In this study we used a previously generated Pol-II ChIP-on-chip dataset to identify miRNAs that are differentially expressed during C2C12 myogenic versus osteogenic differentiation and thus possibly play a role in lineage specification. Overexpression of one of these miRNAs, miR-378, had no apparent effect on myogenesis while enhancing BMP2-induced osteogenesis, suggesting a positive role for this miRNA in the osteogenic differentiation program.
Our finding that miR-378 is strongly upregulated during C2C12 myogenic differentiation corresponds well to other reports demonstrating miR-378 upregulation during myogenesis and high levels of this miRNA in skeletal muscle [29, 30]. This upregulation of mature miR-378 matches an increase in Pol-II occupancy at a region located within the first intron of the Ppargc1b gene, just upstream of the miR-378 gene. This Pol-II enriched area lies adjacent to an E-box containing Myod-binding region previously shown to be important for miR-378 upregulation during myogenesis . Approximately a third of all miRNA genes, including miR-378, lie within introns of protein-coding genes. Such intronic miRNA genes are usually co-regulated with their host genes and subsequently processed to mature miRNAs after splicing of the pre-messenger RNAs . However, the mRNA expression profile of the miR-378 host gene, Ppargc1b, as assessed by our microarray analysis, does not fully correspond to the mature miR-378 expression profile; while miR-378 is upregulated during myogenesis, Ppargc1b mRNA levels do not change (data not shown). Together with the increase in Pol-II and Myod occupancy seen at sites within the first Ppargc1b intron, this might suggest that miR-378 is regulated independently from Ppargc1b and transcribed as an independent transcript, an interesting hypothesis that requires further study.
The upregulation of miR-378 specifically during C2C12 myogenic differentiation suggests a role for this miRNA in this pathway. Indeed, a study by Gagan et al. has shown that miR-378 promotes C2C12 myogenesis by targeting Msc (musculin, also known as myogenic repressor; MyoR), a repressor of myogenic differentiation that inhibits Myod activity by binding to its co-activators or binding directly to Myod target sequences . In addition, miR-378 has been shown to target mitogen-activated protein kinase 1 (Mapk1) and Bmp2, which are relevant to myoblast proliferation and differentiation, respectively, in pigs . Similarly, miR-378 has also been shown to play a role in the repression of cardiac hypertrophy by targeting Mapk1, Igf1r (insulin-like growth factor 1 receptor), Grb2 (growth factor receptor-bound protein 2) and Ksr1 (kinase suppressor of ras 1), components of the MAP kinase pathway, in rat cardiomyocytes . In contrast, we did not observe any significant effect of overexpression of miR-378 on C2C12 myogenesis, as assessed by the expression of several myogenic marker genes and Ck activity. The discrepancy with the work of Gagan et al. might be attributed to a difference in levels of miR-378 overexpression resulting from the use of different overexpression methods (transient lipofectamine transfection versus our stable lentiviral-transduced cell lines). Alternatively, since the positive effects on myogenesis seen by Gagan et al. were at early time points (day 1 and day 3), it is possible that overexpression of miR-378 merely accelerates myogenesis and similar maximal levels have been reached by both miR-378 overexpressing and control cells at the later time points that we investigated (day 3 and day 6). Our observation that miR-378 overexpression has no apparent effect on myogenesis does not rule out that it plays a role in this process; most likely, endogenous levels of this miRNA are sufficient for its biological function, and overexpression has no additional effect on myogenic markers.
It would, however, still be interesting to take a closer look at the genes that are downregulated by miR-378 overexpression in undifferentiated myoblasts (day 0 time point); genes that are downregulated during C2C12 myogenesis, and significantly downregulated by miR-378 overexpression in myoblasts, such as for example (data not shown) Fgf7 (fibroblast growth factor 7), Crlf1 (cytokine receptor-like factor 1), Ereg (epiregulin) and Cck (cholecystokinin), are potential targets of this miRNA and interesting candidates for further study on the role of miR-378 in myogenesis. Unfortunately, we did not observe a significant effect of miR-378 overexpression on mRNA levels of its published targets Msc, Mapk1, Igf1r, Grb2 and Ksr1[29, 30, 32]. This does not contradict the findings in these publications, since it is possible that miR-378 exerts its effect on these targets at the level of protein translation and not by inducing mRNA degradation (see below).
