miR-378 in Differentiation Figure 6. Effect of miR-378 and 39UTR on nephronectin expression, and its biological activity in double-transfected cells. (A) Western blot for Npnt expression in the double-transfected cells. miR-378 elevated Npnt expression only when 39UTR was present. Both glycosylated and core protein bands were observed. (B) Cell differentiation as revealed by ALP staining for Npnt-transfected and Npnt+39-transfected cells. miR-378 expression inhibited differentiation in Npnt- and mock-transfected cells. However, miR-378 promoted osteoblast differentiation extensively in Npnt+39-transfected cells, implying that miR-378 interacted with the 39UTR and exerted its effect. (C) Typical results in Panel B are provided showing that the presence of the 39UTR promoted cell differentiation upon miR-378 transfection. doi:10.1371/journal.pone.0007535.g006 Npnt+39-mu produced lower levels of core protein and secreted product (Fig. 7D), suggesting that the miR-378 binding sites are important for the upregulation of nephronectin production and secretion. The 39UTR of nephronectin modulates miR-378 targeting GalNT7 The initial delay and later increase in the onset of differentiation and nodule formation seen in the Npnt+39-transfected cells as compared with the nephronectin-transfected cells suggests that the nephronectin 39UTR is significant in osteoblast differentiation. We then examined protein expression affected by the 39UTR. HEK293 cells stably transfected with nephronectin or nephronectin contain- ing the 39UTR were harvested and subjected to proteomic analysis performed by WEMB Biochem Inc (Richmond Hill, ON). A large number of proteins were altered (Table S1). Interestingly, expression of UDP-N-acetyl-alpha-D-galactosamine:polypeptide N-acetylgalactosaminyltransferase 7 (GalNT7), an enzyme known to glycosylate proteins, in the Npnt+39-transfected cells increased to 5.3-fold as compared with nephronectin-transfected cells [fold change = (reading Npnt+39 – reading nephronectin)/reading nephronectin] (Fig. 8A). PLoS ONE | www.plosone.org 7 October 2009 | Volume 4 | Issue 10 | e7535