Li Jun, Sun Farui, Zhang Yuanjin, Pan Xian, Li Bo, Zhang Guofu, Zhou Qian
Department of Orthopedics, Huangshi Central Hospital, Affiliated Hospital of Hubei Polytechnic University, Hungshi, 435000, China.
Department of Geriatrics, Huangshi Central Hospital, Affiliated Hospital of Hubei Polytechnic University, Tianjin Avenue No. 141, Huangshigang District, 435000, Hungshi, Hubei Province, China.
J Orthop Surg Res. 2025 Mar 29;20(1):324. doi: 10.1186/s13018-025-05719-x.
Chondrocyte apoptosis is associated with the severity of cartilage destruction and matrix degeneration in the progression of osteoarthritis. Increasing evidence indicates that autophagy has a significant cytoprotective effect against chondrocyte apoptosis. Here, we investigated the role of microRNA-103-3p (miR-103-3p) in regulating chondrocyte function and elucidated the underlying mechanism.
MiR-103-3p expression in interleukin-1β (IL-1β)-stimulated chondrocytes was evaluated using RT-qPCR. The targets of miR-103-3p predicted by online databases were verified using biotin-based pulldown assay and luciferase reporter assay. IL-1β stimulated-chondrocytes were transfected with miR-103-3p inhibitor along with siRNA targeting cytoplasmic polyadenylation element-binding protein3 (siCPEB3), the autophagy inhibitor 3-MA, or the PI3K agonist 740 Y-P. Chondrocyte proliferation was evaluated using cell counting kit-8. Apoptosis was detected by flow cytometry. The levels of apoptosis-, extracellular matrix (ECM)-, autophagy-, and the PI3K/Akt/mTOR pathway-related proteins in chondrocytes were detected using immunoblotting or immunofluorescence.
We found that IL-1β stimulation upregulated miR-103-3p and downregulated CPEB3 in mouse chondrocytes. Inhibiting miR-103-3p reduced IL-1β-induced apoptosis and ECM macromolecule degradation while enhancing autophagy in chondrocytes. MiR-103-3p targeted CPEB3, and its downregulation rescued the expression of level in IL-1β stimulated-chondrocytes. MiR-103-3p downregulation inhibited the PI3K/Akt/mTOR pathway in IL-1β stimulated-chondrocytes by upregulating CPEB3. 3-MA, 740 Y-P, or CPEB3 knockdown counteracted the effect of miR-103-3p downregulation on chondrocyte apoptosis, ECM macromolecule degradation, and autophagy.
Overall, inhibition of miR-103-3p reduces IL-1β-induced apoptosis and ECM macromolecule degradation in chondrocytes by enhancing autophagy through the CPEB3/PI3K/Akt/mTOR pathway.
在骨关节炎进展过程中，软骨细胞凋亡与软骨破坏及基质退变的严重程度相关。越来越多的证据表明，自噬对软骨细胞凋亡具有显著的细胞保护作用。在此，我们研究了微小RNA - 103 - 3p（miR - 103 - 3p）在调节软骨细胞功能中的作用，并阐明其潜在机制。
使用逆转录定量聚合酶链反应（RT - qPCR）评估白细胞介素 - 1β（IL - 1β）刺激的软骨细胞中miR - 103 - 3p的表达。通过基于生物素的下拉试验和荧光素酶报告基因试验验证在线数据库预测的miR - 103 - 3p的靶标。用miR - 103 - 3p抑制剂与靶向细胞质聚腺苷酸化元件结合蛋白3（siCPEB3）的小干扰RNA（siRNA）、自噬抑制剂3 - 甲基腺嘌呤（3 - MA）或磷脂酰肌醇 - 3 - 激酶（PI3K）激动剂740 Y - P转染IL - 1β刺激的软骨细胞。使用细胞计数试剂盒 - 8评估软骨细胞增殖。通过流式细胞术检测细胞凋亡。使用免疫印迹或免疫荧光检测软骨细胞中凋亡、细胞外基质（ECM）、自噬以及PI3K/Akt/mTOR信号通路相关蛋白的水平。
我们发现IL - 1β刺激上调小鼠软骨细胞中miR - 103 - 3p的表达并下调CPEB3的表达。抑制miR - 103 - 3p可减少IL - 1β诱导的软骨细胞凋亡和ECM大分子降解，同时增强软骨细胞的自噬。miR - 103 - 3p靶向CPEB3，其下调可挽救IL - 1β刺激的软骨细胞中CPEB3的表达水平。miR - 103 - 3p下调通过上调CPEB3抑制IL - 1β刺激的软骨细胞中的PI3K/Akt/mTOR信号通路。3 - MA、740 Y - P或CPEB3基因敲低可抵消miR - 103 - 3p下调对软骨细胞凋亡、ECM大分子降解和自噬的影响。
总体而言，抑制miR - 103 - 3p可通过CPEB3/PI3K/Akt/mTOR信号通路增强自噬，从而减少IL - 1β诱导的软骨细胞凋亡和ECM大分子降解。