Besides its putative role in myogenesis, we clearly demonstrate an effect of miR-378 on C2C12 bone differentiation. Our observation that miR-378 overexpression promotes C2C12 osteogenesis in the presence of BMP2, as assessed by Alp activity, calcium deposition and expression of osteogenic marker genes, was surprising considering the lack of changes in its expression level during BMP2-induced osteogenic differentiation. Since this effect of miR-378 overexpression is limited only to BMP2-treated cells, we believe that miR-378 on its own is not a major determinant of the osteogenic cell fate, but more likely plays a role in fine-tuning osteogenic gene expression within the BMP2-induced cellular environment.
A role for miR-378 in modulating osteogenic differentiation has previously been described by Kahai et al. in the context of a nephronectin (Npnt)-3’UTR overexpressing MC3T3-E1 osteo-progenitor cell line . Npnt is an extracellular matrix protein that, when overexpressed, enhances MC3T3-E1 osteoblast differentiation. Npnt secretion depends on its glycosylation by glycosylation-associated enzymes including Galnt7 (UDP-N-acetyl-alpha-D-galactosamine:polypeptide N-acetylgalactosaminyltransferase 7). The 3’UTR of both Npnt and Galnt7 contain a miR-378 binding site. Kahai et al. demonstrated that, during late stages of MC3T3-E1 development (in the presence of ascorbic acid, β-glycerophosphate and dexamethasone), stable cell lines overexpressing Npnt containing its 3’UTR (Npnt-3’UTR) have a higher rate of osteoblast differentiation and bone nodule formation than cell lines overexpressing Npnt without its 3’UTR; this is further enhanced by co-transfection with miR-378. Interestingly, co-transfection of Npnt-3’UTR with miR-378 enhanced production of Npnt and promoted Npnt glycosylation. It was suggested that interaction of the Npnt 3’UTR with miR-378 sequestered this miRNA away from Galnt7, leading to enhanced Galnt7 activity, a subsequent increase in Npnt glycosylation and secretion and, as a result, a higher rate of osteogenesis. In addition, it was proposed that binding of miR-378 to the Npnt 3’UTR resulted in preventing access of other miRNAs, thereby protecting the Npnt mRNA from post-transcriptional regulation and resulting in the observed increase in Npnt synthesis . In line with these findings, we observed significantly higher levels of Npnt mRNA in our C2C12-pMirn378 versus control cells after 6 days of osteogenic differentiation (data not shown). It would therefore be interesting to determine whether a similar Npnt/Galnt7 –mediated mechanism might also play a role in the effect miR-378 overexpression has on BMP2-induced C2C12 osteogenesis. However, the positive effect of miR-378 overexpression on MC3T3-E1 osteoblast differentiation described by Kahai et al. was only observed when co-transfected with Npnt-3’UTR and only during later stages of development. In fact, stable transfection of MC3T3-E1 cells with miR-378 alone actually inhibited osteogenesis . This is in direct contrast with our observation in BMP2-induced C2C12 cells and indicates that the effect of miR-378 may depend on the osteogenic model system used and/or the signaling pathways involved in inducing differentiation: for example, it is conceivable that miR-378 acts specifically on the BMP2 signaling pathway to positively reinforce the BMP2 effect on our C2C12 model system, while this mechanism might not play a role in the differentiation of MC3T3-E1 cells by Kahai et al., which occurred in the absence of BMP2. Further exploration of the mechanism underlying the positive effect of miR-378 on our BMP2-induced C2C12 system may help shed light on this issue.
