In our study, we utilized a simple in vitro model of human neuronal differentiation in which human neuroblastoma SH-SY5Y cells were differentiated into a homogenous population of cells with neuronal morphology. The advantages of this model over other in vitro systems for human neuronal differentiation include its robust differentiation capability (terminal differentiation is obtained within 2 weeks of induction) and the formation of neurons only and not other cell types such as glia (8
). In comparison to previous reports of miRNAs in human neural differentiation (20
), which focused mainly on the profiling of miRNAs, we have advanced well beyond expression profiling and established a number of reliable assays to assess the biological functions of specific miRNAs in the neuronal differentiation of SH-SY5Y cells as well as of human neural progenitor RVM cells. We identified two miRNAs, miR-124a and miR-125b, which promote neurite outgrowth. We further demonstrated how the upregulation of miR-125b during neurogenesis downregulates a set of direct mRNA targets. Since the proteins encoded by these mRNAs normally repress neurogenesis, our model (Fig. ) suggests how miR-125b induction causes an enhanced expression of multiple neuron-important genes.
miR-125b is expressed in many types of tissues, but its highest level of expression is in the brain, especially in mature neurons but not astrocytes (33
). miR-125b is upregulated during mouse neurogenesis (35
), during the neural differentiation of mouse embryonic stem cells (18
), and upon RA treatment of embryonic carcinoma cells (33
) and of neuroblastoma SK-N-BE cells (19
). Adding to these studies, our data demonstrate that miR-125b is not only a marker of differentiation but also a regulator of neuronal differentiation in SH-SY5Y cells. By quantifying the effect of miR-125b ectopic expression and miR-125b knockdown on neurite outgrowth and on the expression of neuronal markers, we demonstrate that miR-125b is both necessary and sufficient to promote the neuronal differentiation of SH-SY5Y cells.
In our functional assays, we examined the effect of miR-125b ectopic expression on differentiation over a short time frame of 4 days and found that only a fraction of the cells differentiated. Importantly, the percentage of “differentiated cells” varies depending on the criteria used for quantification. In the neurite outgrowth assay, we considered only the differentiated cells with apparent neurite outgrowth. Because we used very stringent parameters that allow us to identify only the most mature neurons, βIII-tubulin-positive cells with neurites longer than 30 μm, the percentage of the selected cells was rather small, 1 to 6% (Fig. ). Reducing the stringency by considering a lower minimum neurite length would increase the percentage of selected cells, but the neurite identification then becomes less accurate since cell edges can be mistaken as short neurites. In our immunostaining assay, where differentiation was determined based on the expression of the neuronal protein markers Map2ab, neurofilament, and Syt5, we observed a higher percentage of differentiated cells, 5 to 16% (Fig. ). Hence, the cells appeared to upregulate these markers earlier than the onset of neurite outgrowth.
Because we were concerned with the abnormal karyotype and tumor origin of SH-SY5Y cells, we examined the expression and the function of miR-125b in a more physiologically relevant cell type, human neural progenitor RVM cells. Like primary neural stem cells, RVM cells have a normal karyotype and are able to differentiate into both neurons and glial cells (7
). We showed that, as in SH-SY5Y cells, miR-125b expression was gradually upregulated during the differentiation of RVM cells. miR-125b ectopic expression significantly enhanced the neurite outgrowth of RVM cells in both growth medium and differentiation medium. Thus, our data indicate that miR-125b is important for neuronal differentiation in both RVM cells and SH-SY5Y cells and suggest a common function of miR-125b in neural progenitor cells. Potentially, miR-125b gain of function may be useful to enhance the in vitro neuronal differentiation of primary human neural stem cells for treatments of neurodegenerative diseases. This approach would probably be more advantageous than other types of gene therapy since the miRNA is a small molecule that, in principle, can be delivered more easily into a cell.
On the other hand, we also noted several differences in the effects of miR-125b on SH-SY5Y and RVM cells. miR-125b ectopic expression exhibited a stronger effect on the average neurite length in RVM cells than in SH-SY5Y cells in growth medium, but the reverse was observed for differentiation medium. Hence, in RVM cells, miR-125b alone is sufficient to promote the extension of neurite length, but in SH-SY5Y cells, it requires the addition of RA. Furthermore, the knockdown of miR-125b in SH-SY5Y cells significantly reduced the extension of neurites induced by RA; however, the same effect was not observed when miR-125b was knocked down in RVM cells undergoing differentiation. Since the two cell lines were differentiated by two different methods, the differences in the effects of miR-125b may be more apparent than real, but it does appear as if the role of miR-125b in neurite outgrowth is more necessary for the RA-induced differentiation of SH-SY5Y cells than it is for the differentiation of RVM cells upon the withdrawal of EGF and bFGF. Additionally, the phenotype may also be determined by the intrinsic differences between the two cell lines: as they express different mRNAs, the genes directly and indirectly affected by miR-125b regulation are likely to be different. The physiological functions of miR-125b in vivo may also depend on different extrinsic and intrinsic factors that are regulated in a temporal and spatial manner. Interestingly, we recently showed that the knockdown of miR-125b leads to severe defects in zebrafish brain development, including the malformation of axonal tracts in midbrain and hindbrain, suggesting that miR-125b is required for neuronal differentiation in vivo (our unpublished data). It would be interesting to further study the cell-specific function of miR-125b in vivo.
