Figure 2.

Overexpression of miR-650 decreases ING4 expression and increases IL6 production induced by treatment with IL1B in MG-63 osteosarcoma cells. Following transfection with the miR-650 mimic, the cells were incubated for 48 h and treated with IL1B (10 ng/ml) to induce IL6 production. (A) mRNA and (B) protein expression of ING4 and (C) mRNA expression of IL6 were detected as shown in Fig. 1. (D) Culture medium was collected and used to measure IL6 production with an ELISA. (E and F) A total of 2.0×10⁵ cells were transfected with 200 ng pGL4.32 (luc2p/NF-κB-RE/Hygro) vector and the miR-650 mimic (20 nM). After 1 h, the cells were treated with IL1B (10 ng/ml). Firefly luciferase activity, as a reporter of NFκB activity, was normalized to Renilla activity, as a control for transfection efficiency. Protein expression of IκBα was analyzed with immunoblotting. Significant differences (*P<0.05) compared with the control were calculated with an analysis of variance. ING4, inhibitor of growth family member 4; IL6, interleukin 6; NFκB, nuclear factor κB; IκBα, nuclear factor of κ light polypeptide gene enhancer in B cells inhibitor α.