Department of Stomatology, Xuanwu Hospital, Capital Medical University, Beijing, China.
The affiliated Stomatology Hospital of Kunming medical University, Kunming, Yunnan, China.
J Periodontol. 2019 Apr;90(4):391-399. doi: 10.1002/JPER.18-0190. Epub 2018 Nov 29.
Human periodontal ligament fibroblasts (HPDLFs) represent the first line of defense against pathogens in the periodontal tissue. Porphyromonas gingivialis (P. gingivalis) has been known to be most strongly associated with periodontitis. MicroRNA (miR)-146a is involved in the inflammatory regulation of periodontitis. However, the regulatory mechanism of miR-146a on in P. gingivalis-induced inflammation response in HPDLFs was still unclear. The aim of this study was to investigate whether miR-146a plays a key role in P. gingvalis-induced inflammation responses through regulation of TRAF6 in HPDLFs.
MiR-146a expression was measured by real-time polymerase chain reaction (PCR) in HPDLFs stimulated with P. gingivalis and its lipopolysaccharide (LPS). IL-1ß, IL-6, and IL-8 were determined by enzyme-linked immunosorbent assay (ELISA) in the culture supernatants of HPDLFs after transfected with miR-146a mimic or inhibitor. Meanwhile, the expression of TRAF6 was measured by real-time PCR and Western blot. Then, we used luciferase reporter assay to detect whether miR-146a binds to the 3'-UTR of TRAF6. By using small interfering RNA (siRNA) of TRAF6, the phosphorylation of p38 mitogen-activated protein kinase (MAPK) was measured by Western blot. Finally, after inhibition of TRAF6 and p38 in HPDLFs, we analyzed the expression of miR-146a upon P. gingivalis challenge.
P. gingivalis and its LPS significantly induced miR-146a expression in HPDLFs. Overexpression of miR-146a significantly suppressed the IL-1ß, IL-6 and IL-8 secretion, TRAF6 expression, and p38 phosphorylation. In contrast, the levels of these indexes significantly increased by inhibition of miR-146a. Furthermore, MiR-146a directly binds to the 3'-UTR of TRAF6 in P. gingivalis-induced HPDLFs, but not in P. gingivalis LPS stimulation. Suppression of TRAF6 could inhibit the phosphorylation of p38. Finally, inhibition of TRAF6 and p38 significantly abolished P. gingivalis-induced miR-146a upregulation in HPDLFs.
MiR-146a contribute to negative regulation of P. gingivalis-induced proinflammatory cytokines secretion in HPDLFs though TRAF6/p38 MAPK pathway. Maintaining miR-146a homeostasis plays a key role in controlling inflammatory response in periodontal tissues.
人牙周膜成纤维细胞（HPDLF）是牙周组织中抵御病原体的第一道防线。牙龈卟啉单胞菌（P. gingivalis）与牙周炎的关系最为密切。微小 RNA（miR）-146a 参与牙周炎的炎症调控。然而，miR-146a 在 HPDLF 中对 P. gingivalis 诱导的炎症反应的调控机制尚不清楚。本研究旨在探讨 miR-146a 是否通过调节 HPDLF 中的 TRAF6 在 P. gingivalis 诱导的炎症反应中发挥关键作用。
通过实时聚合酶链反应（PCR）测量 P. gingivalis 及其脂多糖（LPS）刺激的 HPDLF 中 miR-146a 的表达。用酶联免疫吸附试验（ELISA）测量转染 miR-146a 模拟物或抑制剂后 HPDLF 培养上清液中 IL-1β、IL-6 和 IL-8 的含量。同时，用实时 PCR 和 Western blot 测量 TRAF6 的表达。然后，我们用荧光素酶报告基因检测来检测 miR-146a 是否与 TRAF6 的 3'-UTR 结合。用 TRAF6 的小干扰 RNA（siRNA）测量 p38 丝裂原活化蛋白激酶（MAPK）的磷酸化。最后，在 HPDLF 中抑制 TRAF6 和 p38 后，我们分析了 P. gingivalis 刺激后 miR-146a 的表达。
P. gingivalis 和 LPS 显著诱导 HPDLF 中 miR-146a 的表达。miR-146a 的过表达显著抑制了 IL-1β、IL-6 和 IL-8 的分泌、TRAF6 的表达和 p38 的磷酸化。相反，通过抑制 miR-146a，这些指标的水平显著增加。此外，miR-146a 可直接结合 P. gingivalis 诱导的 HPDLFs 中 TRAF6 的 3'-UTR，但不能结合 P. gingivalis LPS 刺激。抑制 TRAF6 可抑制 p38 的磷酸化。最后，抑制 TRAF6 和 p38 显著消除了 P. gingivalis 诱导的 HPDLF 中 miR-146a 的上调。
miR-146a 通过 TRAF6/p38 MAPK 通路负调控 HPDLF 中 P. gingivalis 诱导的促炎细胞因子分泌。维持 miR-146a 平衡在控制牙周组织炎症反应中起着关键作用。
