Differentiation of NS and ES cells in vitro has attracted wide interest as an experimental system for investigating specific molecular pathways of cell development. Here, we have demonstrated neuronal and glial conversion of mouse NS cells. In this model, the neuronal population is formed mainly during the first 2 days, as indicated by the upregulation of β-III tubulin. Nevertheless, commitment to mature neurons was confirmed by the expression of NeuN at 6 days, which indicates the presence of immature postmitotic neurons. Astrocytic population peaked at 6 days, and this was maintained throughout differentiation.
Functional studies of apoptosis-associated miRNAs have focused intensely on cancer research. However, our results demonstrate that specific miRNAs regulating pro-and antiapoptotic genes may also participate in differentiation processes. The involvement of miR-16 in cell differentiation has not been previously reported. Indeed, miR-16 expression was markedly increased throughout mouse, rat and human neural differentiation. Nevertheless, the onset of miR-16 expression during mouse NS cell differentiation was not associated with the appearance of any specific cell type. miR-16 was shown to be implicated in cell cycle regulation, as well as apoptosis induction by targeting Bcl-2 [25
]. Apoptosis is not markedly regulated during mouse NS cell differentiation, and Bcl-2 expression is increased (data not shown). Therefore, a possible role for miR-16 in the context of differentiation may be associated with cell cycle control in downregulating the proliferation potential of differentiating cells. During differentiation of neural precursors, a tightly coordinated regulation of cell cycle exit is crucial for the generation of appropriate number of neurons and proper wiring of neuronal circuits [50
]. Nevertheless, here-to-fore unrecognized miR-16 targets may exert distinct, yet crucial functions during cell differentiation.
The let-7 family consists of eleven very closely related genes [51
]. They are highly conserved in animals from worms to humans, and their expression increases after differentiation and maturation of tissues [52
]. In addition to regulating apoptosis by targeting caspase-3 [10
], it was also demonstrated that let-7 family members regulate RAS and HMGA2 oncogene through the 3'UTR [53
]. In the present study, let-7a expression was shown to be cyclic during mouse NS cells differentiation, and corresponded to the onset of neurogenesis and gliogenesis. This suggests that let-7a expression is associated with a general mechanism of differentiation rather than differentiation of specific cell types. Our results are supported by the previous report showing that let-7a is a critical regulator of neuronal differentiation [12
]. In fact, increased let-7a expression during ES cells, PC12 and NT2N differentiation also underscores the important role of let-7a during general differentiation. Additional studies are warranted to evaluate the mechanism(s) by which let-7a regulates cell differentiation. Importantly, although highly modulated during cell differentiation, both let-7a and miR-16 were significantly expressed in neurospheres (data not shown). Therefore, it is possible that additional mechanisms exist to antagonize let-7a and miR-16 expression during NS cell differentiation.
A specific role for miR-34a during neuronal differentiation has not been reported. Interestingly, herein we showed a significant upregulation of miR-34a during mouse NS cell differentiation, and this paralleled the appearance of postmitotic neurons. In addition, in vitro
transient overexpression of miR-34a increased the proportion of NeuN-positive cells. Given that miR-34a has been shown to regulate genes involved in cell cycle arrest, it is possible that miR-34a upregulation is related to cell cycle exit and the subsequent appearance of immature postmitotic neurons. In fact, miR-34a upregulation in ES cells after LY treatment, as well as in NT2N cells after incubation with the mitosis inhibitor, supports this hypothesis. In addition, it has been previously shown that miR-34a can suppress cell-cycle genes and induce neural phenotype in neuroblastoma tumors [56
]. Importantly, it has been previously reported that miR-34a overexpression had no effect on cell cycle arrest and survival/apoptosis of astrocytes [57
]. However, additional studies are required to confirm the influence of miR-34a in neurogenesis and evaluate whether it mediates p53 effects on cell differentiation. In fact, several potential miR-34a targets might be involved in the transition towards postmitotic neurons. It was previously demonstrated that miR-34a targets Sirt1 and is regulated by p53, which in turn, is activated by Sirt1 suppression [59
]. In addition, miR-34a may also regulate the differentiation process by influencing Notch signaling pathway [57
]. In contrast to let-7a and miR-16, miR-34a was barely detected in undifferentiated cells, supporting its specific involvement in cell differentiation.
miR-19a and miR-20a are members of the miR-17-92 cluster [61
], which consists of seven mature miRNAs, previously linked to tumorigenesis. Recently, additional functions have been assigned to this cluster, particularly to miR-20a and miR-19a. Specifically, miR-20a was shown to control monocyte differentiation [62
]. In fact, transfection of hematopoietic progenitors with miR-20a increased the proliferation of monocytes and blocked differentiation, whereas inhibition of miR-20a caused a decrease in proliferation and more rapid differentiation and maturation. Our results, showing that miR-20a decreases during mouse NS cell differentiation, are in agreement with this previous report.
Only limited information is available regarding the physiological role of miR-19a. It has been demonstrated that it acts as a positive regulator of antiapoptotic Stat3/IL-6 receptor signaling by directly suppressing SOCS-1, thereby facilitating malignant growth of multiple myeloma [63
]. Further, miR-19 appears to affect the level of proapoptotic protein Bim, thereby preventing apoptosis and promoting cell survival. The possible role of miR-19a on cell survival, may explain why this miRNA was upregulated at early stages after the induction of mouse NS cell differentiation. However, additional studies are required to determine the specific role of both miR-20a and miR-19a during cell differentiation, and also evaluate if their expression is restricted to a specific cell type.