Fig 2: Bioinformatics prediction of upstream miRNA of COX-2, and miR-758 expression in synovium tissues and synovial fluid. (A) miR-758-3p can potentially interact with COX-2. Reverse transcription-quantitative PCR analysis was performed to detect miR-758-3p expression in (B) synovium tissues and (C) synovial fluid from patients with acute knee trauma and KOA, respectively. *P<0.05, **P<0.01 vs. control group. miRNA/miR, microRNA; COX-2, cyclooxygenase-2; KOA, knee osteoarthritis.

Fig 3: Effect of the EEAKSs on dental plaque bacterial-LPS-induced prostaglandin E2 and cyclooxygenase-2. (A) The effect of bacterial LPS on PGE2 and COX-2 levels in immortalized gingival fibroblast (IGF), immortalized human oral keratinocyte (IHOK), and RAW264.7 macrophage. Three DPB-LPS (DPB-LPS-1, DPB-LPS-2, DPB-LPS-3) were prepared from dental plaques and each DPB-LPS (1 µg/mL) was treated to media for 24 h and PGE2 and COX-ELISA analysis was performed. PG-LPS was also extracted from P. gingivalis. Moreover, 1 µg/mL PG-LPS was treated to media for 24 h and ELISA analysis was also performed. The data are expressed as the mean ± standard error of the mean (SEM). # p < 0.01, * p < 0.001 vs. without bacterial LPS control medium from each cell line. (B) The effect of EEAKSs on bacterial LPS-induced PGE2 and COX-2 expression. EEAKSs (10 µg/mL) were treated in the media with DPB-LPS or PG-LPS (1 µg/mL) and the PGE2 and COX-2 level was estimated using the ELISA kit. ** p < 0.001 vs. only DPB-LPS treated cells, # p < 0.05, * p < 0.01 vs. only PG-LPS treated cells. (+), add; (-), no add. (C) The effect of EEAKSs on human cell growth. Immortalized human oral keratinocytes (IHOKs), immortalized human gingival fibroblasts (IGFs), and RAW264.7 macrophage cells were treated with indicated concentration of EEAKSs for 24 h and MTT assay was performed. DMSO was treated as the assay control.

Fig 4: Relative luciferase activity of 293T cells co-transfected with agomiR-758-3p and wild-type or mutant 3'-untranslated region of the COX-2 gene. The dual-luciferase reporter assay was performed to confirm the interaction between miR-758-3p and COX-2 mRNA. **P<0.01 vs. NC group. miR, microRNA; COX-2, cyclooxygenase-2; NC, negative control.

Fig 5: Effect of miR-758-3p on COX-2 expression and the proliferation of synovial cells. (A) miR-758-3p expression in synovial cells transfected with agomiR-NC or agomiR-758-3p. Reverse transcription-quantitative PCR and western blot analyses were performed to detect COX-2 (B) mRNA and (C) protein expression levels in synovial cells transfected with agomiR-NC or agomiR-758-3p, respectively. ELISA was performed to detect (D) COX-2 and (E) PGE2 protein expression levels in the supernatant of synovial cells transfected with agomiR-NC or agomiR-758-3p, respectively. (F) The MTT assay was performed to assess the proliferation of synovial cells transfected with agomiR-NC or agomiR-758-3p. *P<0.05, **P<0.01 vs. agomiR-NC group. miR, microRNA; COX-2, cyclooxygenase-2; NC, negative control; PGE2, prostaglandin E2.