METTL3 promotes the osteogenic differentiation of periosteum-derived MSCs via regulation of the HOXD8/ITGA5 axis in congenital pseudarthrosis

IF 3.4 3区环境科学与生态学 Q3 CELL & TISSUE ENGINEERING Regenerative Therapy Pub Date : 2024-05-21 DOI:10.1016/j.reth.2024.04.004

Weihua Ye, Zheng Liu, Yaoxi Liu, Han Xiao, Qian Tan, An Yan, Guanghui Zhu

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Abstract

Background

Congenital pseudarthrosis of the tibia (CPT) is a dominant health challenge in pediatric orthopedics. The essential process in the development of CPT is the limited capacity of mesenchymal stem cells (MSCs) derived from CPT to undergo osteogenic differentiation. Our research aimed to elucidate the role and mechanism of methyltransferase-like 3 (METTL3) in the osteogenic differentiation process of CPT MSCs.

Methods

The osteogenic differentiation medium was used to culture MSCs, and the detection of osteogenic differentiation was performed using Alizarin Red S and alkaline phosphatase (ALP) assays. Gene or protein expression was assessed by quantitative real-time polymerase chain reaction (qRT-PCR), Western blot, or immunofluorescence (IF) staining. The m⁶A modification of Homeobox D8 (HOXD8) was verified by methylated RNA immunoprecipitation (MeRIP) assay. Interactions between METTL3 and HOXD8 or HOXD8 and integrin alpha 5 (ITGA5) promoter were validated by the luciferase reporter gene, RIP, and chromatin immunoprecipitation (ChIP) assays.

Results

METTL3 overexpression enhanced CPT MSCs' osteogenic differentiation. METTL3 stabilized the HOXD8 in an m⁶A-dependent manner. Moreover, the overexpressed ITGA5 up-regulated the CPT MSCs’ osteogenic differentiation. Further, HOXD8 could transcriptionally activate ITGA5. METTL3 increased the transcription of ITGA5 via HOXD8 to enhance the osteogenic differentiation of CPT MSCs.

Conclusion

METTL3 promoted osteogenic differentiation via modulating the HOXD8/ITGA5 axis in CPT MSCs.

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METTL3通过调控HOXD8/ITGA5轴促进先天性假关节中骨膜来源间充质干细胞的成骨分化

背景先天性胫骨假关节（CPT）是小儿骨科的主要健康挑战。先天性胫骨假关节（Congenital pseudarthrosis of tibia，CPT）是小儿骨科面临的主要健康挑战。CPT发病的关键过程是来自CPT的间充质干细胞（MSCs）进行成骨分化的能力有限。我们的研究旨在阐明类似甲基转移酶3（METTL3）在CPT间充质干细胞成骨分化过程中的作用和机制。方法使用成骨分化培养基培养间充质干细胞，并使用茜素红S和碱性磷酸酶（ALP）检测成骨分化。基因或蛋白质表达通过实时定量聚合酶链反应（qRT-PCR）、Western印迹或免疫荧光（IF）染色进行评估。通过甲基化 RNA 免疫沉淀（MeRIP）检测验证了同源染色体 D8（HOXD8）的 m6A 修饰。荧光素酶报告基因、RIP和染色质免疫共沉淀（ChIP）实验验证了METTL3与HOXD8或HOXD8与整合素α5（ITGA5）启动子之间的相互作用。METTL3 以 m6A 依赖性方式稳定了 HOXD8。此外，过表达的 ITGA5 能上调 CPT 间充质干细胞的成骨分化。此外，HOXD8 还能转录激活 ITGA5。结论 METTL3通过调节HOXD8/ITGA5轴促进了CPT间充质干细胞的成骨分化。

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来源期刊

Regenerative Therapy Engineering-Biomedical Engineering

CiteScore

6.00

自引率

2.30%

发文量

106

审稿时长

49 days

期刊介绍： Regenerative Therapy is the official peer-reviewed online journal of the Japanese Society for Regenerative Medicine. Regenerative Therapy is a multidisciplinary journal that publishes original articles and reviews of basic research, clinical translation, industrial development, and regulatory issues focusing on stem cell biology, tissue engineering, and regenerative medicine.