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. 2014 Oct 31;11(9):1105–1112. doi: 10.4161/rna.35508

Mir-23a and mir-125b regulate neural stem/progenitor cell proliferation by targeting Musashi1

Ubaldo Gioia 1,2,†, Valerio Di Carlo 1,3,†, Pasquale Caramanica 1, Camilla Toselli 1,4, Antonella Cinquino 1, Marcella Marchioni 5, Pietro Laneve 4, Stefano Biagioni 1,3,4, Irene Bozzoni 1,3,4,5, Emanuele Cacci 1,3,*, Elisa Caffarelli 4,5,*
PMCID: PMC4615800  PMID: 25483045

Abstract

Musashi1 is an RNA binding protein that controls the neural cell fate, being involved in maintaining neural progenitors in their proliferative state. In particular, its downregulation is needed for triggering early neural differentiation programs. In this study, we profiled microRNA expression during the transition from neural progenitors to differentiated astrocytes and underscored 2 upregulated microRNAs, miR-23a and miR-125b, that sinergically act to restrain Musashi1 expression, thus creating a regulatory module controlling neural progenitor proliferation.

Keywords: astrocyte differentiation, cell proliferation, microRNAs, musashi1, neural progenitors, post-transcriptional gene regulation

Abbreviations

BrdU

5-bromo-2′-deoxyuridine

Msi1

Musashi1

miRNA

microRNA

3′UTR

3′Untranslated Region

NSPCs

Neural Stem/Progenitor Cells

Introduction

Musashi1 (Msi1) is a neural, evolutionary conserved, RNA-binding protein that functions as a key differentiation inhibitor and a pro-proliferative factor.1,2 Consistently, Msi1 contributes to the maintenance of Neural Stem/Progenitor Cells (NSPCs) in the undifferentiated state3-5 and its expression in adult brain is limited to stem and progenitor cells.2,6 Its role in neural cell fate regulation is mainly achieved through translational repression of Numb, a protein that positively modulates neuronal differentiation by binding to Notch1.7-9 Because of that, Msi1 downregulation is required for transitions between cellular programs regulating progenitor cell proliferation and differentiation.10 Moreover, it has also been shown that Msi1 may influence stem cell maintenance and differentiation by cooperating with the protein Lin28 for blocking the biogenesis of let-7 microRNA (miRNA) family,11 which is strongly induced during neural differentiation of embryonic stem cells.12 On the other hand, when expressed at high levels, Msi1 behaves as an oncogenic protein that has been associated with the development of multiple tumours.13,14

Due to its physiological and pathological implications, unveiling the mechanisms controlling the cellular levels of this stem cell regulator is important for better understanding the cell fate decision between proliferation and differentiation. Study of transcriptional regulation of Msi1 gene led to the identification of an intronic enhancer, which controls Msi1 expression in NSPCs.15 However, post-transcriptional control mediated by miRNAs represents a further important regulation level, especially in neural differentiation where the action of these negative modulators of gene expression is pervasive. In particular, our study was addressed to the identification of miRNAs that may control Msi1 expression during the transition from murine embryonic NSPCs to differentiated astrocytes.

We profiled the expression of miRNAs during this process, and identified specific subsets of molecules modulated in response to astrocyte differentiation stimulus. We underscored a relevant function for miR-23a and miR-125b, that display a common pattern of induction, in controlling NSPC proliferation. We show that these molecules act sinergically by repressing Msi1 and demonstrate that the downregulation of this gene is critical for regulating NSPC cell growth.

Results and Discussion

As model system for in vitro astrocyte differentiation, we utilized cultures of NSPCs isolated from E13.5 mouse cerebral cortex.16-19 These cells, expressing neural stem cell markers such as nestin and Msi1 when cultured in expansion medium (Fig. 1A), can efficiently differentiate into astrocytes in the presence of FBS or BMP4.19-22 After six days of FBS- or BMP4-treatment, almost the whole cell population expressed the astrocytic marker GFAP (Fig. 1A), whereas βIII tubulin expressing cells were virtually absent, indicating an astrocyte differentiation efficiency >90% (Supplementary Fig. 1). Based on this result, hereafter the astrocyte differentiation experiments are performed using FBS.

Figure 1.

