Fig. 3: Expression of and relationships among circGCN1L1, miR-330-3p, and TNF-α in TMJ synovial tissues and cells.

a TNF-α and its receptor (TNFR1 and TNFR2) expressions in human TMJOA synovial tissues and control tissue. N = 15 (five different samples in each group for three independent experiments). *p < 0.05. b TargetScan, miRanda, and StarBase databases were used to predicate the possible miRNAs targeting TNF. c HEK-293T cells were transfected with miR-330-3p or NC and luciferase reporter constructs containing MT or WT TNF 3′-UTR. N = 3 (one sample for three replicates). *p < 0.05. d Transfect miR-330-3p mimics or inhibitor into synoviocytes from the control patient at different concentrations (10, 20, 50, and 100 nM). N = 6 (two different samples for three independent experiments). *p < 0.05. e The mRNA levels of circGCN1L1, TNF-α, and p65 in synoviocytes from control patients stimulated with TNF-α at different doses (0, 5, 10, and 20 μg/l) for different time periods (12, 24, and 48 h) were measured by RT-qPCR and normalized to β-actin level. The expression of miR-330-3p was normalized to U6 expression. N = 6 (two different samples for three independent experiments). *p < 0.05. f Supernatants of synoviocyte from control patients transfected with miR-330-3p mimics or inhibitor were collected at different time points (1, 2, 4, 8, 16, 32, and 48 h), and the levels of TNF-α protein were detected using ELISA. N = 6 (two different samples for three independent experiments). *p < 0.05. Data are presented as mean ± S.D. Two-tailed t-test (a) and one-way ANOVA with Bonferroni test (c–f) were performed. TNF tumor necrosis factor, TNFR1 tumor necrosis factor receptor type 1, TNFR2 tumor necrosis factor receptor type 2, RT-qPCR real-time quantitative polymerase chain reaction, NC normal control, MT mutant, WT wide type, ELISA enzyme-linked immunosorbent assay.