Fig. 4

Identification of IL-6 as a target of miR-653-5p. a The secondary structure and the positional entropy for each position of miR-653-5p. b Venn diagram displaying miR-653-5p computationally predicted to target IL-6 by different algorithms. c Schematic representation of IL-6 3′ UTR demonstrating putative miRNA target site, luciferase activities of wild type (WT-UTR) and mutant (MUT-UTR) constructs. d, e The wild or mutant type IL-6 3′ UTR reporter plasmid was co-transfected with mimic-NC or miR-653-5p mimic into human primary chondrocytes and C28/I2 cells. 48 h after transfection, luciferase activity was measured. n = 3 replicates per group. f Western blot assays showed that overexpression of miR-653-5p could decrease IL-6 protein level, while inhibition of miR-653-5p could increase IL-6 protein level in both primary human chondrocytes and C28/I2 cells. n = 3 replicates per group. g Compared with normal controls, IL-6 protein expression was increased in human primary OA chondrocytes and C28/I2 OA cells using the immunofluorescence analysis. n = 3 replicates per group. Scale bar = 50 μm. P values are from one-way ANOVA test by Tukey’s post hoc test (d, e, f) and two-tailed unpaired Student’s t-test (g). miRNA, microRNA; IL-6, interleukin 6; OA, osteoarthritis; NC, normal control