Department of Conservative Dentistry and Endodontology, College of Stomatology, Guangxi Medical University, Nanning, P.R. China.
Laboratory of Tissue Regeneration and Immunology and Department of Periodontics, Beijing Key Laboratory of Tooth Regeneration and Function Reconstruction, Capital Medical University School of Stomatology, Beijing, P.R. China.
J Physiol Pharmacol. 2020 Dec;71(6). doi: 10.26402/jpp.2020.6.14. Epub 2021 Apr 22.
Fibroblast injury and autophagy dysfunction have been shown to contribute to the persistence of oral wounds. Recently, microRNAs have emerged as vital regulators and fine tuners of various pathophysiological cellular processes that influence the wound healing process. This study explored the biological function and regulatory mechanism of miRNA-21 (miR-21) in the healing of oral wounds by interfering with autophagy. Healthy gingival cells derived from wild-type (WT) and from miR-21KO mice were characterized by immunocytofluorescence, and changes in wound healing were subsequently assessed using an in vitro scratch wound healing assay. The roles of critical proteins required for autophagy, autophagy related 5 (ATG5) and Bcl-2 interacting coiled-coil protein 1 (Beclin1) were evaluated by immunohistochemistry. Human gingival fibroblasts (HGFs) were transfected with a miR-21 mimic and a miR-negative control, and the relative expression of miR-21, ATG5, Beclin1 and LC3-I/II was characterized by qRT-PCR and Western blot. Pathological changes were observed in a palatal wound healing model using WT and miR-21KO mice. Immunohistochemistry was used to examine extracellular matrix (ECM) proteins and autophagy markers. Cell migration was delayed in gingiva-derived mesenchymal stem cells (GMSCs) from miR-21KO mice compared with WT mice. The expression of ATG5 and Beclin1 was significantly up-regulated in miR-21KO gingiva. Transfection of a miR-21 mimic into HGFs inhibited autophagy and up-regulated miR-21 expression. Knockdown of miR-21 suppressed the expression of fibronectin and CTGF, enhanced the autophagy effect of fibroblasts, suggesting that autophagy is involved in miR-21 regulated palatal wound healing. Taken together, these results suggest that miR-21 promotes oral wound healing by increasing ECM production through the inhibition of autophagy and facilitates clinical management of wound healing.
成纤维细胞损伤和自噬功能障碍已被证明有助于口腔伤口的持续存在。最近，microRNAs 已成为各种影响伤口愈合过程的病理生理细胞过程的重要调节因子和微调因子。本研究通过干扰自噬来探讨 miRNA-21（miR-21）在口腔伤口愈合中的生物学功能和调节机制。通过免疫细胞荧光法对源自野生型（WT）和 miR-21KO 小鼠的健康牙龈细胞进行特征描述，随后通过体外划痕愈合试验评估伤口愈合的变化。通过免疫组织化学评估自噬所需的关键蛋白，包括自噬相关 5（ATG5）和 Bcl-2 相互作用卷曲螺旋蛋白 1（Beclin1）的作用。用 miR-21 模拟物和 miR-阴性对照转染人牙龈成纤维细胞（HGFs），并通过 qRT-PCR 和 Western blot 分析 miR-21、ATG5、Beclin1 和 LC3-I/II 的相对表达。使用 WT 和 miR-21KO 小鼠的腭伤口愈合模型观察病理变化。通过免疫组织化学检测细胞外基质（ECM）蛋白和自噬标志物。与 WT 小鼠相比，miR-21KO 小鼠的牙龈衍生间充质干细胞（GMSCs）的细胞迁移延迟。miR-21KO 牙龈中的 ATG5 和 Beclin1 表达明显上调。转染 miR-21 模拟物可抑制 HGFs 的自噬并上调 miR-21 的表达。miR-21 的下调抑制了纤维连接蛋白和 CTGF 的表达，增强了成纤维细胞的自噬作用，提示自噬参与 miR-21 调节的腭伤口愈合。综上所述，这些结果表明，miR-21 通过抑制自噬增加 ECM 产生促进口腔伤口愈合，并有助于伤口愈合的临床管理。