Figure 1.

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Analysis of Msi1 mRNA and protein levels during in vitro astrocyte differentiation. (A) Neural progenitors (NSPCs) propagated in expansion medium were virtually all positive for the neural stem/progenitor cell markers nestin and Musashi1; NSPCs differentiated in FBS-containing medium for 6 d (astrocytes) homogeneously expressed the glial marker GFAP. Note that very few differentiated astrocytic cells express Musashi1 (arrows). Nuclei are stained with Hoechst. Scale bar 20 μm. (B) RT-PCR of Msi1 mRNA in NSPCs treated with FBS for 3 and 6 d. The histogram shows the relative quantities of Msi1 mRNA versus the 0 time point, set to a value of 1. Gapdh mRNA was used as a loading control. Data are presented as the mean values ± SEM from 3 independent experiments. *p<0,05; **p<0,01; ***p<0,001. (C) Immunoblotting of Msi1 in NSPCs treated with FBS for 3 and 6 d. The densitometric analysis on the right shows the relative amount of Msi1 protein vs. untreated cells (0 time point), set to a value of 1. Legend details as in panel (B).

The expression of Msi1, at both the mRNA and protein levels, was analyzed at specific time points upon FBS treatment of NSPCs (Fig. 1B-C). Notably, we found that while the Msi1 mRNA levels strongly increased, reaching a 4-fold induction, the levels of the Msi1 protein decreased by about 80% at 6 d of FBS-treatment, according to immunocytochemistry analysis (Fig. 1A). Such inverse correlation is indicative of a translational regulation of Msi1 expression.

To identify miRNAs potentially involved in Msi1 gene control, we carried out a global analysis of miRNA population in NSPCs exposed to FBS-containing medium for 0, 3 and 6 days, by high-throughput qRT-PCR. Evaluation of the expression levels of 345 miRNAs allowed us to produce an atlas of 176 expressed molecules that are graphically represented in the heat map of Figure 2A. Based on their expression profiles, we could identify 3 distinct subsets shown in the pie chart of Figure 2B. The first subset includes downregulated miRNAs (∼18%), the second one comprised the unaffected molecules (∼15%), and the third one the upregulated miRNAs (∼67%). Among the miRNAs whose expression was modulated, we selected several molecules and validated their expression profile by Northern blot. Differently from TaqMan assay, this analysis does not allow to distinguish between closely related mature sequences of miRNA family members, expressed from different precursors. Figure. 3 shows the expression profiles of the downregulated miR-124, and the upregulated miR-26a/b, miR-23a/b, miR-27a/b, miR-24, miR-125b, miR-29a/b, miR-29c. Accordingly with the high-throughput approach results, the expression levels of the neuronal-specific miR-124, which is weakly expressed in NSPCs, became undetectable upon FBS-treatment. Among the upregulated miRNAs, 2 sub-populations with different expression timing can be recognized: i) a first class, containing miR-26a/b, miR-23a/b, miR-27a/b, miR-24 and miR-125b, induced early after FBS treatment (1 day) and accumulating at higher levels at later differentiation stages (3 and 6 days); ii) a second class, including the members of miR-29 family, consistently upregulated at 6 d. While the members of the first class may potentially participate in all stages of differentiation, those of the second class are presumably involved at later stages. Similar miRNA profiles were obtained when NSPCs were differentiated with BMP4 (Supplementary Fig. 2), suggesting that the observed miRNA modulation is not due to the specific NSPC treatment. A further qRT-PCR miRNA expression profile was carried out in parallel in NSPCs treated with the gamma-secretase inhibitor DAPT, that promotes efficient neuronal differentiation while repressing astrocyte differentiation, or with FBS (Supplementary Fig. 3).19,23,24 In agreement with the demonstration that neural cell subtypes differ extensively in their miRNA expression pattern,25 we found that the neuronal-specific miR-124, expressed at very low levels in NSPCs, is induced in neuron-enriched cultures whereas it is downregulated upon astrocytic differentiation stimulus (Supplementary Fig. 3B). Moreover, we found that miR-23a/b, previously reported as an astrocyte-specific miRNA,26 is significantly upregulated upon FBS-treatment, whereas it is not induced in neurons generated from NSPCs (Supplementary Fig. 3B). This result establishes that miR-23a/b may be definitely considered an astrocytic marker.

