title: The negative feedback regulation of microRNA-146a in human periodontal ligament cells after Porphyromonas gingivalis lipopolysaccharide stimulation.
abstract: OBJECTIVE
  Toll-like receptors (TLRs) pathway has been demonstrated to play an important role in periodontitis. However, the regulatory mechanism of microRNAs (miRNAs) on TLRs pathway is still unclear. Hence, this study is to explore the function of miRNA-146a in inflammatory reaction induced by Porphyromonas gingivalis lipopolysaccharide (LPS) in human periodontal ligament cells (hPDLCs).
  
  
  METHODS
  Cells were treated with 1 or 10 μg/ml P. gingivalis LPS. The expression of TLR2, TLR4 and miRNA-146a were measured by real-time polymerase chain reaction (PCR). Enzyme-linked immunosorbent assay (ELISA) was applied to detect nuclear factor (NF)-κ B p65 nuclear activity, interleukin-1β (IL-1β), IL-6, IL-8 and tumor necrosis factor-α (TNF-α). To examine the underlying mechanisms, cells were exposed to anti-TLR2/4 mAb or miRNA-146a inhibitor/mimic and evaluated by real-time PCR and ELISA.
  
  
  RESULTS
  10 μg/ml P. gingivalis LPS increased the expressions of TLR2 (3.79 ± 0.31), TLR4 (2.21 ± 0.31), and miRNA-146a (4.91 ± 0.87), NF-κ B p65 nuclear activity (6.51 ± 0.77 fold) (p < 0.05). 1 μg/ml P. gingivalis LPS induced TLR2 (3.05 ± 0.23), miRNA-146a (3.66 ± 0.83) and NF-κ B p65 nuclear activity (4.06 ± 0.78 fold) (p < 0.05), except TLR4 (1.11 ± 0.30, p > 0.05). Also, cytokines production increased (p < 0.05). The up-regulation of miRNA-146a could be blocked by anti-TLR2/4 mAb (p < 0.05). After the blockage of miRNA-146a, TLR2, TLR4, NF-κ B p65 nuclear activity and proinflammatory cytokines increased. However, after application of miRNA-146a mimic, the levels of these indexes decreased obviously (p < 0.05).
  
  
  CONCLUSIONS
  MiRNA-146a functions as a negative feedback regulator via down-regulating proinflammatory cytokine secretion and blocking TLRs signaling pathway in hPDLCs after P. gingivalis LPS stimulation.
authors.affiliation: Department of Periodontics, Institute of Stomatology, School of Dentistry, Hospital of Stomatology, Tianjin Medical University, Tianjin, 300070, China, jiangshaoyun11@126.com.
authors.forename: Shao-Yun
authors.initials: SY
authors.lastname: Jiang
authors.forename: Dong
authors.initials: D
authors.lastname: Xue
authors.forename: Yu-Feng
authors.initials: YF
authors.lastname: Xie
authors.forename: Dong-Wang
authors.initials: DW
authors.lastname: Zhu
authors.forename: Yun-Yun
authors.initials: YY
authors.lastname: Dong
authors.forename: Cong-Cong
authors.initials: CC
authors.lastname: Wei
authors.forename: Jia-Yin
authors.initials: JY
authors.lastname: Deng
mesh_terms: D000911:Antibodies, Monoclonal / Q000494:pharmacology; D002478:Cells, Cultured; D025461:Feedback, Physiological / Q000502:physiology*; D006801:Humans; D008070:Lipopolysaccharides / Q000737:chemistry / Q000494:pharmacology*; D035683:MicroRNAs / Q000037:antagonists & inhibitors / Q000235:genetics*; D016328:NF-kappa B / Q000187:drug effects; D010513:Periodontal Ligament / Q000187:drug effects* / Q000473:pathology; D016966:Porphyromonas gingivalis / Q000737:chemistry*; D013268:Stimulation, Chemical; D051195:Toll-Like Receptor 2 / Q000235:genetics; D051197:Toll-Like Receptor 4 / Q000235:genetics; D014409:Tumor Necrosis Factor-alpha / Q000037:antagonists & inhibitors / Q000187:drug effects
chemical_list: D000911:Antibodies, Monoclonal; D008070:Lipopolysaccharides; C542542:MIRN146 microRNA, human; D035683:MicroRNAs; D016328:NF-kappa B; C495342:TLR2 protein, human; C495345:TLR4 protein, human; D051195:Toll-Like Receptor 2; D051197:Toll-Like Receptor 4; D014409:Tumor Necrosis Factor-alpha