Qin Jian, Zou Li, Lu Fachao, Liu Fang, Min Qian, Zhu Lilei
Department of Endodontics, Changsha Stomatological Hospital, Hunan Province, Changsha, 410005, People's Republic of China.
Department of Periodontology, Changsha Stomatological Hospital, Hunan Province, No.389, Youyi Road, Changsha, 410005, People's Republic of China.
Odontology. 2025 Jan;113(1):305-317. doi: 10.1007/s10266-024-00977-3. Epub 2024 Jul 5.
Angiogenesis serves as the determinate element of pulp regeneration. Dental pulp stem cell (DPSC) implantation can promote the regeneration of dental pulp tissue. Herein, the role of mA methyltransferase methyltransferase-like 3 (METTL3) in regulating DPSCs-induced angiogenesis during pulp regeneration therapy was investigated. Cell DPSC viability, HUVEC migration, and angiogenesis ability were analyzed by CCK-8 assay, wound healing, Transwell assay, and tube formation assay. The global and EST1 mRNA m6A levels were detected by mA dot blot and Me-RIP. The interactions between E26 transformation-specific proto-oncogene 1(ETS1), human antigen R(HuR), and METTL3 were analyzed by RIP assay. The relationship between METTL3 and the mA site of ETS1 was performed by dual-luciferase reporter assay. ETS1 mRNA stability was examined with actinomycin D. Herein, our results revealed that human immature DPSCs (hIDPSCs) showed stronger ability to induce angiogenesis than human mature DPSCs (hMDPSCs), which might be related to ETS1 upregulation. ETS1 knockdown inhibited DPSCs-induced angiogenesis. Our mechanistic experiments demonstrated that METTL3 increased ETS1 mRNA stability and expression level on DPSCs in an mA-HuR-dependent manner. ETS1 upregulation abolished sh-METTL3's inhibition on DPSCs-induced angiogenesis. METTL3 upregulation promoted DPSCs-induced angiogenesis by enhancing ETS1 mRNA stability in an mA-HuR-dependent manner. This study reveals a new mechanism by which mA methylation regulates angiogenesis in DPSCs, providing new insights for stem cell-based tissue engineering.
血管生成是牙髓再生的决定性因素。牙髓干细胞（DPSC）植入可促进牙髓组织再生。在此，研究了甲基转移酶样3（METTL3）在牙髓再生治疗中调节DPSC诱导的血管生成中的作用。通过CCK-8法、伤口愈合实验、Transwell实验和管腔形成实验分析细胞DPSC活力、人脐静脉内皮细胞（HUVEC）迁移和血管生成能力。通过甲基化（mA）斑点印迹和甲基化RNA免疫沉淀（Me-RIP）检测全局和EST1 mRNA的m6A水平。通过RNA免疫沉淀（RIP）实验分析E26转化特异性原癌基因1（ETS1）、人抗原R（HuR）和METTL3之间的相互作用。通过双荧光素酶报告基因实验研究METTL3与ETS1的mA位点之间的关系。用放线菌素D检测ETS1 mRNA稳定性。在此，我们的结果显示，人未成熟DPSC（hIDPSC）比人成熟DPSC（hMDPSC）具有更强的诱导血管生成能力，这可能与ETS1上调有关。ETS1敲低抑制DPSC诱导的血管生成。我们的机制实验表明，METTL3以mA-HuR依赖的方式增加DPSC上ETS1 mRNA的稳定性和表达水平。ETS1上调消除了sh-METTL3对DPSC诱导的血管生成的抑制作用。METTL3上调通过以mA-HuR依赖的方式增强ETS mRNA稳定性来促进DPSC诱导的血管生成。本研究揭示了一种新的机制，即mA甲基化调节DPSC中的血管生成，为基于干细胞的组织工程提供了新的见解。
