Figure 4
MyD88 is a direct target of miR-3085-3p. (A) SW1353 cells were transiently transfected with the MyD88 3′UTR subcloned into the pmirGLO vector (wild-type) or a construct with miR-3085-3p seed sites mutated (mutant) with miR-3085-3p mimic or non-targeting control (NTC) for 24 h. Firefly luciferase relative light units were normalised to Renilla relative light units to give overall relative light units. (B) Primary human articular chondrocytes were grown in monolayer culture and transiently transfected with miR-3085-3p or a non-targeting control (NTC) for 48 h; MyD88 was measured by (B) qRT-PCR and (C) SW1353 cells were transfected with miR-3085-3p or a non-targeting control (NTC) for 48 h, MyD88 protein level were measured by western blot. (D) Primary human articular chondrocytes were grown in monolayer culture and transiently transfected with MyD88 siRNA or a non-targeting control (NTC) for 24 h prior to stimulation with IL-1β (5 ng/ml) or control for 6 h; MMP13 was measured by qRT-PCR. Mean + /- SEM, n = 3. Student’s t-test; *p < 0.05; **p < 0.01; ***p < 0.001. (E) SW1353 cells were grown in monolayer culture and transiently transfected with MyD88 siRNA or a non-targeting control (NTC) for 24 h prior to stimulation with IL-1β (5 ng/ml) or control for 30 min prior to western blot analysis. Full-length blots are presented in Supplementary data; N.B. the full blot for MyD88 in (C) is over saturated.