We were as yet unable to identify the genes that are directly targeted by miR-378 during BMP2-induced C2C12 osteogenesis. Most effects seen in our mRNA microarray analysis are likely to be secondary to the initial effect of miR-378, making it difficult to identify its direct target(s). Given the overall positive effect of miR-378 on the expression of osteogenic markers, and negative effect on myogenic markers, we expected the initial targeting event to take place early during the differentiation process. To identify direct miR-378 targets, we therefore selected genes a) that were downregulated by miR-378 overexpression early (day 0) and consistently during osteogenesis, b) that contained a predicted miR-378 target site in their 3’UTR and c) that were known to play a role in the regulation of osteoblast differentiation. This led to the selection of Grem1, Wnt5a and Wnt10a as putative targets. Grem1 is a secreted glycoprotein that binds BMP2 and prevents BMP2 signaling and activity in cells of the osteoblast lineage . Targeting of Grem1 by miR-378 could thus increase the levels of BMP2 available for inducing osteogenesis. Wnts are a family of 19 secreted glycoproteins that activate their cell surface receptors to induce specific intracellular signaling cascades controlling gene expression and play a critical role in embryonic development, postnatal development and adult tissue homeostasis . Wnt signaling regulates cellular processes including proliferation, differentiation, and apoptosis through β-catenin-dependent canonical and β-catenin-independent non-canonical pathways and has been shown to play an important role in bone formation . Wnt5a has been found to be the most dominant Wnt expressed during osteogenesis of human mesenchymal stem cells (hMSCs) both in vitro and in vivo and Wnt5a signaling has been shown to be important for BMP2-mediated osteogenesis in MC3T3-E1 cells, though the exact signaling pathways involved remain unclear . Wnt10a has also been shown to stimulate osteogenesis . Given their important role in osteoblast formation, it was interesting to determine whether these Wnts were indeed targeted by miR-378 and subsequently how this could relate to the observed increase in osteogenic differentiation. However, our luciferase-reporter assay demonstrated that miR-378 did not directly target the 3’UTR of any of these selected candidates and further work is thus required to identify the mechanism by which miR-378 exerts its effect.
The imperfect complementarity that may exist between a miRNA and its target, the possibility for combinatorial regulation that depends on the presence of other miRNAs to observe an effect, and the various mechanisms by which miRNAs may act, pose a great challenge common to all studies of miRNA function. In our approach we assumed that miR-378 exerts its effect by mRNA destabilization and/or degradation, resulting in a decrease in mRNA levels of its target(s). It is possible, however, for a miRNA to have only very subtle effects on (multiple) targets that cannot be observed as a change in mRNA levels of its direct targets, or to affect protein translation without affecting mRNA levels [14, 40]. In addition, miRNAs have been shown to be able to affect mRNA levels of their target genes via alternative mechanisms than binding to their 3’UTR, which would not be detected using a luciferase-3’UTR reporter assay. For instance, it has been shown that miRNAs can affect gene transcription by inducing histone modifications at target promoter sites . Interestingly, a study by Gerin et al. has shown that miR-378 can specifically increase the transcriptional activity of Cebpa and Cebpb (CCAAT/enhancer binding protein, alpha and beta) on adipocyte gene promoters, though it could not be excluded that this was an indirect effect through e.g. inhibition of a co-repressor . Given the role of Cebpb in synergizing with Runx2 to regulate bone-specific gene expression , it would be very interesting to investigate whether a similar mechanism underlies the effect of miR-378 on BMP2-induced osteogenesis.
So far, we have attributed the effects seen in C2C12 cells transduced with the miR-378 precursor expression construct to mature miR-378, the 3p strand of the precursor miRNA. However, it should be noted that these cells also overexpress miR-378*, the less-abundant 5p strand. Although present at 10–30 times lower levels than miR-378 (data not shown), it cannot be excluded that the effects seen are (in part) the result of miR-378* overexpression, and it would thus be interesting to also search for putative miR-378* targets within the group of affected genes.
In this study, we used our previous Pol-II ChIP-on-chip dataset to identify lineage-specific miRNA expression. Since the probes on the arrays used for this dataset were restricted to promoter sequences of protein coding genes, the results of this approach do not represent the full picture of Pol-II occupancy at all miRNA gene promoters in the genome. This could explain why several miRNAs known to be specifically upregulated during myogenesis, the so-called myomiRs (miR-1/206 and miR-133 families) , were not identified. However, our approach did provide a first means to identify several miRNAs with differential Pol-II occupancy during myogenic versus osteogenic differentiation. Most of these miRNAs, including miR-21, miR-34b/c, miR-99b, miR-365 and miR-675, have an as yet unknown role in these differentiation pathways and are thus attractive candidates for further investigation.