MicroRNA-29b Enhances Osteoclast Survival by Targeting BCL-2-Modifying Factor after Lipopolysaccharide Stimulation

Figure 1

LPS upregulates the expression of miR-29b. Tibiae from LPS-treated or vehicle-treated (V, PBS) mice were analyzed by qPCR to quantify the expression of miR-29b (a) and TNF-α, IL-1β, and IL-6 (d) (5 mg/kg/d LPS: ; V: ). BMMs were prepared and incubated with M-CSF (30 ng/ml) and RANKL (40 ng/ml) for 40 h, washed thoroughly, incubated further with LPS (50 ng/ml) in the presence of M-CSF (30 ng/ml) for the indicated times, and then analyzed by qPCR to quantify the expression of miR-29b (b), TNF-α, IL-1β, and IL-6 (e). (c) To determine TRAP-positive OCs in vivo, mouse femora were excised free of a soft tissue and decalcified in EDTA. Representative histological sections of the distal femoral metaphysis of mice from each of the two groups were stained for TRAP to identify OCs (indicated by the arrowhead) to calculate OC.N/BS. Scale bar: 50 μm in the representative photos. (f) BMMs were prepared and incubated with M-CSF (30 ng/ml) and RANKL (40 ng/ml) for 40 h, washed thoroughly, incubated further with LPS (50 ng/ml) in the presence of M-CSF (30 ng/ml) and each of neutralizing Abs (anti-TNF-α Ab, 0.5 μg/ml; anti-IL-1β Ab, 2 μg/ml; and anti-IL-6 Ab, 2 μg/ml) or its corresponding IgG (mouse IgG) for the indicated times, and then analyzed by qPCR to quantify the expression of miR-29b. (g) Primary osteoblasts were treated with LPS (50 ng/ml) for the indicated time points and analyzed by qPCR to quantify miR-29b expression. The Ct values of the genes were widely distributed between 17.15 and 28.41 ; compared with each corresponding V-treated group. Similar results were obtained in three independent experiments.

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