Role of miRNA-146 in proliferation and differentiation of mouse neural stem cells

miRNAs are a fundamental class of biological molecules that play a crucial role in development [1]. miRNA dysfunction has been linked to cancer [2], as well as other biological processes. Genes encoding miRNAs, which are found in most eukaryotes, produce short (18–25 nt) RNAs that bind to mRNA transcripts and down-regulate expression either through mRNA destabilization or translational repression [3,4]. miRNAs regulate gene expression by guiding the RNA-induced silencing complex to a target sequence, which is usually located at the 3′-UTR of mRNAs [5]. The widespread involvement of miRNAs in regulating developmental processes, physiological responses and pathological conditions in animals has been amply demonstrated [4,6]. Nonetheless, the specific functions of each miRNA in the various contexts in which it is expressed are only beginning to be discovered. Neural stem cells (NSCs) have the ability to self-renew and are capable of differentiating into neurons, astrocytes, and oligodendrocytes. NSCs have been isolated from the developing brain and more recently from the adult central nervous system. In recent years, a variety of signal pathways have been identified that ensure NSC maintenance and control the development of neural tissues and progenitor cells. Thus, miRNAs are potentially key post-transcriptional regulators in NSCs self-renewal and differentiation. Distinct sets of miRNAs have been shown to be specifically expressed in embryonic stem cells [7,8]. Knockout or knockdown of Dicer causes embryonic lethality and loss of stem cell populations [9]. Argonaute family members, which are key components of the RNA-induced silencing complex, are required for maintaining germ line stem cells in various species [10,11]. Together, these observations support a role for miRNAs in stem cell biology. Several brain-specific miRNAs have recently been identified. Among these miRNAs, miR-146 is a family of miRNA precursors found in mammals, including humans. miR-146 is primarily involved in the regulation of inflammation and other process that involve the innate immune system [12]. miR-146 is thought to be a mediator of inflammation, along with another miRNA, miR-155. Expression of miR-146 is up-regulated by inflammatory factors, such as interleukin 1 and tumour necrosis factor-α and miR-146 dysregulates a number of targets mostly involved in toll-like receptor pathways that bring about a cytokine response as part of the innate immune system [13]. miR-146 operates in a feedback system or ‘negative regulatory loop’ to finely tune inflammatory responses [14]. Loss of functional miR-146 could predispose an individual to experience chromosome 5q deletion syndrome [14]. However, the functions of miR-146 in proliferation and differentiation of NSCs are poorly understood. In the present study, miR-146 precursor was transfected into NSCs to determine its role in proliferation and differentiation of NSCs.

NSCs were collected from brain tissue of embryonic BABLC (BALB/cAnNCrl mouse) mice, supplied by the Laboratory Animal Center of Second Military Medical University. Brain tissue was isolated and rinsed three times and then transferred to a Dulbecco's modified Eagles medium (DMEM)/F12 (D/F12; Gibco) medium-containing plate for cultivation into a monoplast suspension after repetitive pipette and mechanical separation. Single cells were plated at 1000 cells/cm² in untreated 25-cm² tissue culture flasks and cultured in serum-free neural medium [D/F12+2% B27 supplement (Gibco)+20 ng/ml endothelial growth factor (EGF) (PeproTech)+bFGF (fibroblast growth factor; PeproTech)]. Medium (50%) was replaced every 2 days.

Immunofluorescence was used to identify the NSCs in conjunction with specific markers Nestin, Sox 2 (sex determining region Y–box 2) and Pax 6 (paired box protein Pax-6). NSCs were seeded onto 100 μg/ml poly-L-lysine-coated coverslips and allowed to incubate for 24 h. The cells were then fixed with 4% paraformaldehyde for 10 min and washed three times in PBS buffer. The cells were permeabilized with 0.125% Triton X-100 for 10 min and then washed three times in PBS buffer. The cells were then blocked with 10% normal goat serum for 30 min. Mouse NSCs were then incubated with Nestin, Sox 2 and Pax 6 antibodies in a humidified chamber at 4°C overnight. After three PBS buffer washes, the cells were incubated with Cy5.5-labelled secondary antibodies at room temperature for 1 h, the cells were rinsed three times with PBS buffer. Finally, nuclei were labelled by incubating with DAPI (Sigma). The cells were examined using a phase-contrast fluorescence microscope (Olympus).

The 3′-UTR fragments for Notch 1 were generated by PCR using the following primers: 5′-cttggagtgtggtttggttca-3′ and 5′-tcaggctcagagagggcat-3′and cloned into the psiCHECK-2 vector (Promega) downstream from the Renilla luciferase cassette. The predicted miR-146-binding site was mutated using the QuikChange II XL Site-Directed Mutagenesis Kit (Stratagene). Human embryonic kidney (HEK)293T cells were grown in a 96-well plate and co-transfected with the luciferase reporter vector together with a miRNA precursor or a negative control (20 nM; Ambion) using Attractene (Qiagen) according to the manufacturer's instructions. Activities of firefly and Renilla luciferase were analysed using the Dual-Luciferase Reporter Assay System (Promega) 24 h after transfection.

