牙周炎损伤的牙髓干细胞通过分泌携带 miRNA-378a 的细胞外囊泡，靶向 Sufu 激活 Hedgehog/Gli1 信号通路，促进局部血管生成。
Periodontitis-compromised dental pulp stem cells secrete extracellular vesicles carrying miRNA-378a promote local angiogenesis by targeting Sufu to activate the Hedgehog/Gli1 signalling.
机构信息
Department of Periodontology, School of Stomatology, State Key Laboratory of Military Stomatology, National Clinical Research Center for Oral Diseases and Shaanxi Engineering Research Center for Dental Materials and Advanced Manufacture, Fourth Military Medical University, Xi'an, China.
Shaanxi Key Laboratory of Free Radical Biology and Medicine, The Ministry of Education Key Laboratory of Hazard Assessment and Control in Special Operational Environments, Fourth Military Medical University, Xi'an, China.
出版信息
Cell Prolif. 2021 May;54(5):e13026. doi: 10.1111/cpr.13026. Epub 2021 Mar 23.
OBJECTIVES
Previously, our investigations demonstrated robust pro-angiogenic potentials of extracellular vesicles secreted by periodontitis-compromised dental pulp stem cells (P-EVs) when compared to those from healthy DPSCs (H-EVs), but the underlying mechanism remains unknown.
MATERIALS AND METHODS
Here, circulating microRNAs (miRNAs) specifically found in P-EVs (compared with H-EVs) were identified by Agilent miRNA microarray analysis, and the roles of the candidate miRNA in P-EV-enhanced cell angiogenesis were confirmed by cell transfection and RNA interference methods. Next, the direct binding affinity between the candidate miRNA and its target gene was evaluated by luciferase reporter assay. CCK-8, transwell/scratch wound healing and tube formation assays were established to investigate the proliferation, migration, and tube formation abilities of endothelial cells (ECs). Western blot was employed to measure the protein levels of Hedgehog/Gli1 signalling pathway components and angiogenesis-related factors.
RESULTS
The angiogenesis-related miRNA miR-378a was found to be enriched in P-EVs, and its role in P-EV-enhanced cell angiogenesis was confirmed, wherein Sufu was identified as a downstream target gene of miR-378a. Functionally, silencing of Sufu stimulated EC proliferation, migration and tube formation by activating Hedgehog/Gli1 signalling. Further, we found that incubation with P-EVs enabled the transmission of P-EV-contained miR-378a to ECs. Subsequently, the expressions of Sufu, Gli1 and vascular endothelial growth factor in ECs were significantly influenced by P-EV-mediated miR-378a transmission.
CONCLUSIONS
These data suggest that P-EVs carrying miR-378a promote EC angiogenesis by downregulating Sufu to activate the Hedgehog/Gli1 signalling pathway. Our findings reveal a crucial role for EV-derived miR-378a in cell angiogenesis and hence offer a new target for modifying stem cells and their secreted EVs to enhance vessel regenerative potential.
目的
此前，我们的研究表明，与健康牙髓干细胞（H-EVs）来源的细胞外囊泡（EVs）相比，牙周炎受损牙髓干细胞（P-EVs）分泌的 EVs 具有更强的促血管生成潜力，但具体的作用机制尚不清楚。
材料和方法
在这里，通过 Agilent miRNA 微阵列分析鉴定了在 P-EVs（与 H-EVs 相比）中特异性存在的循环 microRNAs（miRNAs），并通过细胞转染和 RNA 干扰方法证实了候选 miRNA 在 P-EV 增强细胞血管生成中的作用。接下来，通过荧光素酶报告基因检测评估候选 miRNA 与其靶基因之间的直接结合亲和力。建立 CCK-8、Transwell/划痕愈合和管形成实验来研究内皮细胞（ECs）的增殖、迁移和管形成能力。Western blot 用于测量 Hedgehog/Gli1 信号通路成分和血管生成相关因子的蛋白水平。
结果
发现与血管生成相关的 miRNA miR-378a 在 P-EVs 中富集，并证实其在 P-EV 增强细胞血管生成中的作用，其中 Sufu 被鉴定为 miR-378a 的下游靶基因。功能上，沉默 Sufu 通过激活 Hedgehog/Gli1 信号通路刺激 EC 的增殖、迁移和管形成。此外，我们发现 P-EVs 孵育可将 P-EV 中包含的 miR-378a 传递给 ECs。随后，P-EV 介导的 miR-378a 传递显著影响 ECs 中 Sufu、Gli1 和血管内皮生长因子的表达。
结论
这些数据表明，携带 miR-378a 的 P-EVs 通过下调 Sufu 来激活 Hedgehog/Gli1 信号通路促进 EC 血管生成。我们的研究结果表明 EV 衍生的 miR-378a 在细胞血管生成中起关键作用，为修饰干细胞及其分泌的 EV 以增强血管再生潜能提供了新的靶点。
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