alkbh5介导的m6A去甲基化修饰RAD51通过促进骨质疏松症DNA损伤抑制成骨分化

IF 3 3区医学 Q2 BIOCHEMISTRY & MOLECULAR BIOLOGY

Molecular immunology Pub Date : 2025-08-16 DOI:10.1016/j.molimm.2025.08.008

Minli Qiu , Zetao Liao , Xinyu Wu , Peili He , Xianghui Wen , Ya Xie , Jun Qi

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引用次数: 0

摘要

骨质疏松症（OP）是一种以骨密度降低和骨组织微结构退化为特征的代谢性疾病。成骨分化通过促进新骨形成、保持骨强度和密度、抑制骨吸收在OP发病中起关键作用。RNA表观遗传修饰越来越多地涉及骨代谢的多个方面。我们之前的研究揭示了RAD51在OP进展中的调节作用。本研究旨在探讨RAD51是否通过RNA甲基化修饰参与OP，并阐明其潜在机制。将MC3T3-E1细胞诱导成骨分化并置于模拟微重力环境下建立体外OP模型。建立了去卵巢（OVX）小鼠OP模型。用点印迹法测定RNA甲基化水平。RT-qPCR检测m6A甲基转移酶和去甲基化酶的mRNA表达。采用茜素红S和碱性磷酸酶（ALP）染色评价成骨分化能力。Western blot检测蛋白表达。RAD51与AlkB同源物5 (ALKBH5)/YTH结构域家族（YTHDF）1的相互作用通过RNA免疫沉淀和双荧光素酶报告基因检测得到验证。结果表明，alkbh5介导的m6A去甲基化可显著抑制MC3T3-E1细胞中RAD51的表达。此外，ALKBH5敲低可通过减轻DNA损伤来增强成骨细胞分化。机制上，ALKBH5/YTHDF1 m6A调控轴通过m6A甲基化动力学调节RAD51 mRNA的稳定性。体内实验表明，ALKBH5缺失通过抑制DNA损伤途径减轻OVX小鼠骨丢失，促进成骨细胞发生。综上所述，这些发现表明alkbh5介导的RAD51的m6A去甲基化通过诱导OP的DNA损伤来抑制成骨分化，提示骨质疏松症治疗的潜在治疗靶点。

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ALKBH5-mediated m6A demethylation modification of RAD51 inhibits osteogenic differentiation via promoting DNA damage in osteoporosis

Osteoporosis (OP) is a metabolic disorder characterized by reduced bone mineral density and degeneration of bone tissue microarchitecture. Osteogenic differentiation plays a pivotal role in OP pathogenesis by facilitating new bone formation, preserving bone strength and density, and counteracting bone resorption. RNA epigenetic modifications have been increasingly implicated in multiple aspects of bone metabolism. Our previous studies revealed the regulatory role of RAD51 in OP progression. This study aimed to investigate whether RAD51 undergoes RNA methylation modification to participate in OP and to elucidate its underlying mechanisms. MC3T3-E1 cells were induced to undergo osteogenic differentiation and exposed to a simulated microgravity environment to establish an in vitro OP model. An ovariectomized (OVX) murine OP model was also established. RNA methylation level was quantified using dot blot assay. RT-qPCR was employed to analyze mRNA expression of m⁶A methyltransferases and demethylases. Osteogenic differentiation capacity was assessed by Alizarin Red S and alkaline phosphatase (ALP) staining. Protein expressions were evaluated by Western blot. The interaction between RAD51 and AlkB Homolog 5 (ALKBH5)/YTH domain family (YTHDF)1 was validated through RNA immunoprecipitation and dual-luciferase reporter assays. Results demonstrated that ALKBH5-mediated m⁶A demethylation significantly suppressed RAD51 expression in MC3T3-E1 cells. Furthermore, ALKBH5 knockdown enhanced osteoblast differentiation by alleviating DNA damage. Mechanistically, the ALKBH5/YTHDF1 m⁶A regulatory axis modulated RAD51 mRNA stability through m⁶A methylation dynamics. In vivo experiments revealed that ALKBH5 deletion mitigated bone loss and promoted osteoblastogenesis in OVX mice through inhibition of DNA damage pathways. Collectively, these findings indicated that ALKBH5-mediated m⁶A demethylation of RAD51 inhibited osteogenic differentiation by inducing DNA damage in OP, suggesting potential therapeutic targets for osteoporosis treatment.

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来源期刊

Molecular immunology 医学-免疫学

CiteScore

6.90

自引率

2.80%

发文量

324

审稿时长

50 days

期刊介绍： Molecular Immunology publishes original articles, reviews and commentaries on all areas of immunology, with a particular focus on description of cellular, biochemical or genetic mechanisms underlying immunological phenomena. Studies on all model organisms, from invertebrates to humans, are suitable. Examples include, but are not restricted to: Infection, autoimmunity, transplantation, immunodeficiencies, inflammation and tumor immunology Mechanisms of induction, regulation and termination of innate and adaptive immunity Intercellular communication, cooperation and regulation Intracellular mechanisms of immunity (endocytosis, protein trafficking, pathogen recognition, antigen presentation, etc) Mechanisms of action of the cells and molecules of the immune system Structural analysis Development of the immune system Comparative immunology and evolution of the immune system "Omics" studies and bioinformatics Vaccines, biotechnology and therapeutic manipulation of the immune system (therapeutic antibodies, cytokines, cellular therapies, etc) Technical developments.