We investigated the ability of microRNAs to regulate osteoblast differentiation by controlling distinct aspects of the differentiation process. We have identified by microRNAs microarray a set of miRNAs that are differentially regulated during differentiation of the two osteoblast cell lines MC3T3-E1 and C3H10T1/2. We focused on miR-468 that is a potential regulator of Runx2.
In our microarray analysis miR-468 was expressed at a high level and was strongly down regulated during osteoblast differentiation. The down regulation of the expression of miR-468 well matches with the regulation of the expression of Runx2 that is increased during the early stage of osteoblast differentiation. After induction of osteoblast differentiation, miR-468 expression is decreasing from day 3 over time and was highly consistent with the microarray data.
One single target site for miR-468 was present in the first 500 bp of the 3′UTR of the Runx2 gene. These first 500 bp are able to mediate a 40 and 60% inhibition of the Runx2 activity in MC3T3-E1 and C3H10T1/2 cells respectively. We demonstrate that Runx2 expression was specifically down-regulated by miR-468. We showed by western blot and using reporter plasmids that miR-468 reduced Runx2 expression, inhibited its transcriptional activity and decreased expression of osteoblastic markers (SP7, BSP, type1 collagen) that are regulated by Runx2. The inhibitory effect of miR-468 on Runx2 expression was abolished when its target site on the 3′UTR of Runx2 is mutated.
All together these results indicate that Runx2 is a direct target of miR-468. Our findings suggest that miR-468 acts as Runx2 attenuators to antagonize osteoblast differentiation in pre-osteoblastic cells and possibly in BMSCs. This further indicates that a down-regulation of miR-468 may be required to release the factor Runx2 from repression and to induce osteoblast differentiation.
17 May 2014 - 20 May 2014