miR-378 in Differentiation Figure 2. The effect of nephronectin 39UTR on osteoblast differentiation. (A) A nephronectin construct with a fragment of the nephronectin 39UTR spanning the target sequence of miR-378 is shown (Npnt+39). Cell lysate and conditioned medium from MC3T3-E1 cells stably transfected with Npnt+39 were analyzed by western blot showing secretion of the product. (B) Cells transfected with the full-length nephronectin (Npnt-1 and Npnt- 2), nephronectin containing the 39UTR (Npnt+39-1 and Npnt+39-2), or with the control vector were grown for 10 days and stained for ALP expression. Multiple fields were examined for each construct, and stained cells were counted per field for quantitative analysis. (C) Typical micrographs of ALP expression are shown using two different phases (top and bottom panels) of the microscope. doi:10.1371/journal.pone.0007535.g002 of nephronectin during osteoblast development. Expression of endogenous miR-378 was examined in MC3T3-E1 osteoblasts under differentiation-inducing conditions. The experiments indi- cated that miR-378 expression increased significantly during cell condensation at around day 10, followed by a sharp decline during cell differentiation (Fig. 5B), suggesting involvement of miR-378 in MC3T3-E1 cell differentiation. To study the role of miR-378 in MC3T3-E1 differentiation, it is essential to ectopically provide the cells with miR-378 constantly. Current methods use chemically synthetic miRNAs for functional studies, which could only last for a few days before degradation. Since nephronectin-mediated MC3T3-E1 cell differentiation takes place at least one week after induction, it is essential to obtain stable cell lines expressing miR-378. We thus developed a method to generate a construct that stably expresses miR-378. The construct contained a H1 promoter driving expression of pre-miR- 378, an antibiotic selection marker, and a GFP for monitoring transfection efficiency [53]. Transient transfection of U87 cells with this construct indicated high efficiency of GFP expression. Using two primers, miR-378N (59agatctagggctcctgactcc) and miR- 378C (59aggccttctgactccaagtcc), which amplify the exogenously transfected miR-378 but not endogenous miR-378, we observed PLoS ONE | www.plosone.org 4 October 2009 | Volume 4 | Issue 10 | e7535