smad signaling pathway inhibitor LDN193189 were delivered into AS fibroblasts to evaluate the osteogenic differentiation of AS fibroblasts and the activation of the BMP/Smad pathway.
miR-92b-3p was highly expressed in AS fibroblasts. AS fibroblasts showed enhanced osteogenic differentiation and proliferation, while inhibition of miR-92b-3p suppressed osteogenic differentiation and proliferation of AS fibroblasts. miR-92b-3p targeted TOB1, and TOB1 was poorly expressed in AS fibroblasts. The concurrent downregulation of TOB1 and inhibition of miR-92b-3p elevated the levels of RUNX2, OPN, OSX, and COL I and ALP activity and further enhanced the proliferation of AS fibroblasts. The BMP/Smad pathway was activated in AS fibroblasts. Silencing miR-92b-3p could inhibit the activation of the BMP/Smad pathway by upregulating TOB1. Inhibition of the BMP/Smad pathway reduced the number of calcified nodules and hindered the osteogenic differentiation and proliferation of AS fibroblasts.
Our findings highlighted that silencing miR-92b-3p inhibited the osteogenic differentiation and proliferation of AS fibroblasts by upregulation of TOB1 and inhibition of the BMP/Smad pathway.
强直性脊柱炎（AS）是一种慢性炎症性关节炎。微小 RNA（miR）-92b-3p 的上调与成骨细胞分化增强有关。本研究旨在探讨 miR-92b-3p 在 AS 成纤维细胞成骨分化中的功能机制。
首先从 AS 和非 AS 患者中分离出成纤维细胞并进行培养。然后观察细胞形态，评估细胞增殖，并确定波形蛋白表达模式。此外，还测量碱性磷酸酶（ALP）活性和骨形成标志物 RUNX2、OPN、OSX 和 COL I 的水平，然后测定 miR-92b-3p 和 TOB1 的水平。预测 miR-92b-3p 和 TOB1 的结合位点，并验证其靶关系。最后，将 miR-92b-3p 抑制剂、si-TOB1 和 BMP/Smad 信号通路抑制剂 LDN193189 导入 AS 成纤维细胞，评估 AS 成纤维细胞的成骨分化和 BMP/Smad 通路的激活。
miR-92b-3p 在 AS 成纤维细胞中高表达。AS 成纤维细胞表现出增强的成骨分化和增殖，而抑制 miR-92b-3p 则抑制 AS 成纤维细胞的成骨分化和增殖。miR-92b-3p 靶向 TOB1，而 TOB1 在 AS 成纤维细胞中表达水平较低。同时下调 TOB1 和抑制 miR-92b-3p 可提高 RUNX2、OPN、OSX 和 COL I 的水平和 ALP 活性，并进一步增强 AS 成纤维细胞的增殖。AS 成纤维细胞中激活了 BMP/Smad 通路。沉默 miR-92b-3p 可通过上调 TOB1 抑制 BMP/Smad 通路的激活。抑制 BMP/Smad 通路减少了钙化结节的数量，并阻碍了 AS 成纤维细胞的成骨分化和增殖。
本研究结果表明，通过上调 TOB1 和抑制 BMP/Smad 通路，沉默 miR-92b-3p 抑制了 AS 成纤维细胞的成骨分化和增殖。
