P53 regulation of osteoblast differentiation is mediated through specific microRNAs

Abstract

In order to understand the role of the p53 tumor suppressor gene in microRNA expression during osteoblast differentiation, we used a screen to identify microRNAs that were altered in a p53-dependent manner. MicroRNAs from MC3T3-E1 preosteoblasts were isolated from day 0 (undifferentiated) and day 4 (differentiating) and compared to a p53 deficient MC3T3-E1 line treated similarly. Overall, one fourth of all the microRNAs tested showed a reduction of 0.6 fold, and a similar number of them were increased 1.7 fold with differentiation. P53 deficiency caused 40% reduction in expression of microRNAs in differentiating cells, while a small percent (0.03%) showed an increase. Changes in microRNAs were validated using real-time PCR and two microRNAs were selected for further analysis (miR-34b and miR-140). These two microRNAs were increased significantly during differentiation but showed a dramatic reduction in expression in a p53 deficient state. Stable expression of miR-34b and miR-140 in MC3T3-E1 cells resulted in decreases in cell proliferation rates when compared to control cells. There was a 4-fold increase in p53 levels with miR-34b expression and a less dramatic increase with miR-140. Putative target binding sites for bone specific transcription factors, Runx2 and Osterix, were found for miR-34b, while Runx2, beta catenin and type 1 collagen were found to be miR-140 targets. Western blot analyses and functional assays for the transcription factors Runx2, Osterix and Beta-catenin confirmed microRNA specific interactions. These studies provide evidence that p53 mediated regulation of osteoblast differentiation can also occur through specific microRNAs such as miR-34b and miR-140 that also directly target important bone specific genes.

Keywords

• p53
• Osteoblast differentiation
• microRNA
• Bone