To understand the mechanism mediating miR-125b function, we conducted global profiling to identify miR-125b-responsive genes. We chose to perform this experiment primarily using SH-SY5Y cells because these cells are more responsive to the modulation of miR-125b levels than RVM cells. Using microarrays, we identified 388 genes repressed by miR-125b ectopic expression and predicted that 164 of these genes are the direct targets of miR-125b. This prediction is supported by two lines of evidences: (i) MEME motif discovery identified a 6-nt motif in the 3′ UTR of 129 genes that is perfectly complementary to the seed sequence of miR-125b, and (ii) an integrative search using four conventional miRNA target prediction methods identified 97 direct targets among the 388 genes repressed by miR-125b. Moreover, we found that 57 (~35%) out of the 164 selected targets were downregulated by RA- or BDNF-induced neuronal differentiation by ≥1.5-fold. The inverse expression pattern of these genes in comparison to the endogenous expression of miR-125b implies that they are targeted by miR-125b during differentiation. Although the actual number of endogenous targets is subject to a further validation of our predictions, we do expect the complex function of miR-125b to be mediated by multiple mRNA targets. Previous profiling studies of miRNA targets by microarrays and proteomics demonstrated that miRNAs usually downregulate several hundred genes; the targets are mostly repressed at both mRNA and protein levels, although a number of them are regulated only at the protein level (1
). Our microarray data for SH-SY5Y cells were able to identify only the targets regulated by miR-125b through mRNA degradation and/or deadenylation. In a separate study, we found that p53 is a bona fide target of miR-125b; a modulation of miR-125b largely affects the p53 protein level but did not show any significant change in the transcript level of p53 in SH-SY5Y cells (19a
). Besides p53, it is possible that our microarray analysis also missed other targets that are regulated only by translational inhibition.
We next asked how miR-125b mediates neuronal differentiation by suppressing the 164 predicted targets. Data from IPA suggest that a subset of these targets is connected to the neuronal genes that were indirectly upregulated by miR-125b gain of function. We propose a simple model to explain how miR-125b enhances differentiation. In constructing the model, we assumed that the direct targets of miR-125b inhibit pathways that promote the expression of neuronal genes. Hence, from the network connecting the predicted downregulated direct mRNA targets and the upregulated indirect neuronal effectors, we selected the pathways relevant to neurogenesis and the direct targets with known inhibitory effects or known binding to the components of these pathways. The model focused on 10 predicted direct targets of miR-125b, and we validated these both by real-time PCR analysis of mRNA expression after the ectopic expression of miR-125b and by a luciferase reporter assay (Table ). IPA also revealed that many genes in the modeled pathways are regulated by RA in the same manner as by miR-125b. This relationship, and the fact that RA upregulates miR-125b during differentiation, suggests that miR-125b mediates RA-induced differentiation in SH-SY5Y cells. Our proposed model of the miR-125b network supports this hypothesis, since the ERK signaling pathway featured prominently in our model is also known to mediate RA-induced differentiation in SH-SY5Y cells (27
). Indeed, the model also predicts that miR-125b exerts positive feedback on RXRA, the receptor for RA.
In addition, IPA shows that the predicted targets of miR-125b are also connected to the repressed indirect effectors (genes downregulated 4 days after the transfection of 125b-DP), mainly with positive regulatory effects. These networks are involved in metabolism, proliferation, and apoptosis; thus, in part, miR-125b may enhance differentiation by reducing cell metabolism and proliferation. Experimentally, we did not find any significant effect of miR-125b gain of function on proliferation (using Ki67 staining) (data not shown). Laneve et al. also previously found that miR-125b alone has very little effect on proliferation, although the ectopic expression of miR-125b together with miR-125a and miR-9 inhibits cell cycling in neuroblastoma cells (19
). Hence, the withdrawal of SH-SY5Y cells from the cell cycle during differentiation may require a synergistic effect between miR-125b and other miRNAs. On the other hand, the negative regulation by miR-125b on a number of apoptotic genes, including the four targets BAK1, TP53INP1, PPP1CA, and PRKRA in the p53 pathway, suggests that miR-125b has an antiapoptotic effect. In a separate study, we found that miR-125b downregulates the p53 pathway and that miR-125b gain of function represses apoptosis induced by H7 in SH-SY5Y cells (19a
). These results suggest that miR-125b may promote the survival of differentiated neuronal cells by suppressing apoptosis.
In conclusion, we report here several important and novel functions of miR-125b in neuronal differentiation. Our results demonstrate that this miRNA promotes the differentiation of human neuroblastoma SH-SY5Y cells and human neural progenitor RVM cells toward the neuronal phenotype. In SH-SY5Y cells, we propose a model where the action of miR-125b is mediated by 10 targets that repress multiple pathways involved in neuronal differentiation.