Department of Orthopedics, The Second Affiliated Hospital of Soochow University, Suzhou, China.
Department of Orthopedics, Yancheng Third People's Hospital, Yancheng, China.
IUBMB Life. 2021 Feb;73(2):432-443. doi: 10.1002/iub.2440.
Postmenopausal osteoporosis (PMOP) is mainly caused by multiple factors. Recent studies have suggested that iron accumulation (IA) was closely related to PMOP. However, the detailed molecular mechanisms have not been well demonstrated. We constructed the IA mouse model by intraperitoneal injections of ferric ammonium citrate (FAC) and cell model by culturing with the medium containing FAC. Osteoporosis was confirmed in mouse bone tissues using H&E staining, and the level of serum ferritin, alkaline phosphatase (ALP), procollagen-1 N-terminal peptide (P1NP), and osteocalcin in mice was examined by ELISA. The expressions of XIST and miR-758-3p were detected by qRT-PCR. Cell proliferation and apoptosis were measured by CCK-8, TUNEL, and flow cytometry. The expression levels of apoptotic-related proteins were evaluated by western blot. Dual luciferase reporter assay was used to examine the molecular interaction. The expressions of ALP, P1NP, and osteocalcin, and the H&E staining of bone tissues in mice were analyzed to confirm the biological function of XIST and miR-758-3p in vivo. XIST was up-regulated while miR-758-3p was down-regulated in IA mouse and cell models. XIST knockdown significantly reduced FAC-induced osteoblast apoptosis, which was mimicked by transfection with miR-758-3p mimics. XIST acted as a sponge of miR-758-3p, which targeted caspase 3. IA led to the high expression of XIST and promoted osteoblast apoptosis through miR-758-3p/caspase 3. Transfection with shXIST or miR-758-3p mimics alleviated IA-induced mouse osteoporosis. IA regulated osteoblast apoptosis through XIST/miR-758-3p/caspase 3 axis, which might provide alternative targets for the treatment of osteoporosis.
绝经后骨质疏松症（PMOP）主要由多种因素引起。最近的研究表明，铁积累（IA）与 PMOP 密切相关。然而，其详细的分子机制尚未得到很好的证明。我们通过腹腔注射柠檬酸铁铵（FAC）构建 IA 小鼠模型，并通过用含有 FAC 的培养基培养构建细胞模型。通过 H&E 染色确认小鼠骨组织中的骨质疏松症，并通过 ELISA 检测小鼠血清铁蛋白、碱性磷酸酶（ALP）、前胶原 1 N 端肽（P1NP）和骨钙素的水平。通过 qRT-PCR 检测 XIST 和 miR-758-3p 的表达。通过 CCK-8、TUNEL 和流式细胞术测量细胞增殖和凋亡。通过 Western blot 评估凋亡相关蛋白的表达水平。使用双荧光素酶报告基因检测来检查分子相互作用。分析小鼠中 XIST 和 miR-758-3p 的表达以及骨组织的 H&E 染色，以确认 XIST 和 miR-758-3p 在体内的生物学功能。在 IA 小鼠和细胞模型中，XIST 上调而 miR-758-3p 下调。转染 miR-758-3p 模拟物可显著减少 FAC 诱导的成骨细胞凋亡，从而模拟 XIST 的敲低。XIST 作为 miR-758-3p 的海绵，靶向半胱氨酸蛋白酶 3。IA 导致 XIST 的高表达，并通过 miR-758-3p/caspase 3 促进成骨细胞凋亡。转染 shXIST 或 miR-758-3p 模拟物可缓解 IA 诱导的小鼠骨质疏松症。IA 通过 XIST/miR-758-3p/caspase 3 轴调节成骨细胞凋亡，这可能为骨质疏松症的治疗提供替代靶点。
