Department of Orthopaedics, Qilu Hospital of Shandong University, 250012, Jinan, Shandong, China.
The First Clinical College of Cheeloo College of Medicine, Shandong University, 250012, Jinan, Shandong, China.
Cell Death Dis. 2023 Jan 17;14(1):33. doi: 10.1038/s41419-023-05565-x.
An imbalance in the differentiation potential of bone marrow mesenchymal stem cells (BMSCs) is an important pathogenic mechanism underlying osteoporosis (OP). N6-methyladenosine (mA) is the most common post-transcriptional modification in eukaryotic cells. The role of the Wilms' tumor 1-associated protein (WTAP), a member of the mA functional protein family, in regulating BMSCs differentiation remains unknown. We used patient-derived and mouse model-derived samples, qRT-PCR, western blot assays, ALP activity assay, ALP, and Alizarin Red staining to determine the changes in mRNA and protein levels of genes and proteins associated with BMSCs differentiation. Histological analysis and micro-CT were used to evaluate developmental changes in the bone. The results determined that WTAP promoted osteogenic differentiation and inhibited adipogenic differentiation of BMSCs. We used co-immunoprecipitation (co-IP), RNA immunoprecipitation (RIP), methylated RNA immunoprecipitation (MeRIP), RNA pulldown, and dual-luciferase assay to explore the direct mechanism. Mechanistically, the expression of WTAP increased during osteogenic differentiation and significantly promoted pri-miR-181a and pri-miR-181c methylation, which was recognized by YTHDC1, and increased the maturation to miR-181a and miR-181c. MiR-181a and miR-181c inhibited the mRNA expression of SFRP1, promoting the osteogenic differentiation of BMSCs. Our results demonstrated that the WTAP/YTHDC1/miR-181a and miR-181c/SFRP1 axis regulated the differentiation fate of BMSCs, suggesting that it might be a potential therapeutic target for osteoporosis.
骨髓间充质干细胞（BMSCs）分化潜能失衡是骨质疏松症（OP）的重要发病机制。N6-甲基腺苷（mA）是真核细胞中最常见的转录后修饰。Wilms 瘤 1 相关蛋白（WTAP）作为 mA 功能蛋白家族的一员，在调节 BMSCs 分化中的作用尚不清楚。我们使用患者来源和小鼠模型来源的样本、qRT-PCR、western blot 分析、ALP 活性测定、ALP 和茜素红染色来确定与 BMSCs 分化相关的基因和蛋白的 mRNA 和蛋白水平的变化。组织学分析和 micro-CT 用于评估骨的发育变化。结果确定 WTAP 促进 BMSCs 的成骨分化并抑制成脂分化。我们使用免疫共沉淀（co-IP）、RNA 免疫沉淀（RIP）、甲基化 RNA 免疫沉淀（MeRIP）、RNA 下拉和双荧光素酶测定来探索直接机制。从机制上讲，WTAP 的表达在成骨分化过程中增加，显著促进 pri-miR-181a 和 pri-miR-181c 的甲基化，被 YTHDC1 识别，并增加成熟到 miR-181a 和 miR-181c。miR-181a 和 miR-181c 抑制 SFRP1 的 mRNA 表达，促进 BMSCs 的成骨分化。我们的研究结果表明，WTAP/YTHDC1/miR-181a 和 miR-181c/SFRP1 轴调节 BMSCs 的分化命运，提示其可能成为骨质疏松症的潜在治疗靶点。
