FOXO3/ZC3H13/SLC3A2级联反应调控骨髓间充质干细胞的成骨分化和铁死亡

摘要(中文译文)

骨质疏松症发病机制涉及骨髓间充质干细胞（BMSCs）成骨分化受损。ZC3H13作为一种关键的m6A写入蛋白，通过抑制铁死亡促进BMSC成骨分化。然而，其分子机制在很大程度上仍未被探索。通过定量PCR和免疫印迹进行表达分析。采用EdU实验评估细胞增殖。使用碱性磷酸酶（ALP）表达/活性实验和茜素红染色评估成骨分化。通过检测ROS、Fe和MDA水平评估铁死亡变化。采用RNA免疫沉淀（RIP）、甲基化RNA免疫沉淀（MeRIP）、荧光素酶和mRNA稳定性实验验证ZC3H13对SLC3A2的调控。通过染色质免疫沉淀（ChIP）和荧光素酶实验证实FOXO3与ZC3H13之间的关系。ZC3H13在骨质疏松患者骨髓中表达下调。ZC3H13缺失抑制BMSC增殖和成骨分化，同时增强铁死亡。相反，ZC3H13过表达促进增殖、分化并抑制铁死亡。机制上，ZC3H13以IGF2BP2依赖的方式介导SLC3A2的m6A修饰和稳定性。沉默ZC3H13通过减少SLC3A2抑制BMSC增殖和成骨分化，同时增强铁死亡。此外，FOXO3作为ZC3H13的转录激活因子。敲低FOXO3通过下调ZC3H13抑制BMSC增殖和成骨分化，同时增强铁死亡。另外，FOXO3通过ZC3H13调控SLC3A2表达。本研究揭示了一条新的FOXO3/ZC3H13/SLC3A2调控级联通路，该通路保护BMSCs免受铁死亡，且在骨质疏松症中受损，为治疗开发提供了新的概念框架。

Abstract

Impaired osteogenic differentiation in bone marrow-derived mesenchymal stem cells (BMSCs) is involved in the pathogenesis of osteoporosis. ZC3H13, a crucial m6A writer, promotes BMSC osteogenic differentiation by inhibiting ferroptosis. However, its molecular underpinnings remain largely unexplored. Expression analysis was performed by quantitative PCR and immunoblotting. Cell proliferation was assessed by EdU assay. Osteogenic differentiation was assessed using alkaline phosphatase (ALP) expression/activity assays and Alizarin Red staining. Ferroptosis alteration was evaluated by detecting ROS, Fe, and MDA levels. RNA immunoprecipitation (RIP), methylated RIP (MeRIP), luciferase, and mRNA stability assays were used to validate the regulation of ZC3H13 in SLC3A2. The relationship between FOXO3 and ZC3H13 was confirmed by chromatin immunoprecipitation (ChIP) and luciferase experiments. ZC3H13 was downregulated in the bone marrow of osteoporosis patients. ZC3H13 deficiency inhibited BMSC proliferation and osteogenic differentiation while enhancing ferroptosis. Conversely, ZC3H13 overexpression promoted proliferation, differentiation and suppressed ferroptosis. Mechanistically, ZC3H13 mediated the m6A modification and stability of SLC3A2 in an IGF2BP2-dependent manner. ZC3H13 silencing suppressed BMSC proliferation and osteogenic differentiation while enhancing ferroptosis through SLC3A2 reduction. Furthermore, FOXO3 acted as a transcriptional activator of ZC3H13. Depletion of FOXO3 suppressed BMSC proliferation and osteogenic differentiation while enhancing ferroptosis through downregulation of ZC3H13. Additionally, FOXO3 regulated SLC3A2 expression via ZC3H13. This study delineates a novel FOXO3/ZC3H13/SLC3A2 regulatory cascade that safeguards BMSCs from ferroptosis and is compromised in osteoporosis, providing a new conceptual framework for therapeutic development.

如何引用