miR-378 in Differentiation Figure 4. The 39UTR of nephronectin promoted nephronectin protein glycosylation. (A) Cell lysate from the two MC3T3-E1 stable cells lines (over-expressing the nephronectin protein with and without the 39UTR) was prepared from 48 hour cultures and 10 day cultures. The lysate and conditioned medium was analyzed on western blot probed with the 4B6 antibody. The bands representing glycosylated nephronectin and the nephronectin core protein have been marked separately. (B) Conditioned medium was treated with different de- glycosylation enzymes to reduce the observed molecular size in western blot to confirm nephronectin glycosylation. The cells trans- fected with either the full-length nephronectin or just the MAM domain possessed potential O-glycosylation, which accounts for the significant size-difference in Fig. 4A. Filled arrows indicate the de-O-glycosylated product; and open arrows show the size of core protein. doi:10.1371/journal.pone.0007535.g004 increase in the levels of nephronectin glycosylation, but also observed an increased level of nephronectin core protein in MC3T3-E1 osteoblasts stably transfected with Npnt+39 and miR- 378 (Fig. 6A). This result was then confirmed using multiple transient and stable transfections of HEK293 cells (data not shown). To better understand the increase in nephronectin production by miR-378 expression, we performed differentiation experiments in the same combination as those performed in the western blot analyses. Upon miR-378 transfection, differentiation in nephro- nectin-transfected cells was inhibited in a similar manner as in the mock-transfected cells (Fig. 6B). Co-expression of miR-378 with Npnt+39 significantly promoted cell differentiation, consistent with enhanced nephronectin synthesis, glycosylation, and product secretion. The Npnt+39-transfected cells not only induced a greater number of cells to undergo differentiation, but also produced stronger ALP staining seen by the red color (Fig. 6C). To corroborate these results, we carried out proliferation experiments on the MC3T3-E1 cells stably co-transfected with miR-378/GFP and nephronection/Npnt+39. The experiments showed that co- transfection of miR-378 and nephronectin increased cell prolifer- ation (Fig. S1C), while co-transfection of miR-378 and Npnt+39 Figure 5. Targeting of nephronectin with miR-378. (A) Compu- tational algorithms were used to predict the miRNA that binds to a target sequence in the 39-UTR of nephronectin. miR-378 was found to have the strongest binding to the 39UTR of nephronectin from nucleotides 1902–1923. (B) Expression of endogenous miR-378 in MC3T3-E1 cells under differentiation-inducing conditions was measured by real-time PCR. (C) Left, MC3T3-E1 cells stably transfected with the miR-378 construct were grown in parallel with cells transfected with the control vector for 10 days and stained for ALP activity. Multiple fields were examined for each construct, and stained cells were counted per field for quantitative analysis. Right, typical micrographs of ALP expression are shown in two separate stable clones. doi:10.1371/journal.pone.0007535.g005 inhibited cell proliferation (Fig. S1D) as compared with the control groups. To test the significance of the putative binding sites on nephronectin expression, we generated mutations on both binding sites to disrupt the base pairing structures between miR-378 and nephronectin 39UTR (Fig. 7A). PCR assays were used to test the potential interactions as previously described [56]. Mature miR- 378 was used as a primer in the PCR paring with another primer located in the vector. A plasmid (39UTR) containing a small fragment of Npnt coding sequence and the 39UTR and a plasmid (39UTR-mu) containing the mutation as shown in Fig. Fig. 7A served as PCR-templates (Fig. 7B). PCR product was produced with 39UTR as a template but could not be detected with 39UTR- mu as a template (Fig. 7C). Cells were also transfected with nephronectin containing the 39UTR (Npnt+39) or the mutant 39UTR (Npnt+39-mu). Expression of the mutant construct PLoS ONE | www.plosone.org 6 October 2009 | Volume 4 | Issue 10 | e7535