Figure 2.

Figure 2.

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miRNA profiling during in vitro astrocyte differentiation. (A) The relative expression of each miRNA upon treatment of NPSCs with FBS for 3 and 6 d was determined by high-throughput qRT-PCR. A green-red color scale (-3 to +3) depicts normalized miRNA expression level on a log scale. (B) Pie chart summarizing the data obtained by the qRT-PCR analysis.

Figure 3.

Figure 3.

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Validation of miRNA profiling. (A) Northern blot of a subset of miRNAs in NSPCs treated with FBS for the indicated times (days). (B) The histograms show the relative quantities of miRNAs versus the 0 time point, set to a value of 1. Data were normalized to 5S-rRNA hybridization signal.

To identify the miRNAs potentially targeting mouse Msi1 mRNA, we carried out an in silico analysis centered on the molecules upregulated during astrocyte differentiation. We interrogated different databases (PicTar, TargetScan and Diana tools) and selected miR-125a/b, as the miRNAs displaying one of the highest scores, and miR-23a, as the miRNA with the highest level of induction. Notably, we found that miR-125a/b, so far regarded as neuronal miRNAs, are also induced at high levels along astrocyte differentiation, which highlightes their wide involvement in neural pathways. The putative target sites for the 2 miRNAs, that are 600 nucleotides far from each other, are shown in the schematic representation of Figure 4A. To validate Msi1 3’UTR as the target of miR-23a and miR-125a/b, we set up a luciferase reporter assay. For this purpose, the coding regions of the selected miRNAs were cloned between the polII promoter and termination regions of the U1 snRNA gene (Fig. 4B). Since miR-125a and miR-125b share the same seed (they only differ for 4 nucleotides), we cloned the coding region of miR-125b that is induced at higher levels with respect to miR-125a. Transfection of these constructs into HeLa cells produced miRNA constitutive expression, as verified by Northern blot (Fig. 4C). In parallel, a portion of the 3’UTR of Msi1 mRNA, including the 2 miRNA target sites, was cloned downstream of Renilla luciferase ORF (Fig. 4A). As control, a construct derivative deleted of the miRNA binding sites (mutant 3’UTR), thus precluding miRNA interactions, was generated. The miR-23a and miR-125b expressing vectors were transfected, individually or in combination, along with the expression plasmids containing wild type or mutated Msi1 3’UTR region. The histogram of Figure 4D shows a reduction in luciferase activity of about 30% after transfection of the plasmid expressing wt Msi1 3’UTR along with the single plasmids coding for miR-23a or miR-125b. Luciferase activity decreased by about 40% when the 2 miRNAs were co-expressed. Since half of the amount of each miRNA was used in the co-expression experiments, a miRNA synergic activity on Msi1 can be argued. The transfection of the 2 miRNAs together with the mutated version of Msi1 3’UTR showed no reduction of luciferase activity, demonstrating the specificity of miRNA interaction.

Figure 4.

Figure 4.

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miR-23a and miR-125b may specifically interact with Msi1 3 ’UTR. (A) Schematic representation of the binding sites for miR-23a and miR-125b on Msi1 3 ’UTR. (B) Schematic representation of the constructs expressing the miRNAs. (C) Northern blot of the ectopically expressed miRNAs. 5S-rRNA was used as a loading control. Control cells (CTRL) were transfected with the empty vector. (D) The histogram reports the levels of luciferase activity in cells overexpressing the miRNAs indicated below and transfected with the wild type 3 ’UTR (gray bars) or with its mutant derivative (black bar) lacking the miRNA binding sites. Control cells (CTRL) were transfected with the empty vector. Data are presented as the mean values ± SEM from 3 independent experiments. *p < 0,05; **p < 0,01; ***p < 0,001.

