miR-378 in Differentiation Figure 7. Mutation of the miR-378 binding sites inhibits nephronectin glycosylation. (A) Mutation was generated in the 39UTR of Npnt+39 construct as shown (Npnt+39-mu, upper panel). This impaired both putative binding sites for miR-378 (bottom). (B) Strategy of PCR showing interaction of miR-378 with Npnt 39UTR, which was abolished by sited-directed mutagenesis. (C) Typical PCR products are shown using the mature miR-378 as a primer for the PCR (arrow). The lower bands were primers used for the PCR assays. (D) Cell lysate prepared from cells transfected with Npnt+39 and the mutant Npnt+39-mu was analyzed with western blot showing decreased expression of the mutant construct. doi:10.1371/journal.pone.0007535.g007 To unravel the mechanism, we undertook computational approaches and identified a putative binding site for miR-378 at GalNT7 (Fig. 8B). The GalNT7 39UTR containing the miR-378 target site was inserted into the luciferase reporter vector producing a construct named luc-Gal-UTR (Fig. S2A). The miR-378 target site was mutated producing a mutant construct named luc-Gal-mut. Luciferase activity assays were performed to confirm the targeting. U343 cells were co-transfected with miR-378 and luc-Gal-UTR or luc-Gal-mut. Figure 8C shows that miR-378 significantly decreased luciferase activity in luc-Gal-UTR-transfected cells, but did not affect luciferase activity in the luc-Gal-mut-transfected cells, indicating targeting of GalNT7 39UTR by miR-378. To confirm the down-regulation of GalNT7 by miR-378, cell lysate was prepared from the cells transfected with miR-378 or GFP and analysed by western blot. Repression of GalNT7 was detected in the cells transfected with miR-378 as compared with the cells transfected with GFP (Fig. 8D). U87 cells transfected with GFP (Fig. 8E) or miR-378 (Fig. 8F) were subjected to immunocytometry probed with anti-GalNT7 antibody, followed by confocal microscopic examination. MiR-378-transfected cells expressed lower levels of GalNT7 than the GFP-transfected cells. These results were also confirmed in MT-1 cell line (Fig. S2B). On the other hand, we transfected MC3T3 cells, which express high level of endogenous miR-378 as demonstrated above, with anti-miR-378 expression construct or a control vector. Cell lysates were prepared and analyzed on western blot probed with anti- GalNT7 antibody. The experiments showed that GalNT7 expression was promoted by anti-miR-378 transfection (Fig. 8G), suggesting that miR-378 is sufficient in regulating GalNT7 expression. Furthermore, we transfected MC3T3 cells with siRNAs against GalNT7 designed and synthesized by Gene- Pharma (Shanghai). We found that all four siRNAs significantly silenced GalNT7 expression (Fig. S2C). The effects of these siRNAs on nephronectin post-translational modification and secretion were tested by co-transfecting the MC3T3 cells with one of these four siRNA or an RNA oligo of random sequence with nephronectin expression construct. We found that co- transfection with the siRNAs against GalNT7 produced lower levels of nephronectin in the culture medium as compared with the transfection using the RNA oligo with random sequence (Fig. 8H, arrow), suggesting decreased secretion of nephronectin product. Since glycosaminoglycan of a core protein by modifying enzymes is essential for proteoglycan secretion [57–59], these results confirm that GalNT7 plays a role in nephronectin synthesis. We PLoS ONE | www.plosone.org 8 October 2009 | Volume 4 | Issue 10 | e7535