Institute of Sports Medicine, Shandong First Medical University and Shandong Academy of Medical Sciences, Taian, Shandong 271016, P.R. China.
School of Nursing, Shandong First Medical University and Shandong Academy of Medical Sciences, Taian, Shandong 271016, P.R. China.
Mol Med Rep. 2023 Nov;28(5). doi: 10.3892/mmr.2023.13086. Epub 2023 Sep 15.
Hypoxia can lead to programmed osteoblast death. Prevention of osteoblast apoptosis caused by hypoxia is of great significance in the study of the occurrence and development of bone necrosis. The present study aimed to investigate the effects and mechanism of fibroblast growth factor 23 (FGF23) on hypoxia‑induced apoptosis in primary osteoblasts and MC3T3‑E1 cells osteoblasts. Cells were transfected with a plasmid carrying the FGF23 gene and a cell model of hypoxia‑induced apoptosis was established. FGF23 mRNA levels were measured using reverse transcription‑quantitative (RT‑q) PCR and western blotting was used to assess protein levels. Apoptosis was analyzed by MTT assay, fluorescein diacetate and ethidium bromide staining, flow cytometry and RT‑qPCR and western blotting were used to verify the mRNA and protein levels of apoptosis‑ and autophagy‑related gene mRNA. The targeted relationship between miR‑17‑5p and FGF23 was confirmed using the StarBase database, TargetScan database and a luciferase reporter assay. FGF23 decreased cell survival and increased the rate of apoptosis. The mRNA and protein expression of the pro‑apoptotic genes Bax and caspases 3 and 9 increased, whereas that of the anti‑apoptotic Bcl‑2 decreased. The expressions of the autophagy‑associated proteins beclin‑1, light chain 3‑II (LC3‑II) and the LC3‑II/LC3‑I ratio were significantly increased. In addition, a luciferase reporter assay confirmed that FGF23 directly regulated micro RNA (miR)‑17‑5p. The effects of FGF23 silencing were reversed by miR‑17‑5p inhibition. FGF23 may regulate hypoxia‑induced osteoblast apoptosis by targeting miR‑17‑5p through the autophagy‑signaling pathway. This provides a rationale for FGF23 as a potential therapeutic target for osteonecrosis of the femoral head.
缺氧可导致成骨细胞程序性死亡。预防缺氧引起的成骨细胞凋亡在研究骨坏死的发生和发展中具有重要意义。本研究旨在探讨成纤维细胞生长因子 23(FGF23)对原代成骨细胞和 MC3T3-E1 细胞成骨细胞缺氧诱导凋亡的影响及其机制。转染携带 FGF23 基因的质粒,建立缺氧诱导细胞凋亡模型。采用逆转录-定量(RT-q)PCR 检测 FGF23mRNA 水平,Western blot 检测蛋白水平。MTT 法检测细胞存活率,荧光二乙酸酯和溴化乙锭染色、流式细胞术检测细胞凋亡,RT-qPCR 和 Western blot 检测凋亡和自噬相关基因 mRNA 的表达。利用 StarBase 数据库、TargetScan 数据库和荧光素酶报告基因检测法证实 miR-17-5p 与 FGF23 的靶向关系。FGF23 降低细胞存活率,增加细胞凋亡率。促凋亡基因 Bax 和 caspase-3、caspase-9 的 mRNA 和蛋白表达上调,而抗凋亡基因 Bcl-2 的表达下调。自噬相关蛋白 beclin-1、微管相关蛋白轻链 3-II(LC3-II)和 LC3-II/LC3-I 比值明显增加。此外,荧光素酶报告基因检测证实 FGF23 可直接调控 microRNA(miR)-17-5p。miR-17-5p 抑制可逆转 FGF23 沉默的作用。FGF23 可能通过自噬信号通路靶向 miR-17-5p 调节缺氧诱导的成骨细胞凋亡。这为 FGF23 作为股骨头坏死潜在治疗靶点提供了依据。