To assess the capability of the miRNAs to target endogenous Msi1 mRNA, we ectopically expressed miR-23a and miR-125b in NSPCs and measured endogenous Msi1 protein levels by immunoblotting. Each of them caused a decrease of the Msi1 signal of about 30%, whereas their combined expression caused a stronger Msi1 reduction (∼65%), confirming they act in synergy (Fig. 5A). Since miRNAs can mediate translational repression and/or mRNA degradation, we tested whether miR-23a and miR-125b overexpression may influence Msi1 mRNA stability. qRT-PCR analysis was carried out on total RNA extracted from NSPCs transfected with the plasmids expressing the 2 miRNAs. As shown in the histogram of Figure 5B the levels of Msi1 mRNA were unaffected, indicating that the 2 miRNAs regulate Msi1 expression at the translational level only.

Figure 5.

Figure 5.

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miR-23a and miR-125b control NSPC proliferation through regulation of Msi1 expression. (A) Immunoblotting of Msi1 in NSPCs ectopically expressing miR-23a, miR-125b, both of them, or the control vector (CTRL). Actin was used as a loading control. Data in the histogram on the right show the relative quantities of Msi1 vs. control cells. Data are presented as mean values ± SEM from at least 3 independent experiments. *p<0,05; **p<0,01; ***p < 0,001. (B) Analysis of Msi1 mRNA levels by qRT-PCR in NPSCs overexpressing the 2 miRNAs: the histogram shows the relative amount of Msi1 mRNA versus control cells (CTRL), set to a value of 1. Legend details as in (A). (C) BrdU incorporation assay in NSPCs ectopically expressing miR-23a and miR-125b or untransfected cells (CTRL). Legend details as in (A). (D) Immunoblotting of Msi1 in NPSCs knocked down for Msi1. Cells were transfected with anti-Msi1 siRNAs (siMsi1) or with a non-targeting siRNA as a control (CTRL). Actin was used as a loading control. The histogram on the right quantifies the BrdU incorporation upon Msi1 knockdown. *p<0,05; **p<0,01; ***p<0,001. (E) Rescue assay: BrdU incorporation was quantified in NSPCs after ectopic expression of miR-23a and miR-125b alone (bar miRNAs+/Msi1-) or upon further expression of exogenous Msi1, lacking 3 ’UTR (bar miRNAs+/Msi1+). Proliferation was fully recovered to levels of mock-electroporated control cells (bar miRNAs-/Msi1-). Data are presented as mean values ± SEM from 3 independent experiments. At least 300 cells were counted for each sample in each experiment. *p < 0,05; **p < 0,01; ***p<0,001.

Next, we wondered whether miRNA-mediated Msi1 downregulation may affect NSPCs proliferation. We found that BrdU incorporation dropped by 30% following miR-23a and miR-125b ectopic expression in NSPCs maintained for 72hrs in expansion medium, compared with control cells (Fig. 5C). These data indicate that the 2 miRNAs may contribute to cell growth decrease. To verify that the miRNA target Msi1 is directly involved in such cell growth control, we knocked down Msi1 mRNA in NSPCs by RNAi and analyzed the effect on cell proliferation. Following almost complete ablation of Msi1 protein, a 75% reduction in BrdU incorporation was observed (Fig. 5D). Finally, we also demonstrated that cell proliferation, reduced upon miR-23a and miR-125b ectopic expression, was significantly rescued by the re-introduction of a miRNA-insentitive Msi1 mRNA variant (Fig. 5E). This result definitely links the microRNA-mediated Msi1 downregulation to the decrease of cell proliferation.

We also asked whether miRNA overexpression could affect differentiation. We analyzed the late astrocyte differentiation markers S100β and GFAP and found that their expression were not altered either at the mRNA or at the protein level (Supplementary Fig. 4). However, due to the lack of early astrocyte fate markers,27,28 we cannot rule out the possibility that miRNA-mediated Msi1 downregulation can affect such early event.

In conclusion, we unveiled a new regulatory circuitry made by 2 miRNAs and their common target gene Msi1, which operates to address precursor cells to neural fate.

Materials and Methods

Cell culture and transfection

NSPCs were isolated from E13.5 mouse embryonic cortical tissues and grown in basal medium with the addition of 20 ng/ml human EGF (R&D), 10 ng/ml bFGF (R&D), and 1% N2 (Gibco) supplement. This medium was termed expansion medium.16

To differentiate NSPCs into homogeneous astrocyte cultures, cells were maintained in basal medium supplemented with 2% B27, 1% N2 and 5% Fetal Bovine Serum (FBS) or with 2% B27, 1% N2, and 50 ng/ml BMP4. To obtain neuron-enriched cultures, NSPCs were differentiated with bFGF/DAPT-containing medium consisting of a basal medium, supplemented with 2% B27, 1% N2, 10 ng/ml bFGF and 0.5 mM N-[(3,5-difluorophenyl)acetyl]-lalanyl-2-phenyl]glycine-1,1-dimethylethyl ester (DAPT; Tocris).

