was evident in bone nodule formation as well: after an initial delay in bone nodule formation, the Npnt+39-transfected cells produced significantly higher densities of bone nodules than the nephro- nectin-transfected cells. We hypothesized that over-expression of the 39UTR might have attracted certain miRNAs binding to this fragment and, thereby making them unavailable for targeting some glycosylation- associated enzymes, which are essential for glycosylation of nephronectin. This in turn would increase the activity of glycosylation-associated enzymes, leading to produce more glycosylated nephronectin. Consequently, more nephronectin is secreted to promote osteoblast differentiation. To test this hypothesis, we investigated the differences in glycosylation of the nephronectin core protein in the presence and absence of the nephronectin 39UTR using the two stable cell lines transfected with nephronectin or Npnt+39. Western blot analysis showed that the Npnt+39-transfected cells had a much higher level of glycosylated nephronectin compared with the nephronectin- transfected cells. These results therefore support our hypothesis. The post-transcriptional regulation of nephronectin by miRNA was then examined. The use of bioinformatics algorithms showed that the 39UTR of nephronectin was a potential target of miRNA miR-378. To examine the effect of miR-378, we generated a construct expressing dual fragments of pre-miR-378. Stable over- expression of the miR-378 construct strongly inhibited MC3T3- E1 differentiation. We have previously demonstrated that miR- 378 enhances cell survival and tumor growth [53]. The report presented here that miR-378 inhibits cell differentiation is in consistent with our previous finding. To examine the probability that translation of nephronectin was repressed by miR-378, we performed a number of co-transfection experiments including transient transfection and stable transfec- tion of Npnt+39 with the miR-378 construct in MC3T3-E1 osteoblasts and Hek293 cells. Unexpectedly, co-transfection of Npnt+39 with miR-378 not only promoted nephronectin glyco- sylation, but also enhanced production of nephronectin, perhaps through enhanced translation, while miR-378 showed little effect on nephronectin (no 39UTR) expression, as expected. We could explain this phenomenon by the fact that nephronectin might not be a true target of miR-378 although the sequence in its 39UTR matches well with the sequence of miR-378. The ability to reliably identify the targets of a given miRNA has been known to be a major problem in the miRNA field. Most of the computational predictions of miRNA targets focus on base pairing between the miRNA ‘‘seed’’ region and its target mRNA. Despite the presence of the same target sites, it remains unclear why miR-378 was able to repress luciferase activity while the nephronectin 39UTR when placed in the luciferase construct was not able to repress nephronectin expression. Zhao and co-workers have presented evidence for target selectivity which depends on the free energy surrounding the target site [60]. They proposed that a stable environment with lower free energy may not be accessible to the miRNA silencing complex as compared with the one with higher free energy (or unstable environment). We have also found that the secondary structure of the common miRNA binding site and its flanking regions is normally located in an unstable region with a multi-branching loop-like RNA structure [8]. How the average free energy flanking regions in a miRNA binding site affect targeting by a particular miRNA as well as the efficiencies of miRNA repression of gene expression await further investigation. Bearing this in mind, we propose that binding of miR-378 to the 39UTR of nephronectin not only did not exert direct repression of nephronectin synthesis, but it also created a secondary structure which is less accessible to other miRNAs which normally target miR-378 in Differentiation nephronectin and regulate its expression. This space hindrance resulted in increased synthesis of nephronectin. Furthermore, the binding interaction of miR-378 with nephronectin 39UTR would reduce free miR-378 for targeting nephronectin glycosylation- associated enzyme(s). As a consequence, the enzyme(s) expression would be enhanced resulting in increased nephronectin glycosyl- ation and product secretion. We have previously demonstrated that glycosylation is an essential step for product secretion [57–59]. Concurrent with increased nephronectin protein synthesis, glycosylation, and secretion was the inhibition of proliferation and promotion of differentiation in MC3T3-E1 cells that were co- transfected with miR-378 and the Npnt+39 construct. To test whether some glycosylation-associated enzymes were affected, total proteins in MC3T3-E1 cells stably transfected with nephronectin or Npnt+39 were subjected to proteomic and computational analyses. Indeed, GalNT7, an enzyme known to glycosylate proteins, was up-regulated in Npnt+39-transfected cells and was downregulated by miR-378 expression. These results explained why expression of nephronectin 39UTR was able to promote MC3T3-E1 differentiation and bone nodule formation in the late stages. However, it is not clear why these phenomena were not seen in the early stages. It is possible that the over-expressed nephronectin, although not secreted due to lack of glycosylation, interfered with some signalling molecules inside the cells, playing a role in cell differentiation. This awaits further investigation. Taken together, we have reported a mechanism by which a miRNA can function in promoting protein expression in two ways. It can be achieved by binding to the 39UTR and resulting in space hindrance preventing easy access of other miRNAs, thereby protecting the mRNA from being regulated post-transcriptionally. Secondly, a miRNA can promote protein expression by binding to the 39UTR and freeing the true target of the miRNA, thereby increasing the levels of the target, which in turn enhances protein modification and secretion. Materials and Methods Materials Dulbecco’s modified Eagle’s medium (DMEM), alpha modified Eagle’s medium (AMEM), fetal bovine serum (FBS), trypsin/ EDTA, Lipofectamine 2000, geneticin (G418), hygromycin were purchased from Wisent Inc. T4 DNA ligase, platinum Pfx DNA polymerase, SuperScript II reverse transcriptase, DH5a, and tissue culture supplies were purchased from Invitrogen. Restriction enzymes were purchased from New England Biolabs. RedTaq DNA polymerase, anti-actin and HRP-conjugated secondary antibodies, de-glycosylation enzymes (PNGase F, O-glycosidase, sialidase, and keratanase), horseradish peroxidase-conjugated goat anti-mouse IgG secondary antibody, and other general chemicals were purchased from Sigma. Tissue culture plates were purchased from Sarstedt (Montreal, QC, Canada). ECL Western blot detection kit was from Amersham Life Science (Baie d’Urfe´, QC, Canada). The MC3T3-E1 cell line was obtained from the American Type Culture Collection (Rockville, MD). Cell culture MC3T3-E1 and Hek293 cells were maintained in alpha-MEM and DMEM, respectively, supplemented with 10 (v/v) % fetal bovine serum, 100 units/ml penicillin, and 100 mg/ml streptomycin. Plasmid transfection was done using Lipofectamine 2000 according to the manufacturer’s instruction. Transfected cells were selected under growth medium with 1 mg/ml G418 and were stable for more than one month before experiments. PLoS ONE | www.plosone.org 11 October 2009 | Volume 4 | Issue 10 | e7535