Abstract：
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 m6A 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 m6A 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 m6A 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 m6A-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 m6A-HuR-dependent manner. This study reveals a new mechanism by which m6A methylation regulates angiogenesis in DPSCs, providing new insights for stem cell-based tissue engineering.
摘要：
血管生成是纸浆再生的决定因素。牙髓干细胞(DPSC)植入可以促进牙髓组织的再生。在这里，研究了m6A甲基转移酶甲基转移酶样3(METTL3)在牙髓再生治疗期间调节DPSC诱导的血管生成中的作用.细胞DPSC活力，HUVEC迁移,和血管生成能力通过CCK-8分析，伤口愈合，Transwell分析，和试管形成测定。通过m6A斑点印迹和Me-RIP检测全局和EST1mRNAm6A水平。E26转化特异性原癌基因1（ETS1）之间的相互作用，人抗原R(HuR),和METTL3通过RIP测定进行分析。METTL3与ETS1的m6A位点之间的关系通过双荧光素酶报告基因测定进行。用放线菌素D检查ETS1mRNA的稳定性。我们的结果表明，人类未成熟DPSC（hIDPSC）显示出比人类成熟DPSC（hMDPSC）更强的诱导血管生成的能力，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m6A-HuR依赖性方式增加了DPSC上的ETS1mRNA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m6A-HuR依赖性方式增强ETS1mRNA稳定性来促进DPSC诱导的血管生成。这项研究揭示了m6A甲基化调节DPSC血管生成的新机制。为基于干细胞的组织工程提供新的见解。
