Ye Weihua, Liu Zheng, Liu Yaoxi, Xiao Han, Tan Qian, Yan An, Zhu Guanghui
Orthopedic Department, Hunan Provincial Key Laboratory of Pediatric Orthopedics, Hunan Children's Hospital, Children's Hospital Affiliated to Xiangya Medical College of Central South University, 86# Ziyuan Road, Changsha, Hunan 410007, China.
Regen Ther. 2024 May 21;26:42-49. doi: 10.1016/j.reth.2024.04.004. eCollection 2024 Jun.
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.
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 mA 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.
METTL3 overexpression enhanced CPT MSCs' osteogenic differentiation. METTL3 stabilized the HOXD8 in an mA-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.
METTL3 promoted osteogenic differentiation via modulating the HOXD8/ITGA5 axis in CPT MSCs.
先天性胫骨假关节（CPT）是小儿骨科领域的一项重大健康挑战。CPT发生发展的关键过程是源自CPT的间充质干细胞（MSC）进行成骨分化的能力有限。我们的研究旨在阐明甲基转移酶样3（METTL3）在CPT MSC成骨分化过程中的作用及机制。
使用成骨分化培养基培养MSC，并通过茜素红S和碱性磷酸酶（ALP）检测法进行成骨分化检测。通过定量实时聚合酶链反应（qRT-PCR）、蛋白质印迹法或免疫荧光（IF）染色评估基因或蛋白质表达。通过甲基化RNA免疫沉淀（MeRIP）检测法验证同源盒D8（HOXD8）的mA修饰。通过荧光素酶报告基因、RNA免疫沉淀（RIP）和染色质免疫沉淀（ChIP）检测法验证METTL3与HOXD8或HOXD8与整合素α5（ITGA5）启动子之间的相互作用。
METTL3过表达增强了CPT MSC的成骨分化。METTL3以mA依赖的方式稳定HOXD8。此外，过表达的ITGA5上调了CPT MSC的成骨分化。此外，HOXD8可转录激活ITGA5。METTL3通过HOXD8增加ITGA5的转录，以增强CPT MSC的成骨分化。
METTL3通过调节CPT MSC中的HOXD8/ITGA5轴促进成骨分化。
