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

Figure 3

Identification of target for LPS-induced miR-29b in OCs. BMMs were incubated with M-CSF (30 ng/ml) and RANKL (40 ng/ml) for 40 h, washed thoroughly, and incubated further with LPS (50 ng/ml) in the presence of M-CSF (30 ng/ml) for 48 h. (a) Cells were transfected with 30 nM of miR-29b mimic or con mimic in the presence of M-CSF (30 ng/ml) for 6 h. Total RNA was analyzed by qPCR to quantify the expression of BMF, PUMA, BAK1, BIM, and HRK. Expression levels with con mimic treatment were set at 1. Cell lysates were subjected to Western blot analysis with antibodies against BMF, PUMA, BAK1, BIM, HRK, and BCL-2. Antibodies against β-actin were used for normalization. (b) Without transfection, total RNA was analyzed by qPCR to quantify the expression of BMF, and cell lysates were subjected to Western blot analysis with anti-BMF Ab. (c) Total RNA and tissue lysate of tibiae from LPS-treated or vehicle-treated (V, PBS) mice were analyzed by qPCR to quantify the expression of BMF and were subjected to Western blot analysis with antibodies against BMF. (d) Cells were thoroughly washed, transfected with 30 nM of anti-miR-29b or con inh, and stimulated with LPS (50 ng/ml) in the presence of M-CSF. After 48 h, total RNA was analyzed by qPCR to quantify BMF expression. Expression levels with con inh treatment were set at 1.0. Cell lysates were subjected to Western blot analysis with anti-BMF and anti-BCL-2 Ab. (e) Cells were thoroughly washed, transfected with 50 nM of scRNA or siBMF, stimulated with LPS (50 ng/ml) in the presence of M-CSF for 48 h, and analyzed to measure TRAP-positive MNCs and annexin V-positive cells. siRNA-mediated silencing of BMF was confirmed by RT-PCR and qPCR. The Ct values of the genes were widely distributed between 17.33 and 30.92. ; ; compared with each corresponding control. Similar results were obtained from three independent experiments.

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