FIGURE 5.

miR-29b regulates collagen expression in differentiated osteoblasts. A and B, miR-29b has no effect on collagen gene expression in early osteoblast differentiation. MC3T3 cells were transfected with 50 or 100 nm miR-29b RNA mimics or miRNA Control (miR-C) and cultured with differentiation-inducing medium described as in Fig. 2C. Col1a1 protein was monitored by Western blot (A), and mRNA levels detected by quantitative PCR normalized by GAPDH (B). C, mRNA expression levels of Col1a1, Col5a3, and Col4a2 during MC3T3 osteogenic differentiation. Differentiation was initiated 3 days after plating cells (day 0). The cells were harvested at indicated time points for quantitative RT-PCR normalized to GAPDH. D, reciprocal expression of miR-29b and COL Type I (days 21–28); miR-29b quantitated from Northern blot analyses; total collagen measured from densitometry of Western blots; COL Type I mRNA determined by quantitative PCR.