骨髓间充质干细胞（BMSCs）来源的外泌体METTL3调节SMAD5的m6A甲基化以促进成骨细胞的成骨分化。

Bone marrow mesenchymal stem cells (BMSCs)-derived exosomal METTL3 regulates the m6A methylation of SMAD5 to promote osteogenic differentiation of osteoblasts.

作者信息

Li Zhenhua, Liu Yifei, Zhao Xiulan, Xu Guohua

机构信息

Department of Outpatient, Shanghai Changzheng Hospital, Naval Medical University, Shanghai, China.

Department of spine surgery, Shanghai Changzheng Hospital, Naval Medical University, Shanghai, China.

出版信息

Connect Tissue Res. 2025 Apr 29:1-12. doi: 10.1080/03008207.2025.2496832.

DOI:10.1080/03008207.2025.2496832

PMID:40298375

Abstract

BACKGROUND

Methyltransferase-like 3 (METTL3) is implicated in human diseases, including osteoporosis (OP). In this study, we aimed to explore the functions and mechanisms of METTL3 in OP using bone marrow mesenchymal stem cells (BMSCs).

METHODS

The identification of BMSCs-derived exosomes was conducted by transmission electron microscope (TEM), Nanoparticle Tracking Analysis (NTA) and western blot. The osteogenic differentiation of osteoblasts (hFOB1.19) was analyzed by Alizarin red staining assay, Alkaline phosphatase (ALP) staining assay and western blot. The relationship between METTL3 and SMAD family member 5 (SMAD5) was analyzed by Methylated RNA Immunoprecipitation (MeRIP) assay and dual-luciferase reporter assay.

RESULTS

BMSCs-derived exosomes (BMSC-Exos) promoted the osteogenic differentiation and elevated METTL3 expression in hFOB1.19 cells. Exosomal METTL3 knockdown repressed the osteogenic differentiation in hFOB1.19 cells. METTL3 could stabilize and regulate SMAD5 expression by N6-methyladenosine (m6A) modification. Moreover, SMAD5 overexpression restored exosomal METTL3 knockdown-mediated effect on the osteogenic differentiation in hFOB1.19 cells.

CONCLUSION

BMSCs-derived exosomal METTL3 mediated the m6A methylation of SMAD5 to facilitate osteogenic differentiation of hFOB1.19 cells.

摘要

背景

甲基转移酶样3（METTL3）与包括骨质疏松症（OP）在内的人类疾病有关。在本研究中，我们旨在利用骨髓间充质干细胞（BMSC）探索METTL3在OP中的功能和机制。

方法

通过透射电子显微镜（TEM）、纳米颗粒跟踪分析（NTA）和蛋白质印迹法对BMSC来源的外泌体进行鉴定。通过茜素红染色试验、碱性磷酸酶（ALP）染色试验和蛋白质印迹法分析成骨细胞（hFOB1.19）的成骨分化情况。通过甲基化RNA免疫沉淀（MeRIP）试验和双荧光素酶报告基因试验分析METTL3与SMAD家族成员5（SMAD5）之间的关系。

结果

BMSC来源的外泌体（BMSC-Exos）促进hFOB1.19细胞的成骨分化并提高METTL3表达。外泌体METTL3敲低抑制hFOB1.19细胞的成骨分化。METTL3可通过N6-甲基腺苷（m6A）修饰稳定并调节SMAD5表达。此外，SMAD5过表达恢复了外泌体METTL3敲低介导的对hFOB,1.19细胞成骨分化的影响。