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. 2021 Aug 27;22(5):1227. doi: 10.3892/etm.2021.10661

Figure 3.

Figure 3

miR-27a-3p negatively regulates TLR5 expression by target binding. (A) Schematic diagram demonstrating the potential miR-27a-3p responsive elements in wild-type and mutant of TLR5 3'-UTR (TLR5 3'-UTR-WT and TLR5 3'-UTR-MUT). (B and C) Dual-luciferase reporter assay detected the luciferase of pGL4-TLR5 3'-UTR-WT/MUT vectors in RASFs co-transfected with miR-27a-3p/NC or miR-27a-3p/NC inhibitors (in-miR-27a-3p/NC). *P<0.05, compared with the miR-NC group or in-miR-NC group. (D) RNA pull-down assay measured the TLR5 level in RASFs transfected with biotin-labelled miR-27a-3p (Bio-miR-27a-3p) or its mutant (Bio-miR-27a-3p MUT). *P<0.05, compared with biotin-labelled miR-NC (Bio-miR-NC). (E) Reverse transcription-quantitative polymerase chain reaction detected the TLR5 mRNA level in RA synovial tissue (n=27) and Control synovial tissue (n=27). *P<0.05, compared with the Control. (F) Western blotting investigated the TLR5 protein level in RASFs and CSFs. *P<0.05, compared with CSFs. (G) Western blotting investigated the TLR5 protein level in RASFs transfected with miR-27a-3p/NC or in-miR-27a-3p/NC. *P<0.05, compared with miR-NC or in-miR-NC. miR, microRNA; TLR5, toll-like receptor 5; UTR, untranslated region; WT, wild-type; MUT, mutant; RASFs, RA synovial fibroblasts; NC, negative control; in, inhibitor; RA, rheumatoid arthritis; CSFs, control synovial fibroblasts.