miR-378 in Differentiation Figure 3. Nephronectin 39UTR promotes nodule formation. (A) Time course analysis showing the different effects of Npnt and Npnt+39 transfection on MC3T3-E1 differentiation: early stages, Npnt.Npnt+39; late stages, Npnt,Npnt+39. (B) Multiple fields were examined for each construct, and stained cells were counted per field for quantitative analysis. (C) The cell lines were cultured under nodule-inducing conditions over the three different phases of osteoblast development. Magnified images of bone nodules formed on days 52 and 56 are shown after silver nitrate staining for the detection of bone nodules. (D) Photographs of the whole plates showing bone nodules on day 60 are shown in the lower panel. The total number of bone nodules was statistically analyzed and have been represented in the upper panel. ** p,0.01. doi:10.1371/journal.pone.0007535.g003 expression of pre-miR-378 (data not shown). This pre-miR-378 was processed to produce mature miR-378 as expected (data not shown). This expression system has been successfully used in our previous studies [53,54]. MC3T3-E1 cells were stably transfected with the miR-378 construct or a control vector expressing GFP alone. Two sets of clones were cultured in parallel under nodule-inducing conditions. The cells were cultured for 6 days and then stained for the expression of ALP to identify differentiating cells. We observed minimal ALP expression in the MC3T3-E1 cells over-expressing miR-378 as compared with the two empty vector-transfected cell lines. Typical photographs are shown (Fig. 5C, right). Using a different phase on the same microscope, only the cells stained for ALP were visualized and counted for quantification of ALP expression (Fig. 5C, left). Based on the significant difference in ALP expression between MC3T3-E1 cells transfected with the empty vector and those expressing miR-378, our results indicate that over-expression of miR-378 inhibits osteoblast differentiation. Up regulation of nephronectin expression by miR-378 It has been known that miRNAs repress mRNA translation by targeting the 39UTR. We performed experiments to test whether co-transfection of Npnt+39 with miR-378 would reduce ectopic nephronectin expression. Unexpectedly, we not only detected an PLoS ONE | www.plosone.org 5 October 2009 | Volume 4 | Issue 10 | e7535