When required, NSPCs were cultured in a non-proliferative condition for 3 or 6 d in the presence of basal medium supplemented with 10 ng/ml bFGF (R&D), 1% N2 (Gibco) and 2% B27.

NSPCs were transfected either with plasmids overexpressing miRNAs or with anti-Msi1 siRNAs (L-047549–00–0005, Dharmacon), by using the Amaxa system (Nucleofector, Lonza). As controls, the empty vector and non-targeting siRNAs (D-001810–10–05, Dharmacon) were used. Under these conditions 80–85% of NSPCs were transfected. RNA or protein extraction or immunocytochemistry analyses were performed 72hrs after transfection.

HeLa cells were cultured and transfected as in.29

Immunocytochemistry

Cells were fixed with 4% paraformaldehyde for 20min, permeabilized in 0.025% Triton X-100 for 1hr and incubated for 2hrs at room temperature with the primary antibodies listed in supplemental material. Cells were then incubated for 30min in a solution containing 10 mg/ml Hoechst 33342 and secondary antibodies FITC/Cy3-conjugated (Jackson Immuno-Research, West Grove, PA, USA) at 1:300. Cells were coverslipped with DAKO mounting medium (Dako).

RNA isolation and analysis

Total RNA was extracted with QIAzol Lysis Reagent (Qiagen) and analyzed by semi-quantitative RT-PCR, quantitative RT-PCR (qRT-PCR) and Northern blot as previously described.30

The expression profiling of 345 mouse miRNAs during in vitro astrocyte differentiation was carried out with TaqMan® microRNA Arrays (Megaplex™ Rodent Primer Pool A, Applied Biosystems) as previously described.31 Data are presented as the natural logarithm of fold changes in miRNA expression relative to undifferentiated NSPCs and ordered by ratio with MeV software (http://www.tm4.org/).

Protein extraction and immunoblotting

Protein extraction, immunoblotting and quantification were performed as already described.29 Msi1 antibody was purchased from Santa Cruz Biotechnology (sc-135721), Actin antibody from Sigma (A2066).

Luciferase-based reporter assay

48hrs after HeLa cell transfection, luciferase activities were measured in Glomax multi+ detection system (Promega), using Dual-Luciferase® Reporter Assay System (Promega) according to the manufacturer's protocol.

BrdU-based cell proliferation assay

Electroporated NSPCs were stained with BrdU-specific antibody, following the Labeling and Detection Kit I (Roche) directions. Goat anti-mouse Cy3®-conjugated IgG (Jackson ImmunoResearch) was used as a secondary antibody. Nuclei were counterstained with DAPI. Images were acquired using the Zeiss AxioObserver A1 inverted fluorescence microscope and the AxioVision Rel.4.8 imaging software (Zeiss). At least 2 hundred nuclei were counted in triplicate and the number of BrdU-positive nuclei was recorded.

Rescue assay

24hrs after miR-23a and miR-125b ectopic expression, cells were electroporated with the Msi Δ3’UTR construct, for Msi1 expression recovery. Sample and control cells were cultured for the following 48hrs before BrdU incorporation assay.

Statistics

Statistical significance was determined by 2-tailed Student's t-tests. Quantitative data significance is indicated as follows: *p<0.05; **p<0.01; ***p<0.001. Data shown here are the mean ± SEM from at least 3 experiments unless otherwise indicated.

Supplementary Material

Supplemental Material

Disclosure of Potential Conflicts of Interest

No potential conflicts of interest were disclosed.

Acknowledgments

We thank M. Arceci for technical support.

Funding

This work was supported by project Epigen, IIT-“SEED," FIRB, PRIN. VDC was supported by an Institue Pasteur Fondazione Cenci-Bolognetti fellowship.

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krnb-11-09-977674-s001.zip (4.4MB, zip)

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