FIGURE 2.

miR-29b activates osteogenic differentiation in osteoblasts. A and B, MC3T3 osteoblast cells were transfected with miR-29b mature miRNA oligonucleotides (50 nm), miRNA negative Control 1 (miR-C) (50 nm), or transfection reagent only (Mock) for 48 h for protein and RNA analysis. Both expression of Runx2 protein (A) and Runx2 and AlkP mRNA were up-regulated in miR-29b overexpressing cells (B). C–E, miR-29b promotes osteoblast differentiation. After transfection with 50 or 100 nm miR-29b or miRNA Control (miR-C) for 72 h, confluent MC3T3 cells were treated with α-minimum essential medium containing 10 mm β-glycerophosphate and 50 μg/ml ascorbic acid for 4 or 7 days. The cells were fixed in 2% paraformaldehyde for histochemical detection of alkaline phosphatase (C). The mRNA level of AlkP was detected by quantitative PCR normalized by GAPDH (D). Runx2 and β-actin protein (as control) were detected by Western blot (E). F, Northern blot analysis shows endogenous (miR-C lanes) and overexpressed miR-29b levels in the samples shown in E. U6RNA is the loading control.