The pre-miR-146 sequence and Notch 1 shRNA were synthesized by Sangon Biotech (Shanghai), cloned into a lentiviral vector and then used to recombine lentivirus in HEK293F cells. The lentivirus, designated as pCMV-SM30, also expressed GFP as a marker for monitoring infection efficiency. Analogous lentivirus expressing only monomeric GFP was used as a control. Real-time PCR was used to evaluate the expression level of miR-146 after virus infection. miRNAs were isolated from cells using a microRNA isolation kit (Applied Biosystems) according to the manufacturer's instructions. cDNA synthesis was performed using the High-Capacity cDNA Synthesis Kit (Applied Biosystems) with 2 ng of RNA as a template. The miRNA sequence-specific reverse-transcription PCR primers for miR-146 and endogenous control U6 were purchased from Ambion. Real-time PCR analysis was performed using the Applied Biosystems 7500 real-time PCR system. The gene expression threshold cycle (CT) values of miRNAs from each sample were calculated by normalizing the samples to the internal control U6 and relative quantitation values were plotted.

Notch 1, the target gene of miR-146, was detected by western blot analysis following overexpression of miR-146 and shRNA. Cells were lysed using M-PER Protein Extraction Reagent (Pierce) supplemented with a protease inhibitor cocktail (PMSF). Protein concentrations of the extracts were measured using the BCA assays (Pierce) and equalized with extraction reagent. Equal amounts of extracts were loaded and subjected to SDS/PAGE, followed by transfer onto nitrocellulose membranes. Specific antibodies and horseradish peroxidase-coupled secondary antibodies were purchased from Santa Cruz. Membranes were probed using ultra-ECL western blotting detection reagents. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as an internal control.

Floating neural spheres was the primary morphology of NSCs in vitro. The neural sphere diameter of 1- and 8-day cultures was measured using fluorescence microscope in normal NSCs, miR-146-overexpressed NSCs and shRNA-overexpressed NSCs respectively.

Spontaneously differentiating cells were divided into three groups: normal NSCs (group A), NSCs after miR-146 overexpression (group B) and NSCs after shRNA overexpression (group C). The cells were seeded onto plates with D/F12 containing 10% FBS (Gibco). After 10 days, the cells were harvested and neural specific makers (β-III-tubulin and GFAP) were detected and analysed by immunocytochemical staining and flow cytometry. The primary antibodies were β-III-tubulin and GFAP (Glial fibrillary acidic protein, Abcam). The secondary antibodies were conjugated with Cy5.5.

RNA was extracted from cells using Trizol reagent (Invitrogen). Total RNA was reverse transcribed, followed by 30 PCR cycles using RNA PCR kit ver 3.0 (TARAKA). Information of gene specific primer pairs was listed in Table 1. PCR was performed in 50 μl of mixture containing 10 μl of 5× PCR Buffer (TARAKA), 28.5 μl of double distilled water (ddH₂O), 0.25 μl of Ex-Taq (TARAKA), 0.5μl of forward and reverse primers and 1.5 μl of template cDNA. The cycling conditions consisted of one initial 2-min cycle at 94°C, followed by 30 30-s cycles at 94°C (denaturation), one 30-s cycle at 50°C–60°C (annealing) and one 2-min cycle at 72°C (extension). PCR products were detected by 2.5% agarose gel electrophoresis. Real-time PCR was performed in a 20 μl of mixture containing 10 μl of SYBR premix Ex Taq buffer (Takara), 0.4 μl of fluorescent dye used in PCR (ROX) Reference Dye, 0.8 μM each of forward and reverse primers (Table 1), 1 μl of template cDNA and 7 μl of ddH2O. The cycling conditions consisted of initial 10 s at 95°C followed by 40 cycles of two-temperature cycling: 5 s at 95°C (for denaturation) and 34 s at 60°C (for annealing and polymerization). Each experiment was performed with duplicates in 96-well plate and repeated three times. Gene expression was detected on an ABI 7500 real-time PCR system. The expression level was calculated by the 2-ΔΔCt method to compare the relative expression.

NSCs derived from the dissociation of mouse forebrain samples were suspended in culture medium. The neurospheres were slow to form and the spheres were relatively small. In culture, there were many single cells attached to the flasks. However, as the cells proliferated into small clusters, they detached from the plastic and floated in suspension. Nestin, Sox 2, and Pax 6 protein expressions were detected by immunofluorescence assay. Results demonstrated that different passages of mouse NSCs expressed Nestin, Sox 2 and Pax 6 (Figure 1).

miRNA target genes are likely to have relatively long and conserved 3′-UTR [4]. We noticed that Notch 1 has a long evolutionarily conserved 3′-UTR, so we used the TargetScan algorithm [15] to search for miRNAs that could potentially regulate Notch 1. The miR-146 family (miR-146a and miR-146b) has the same putative target binding sites in Notch 1 in the mouse genome (Figure 2). To directly test whether miR-146 targets Notch 1, we cloned the 3′-UTRs of Notch 1 downstream of a luciferase reporter, and co-transfected these reporter constructs along with miRNA precursors into the human cell line HEK293T. Co-expression of miR-146 was found to effectively down-regulate luciferase expression in constructs with these 3′-UTRs (Figure 2). Mutations in the seed sequence of the predicted miR-146-binding sites within Notch 1 abolished the inhibitory effects of miR-146 on luciferase expression. The pre-miR-146 sequence and Notch 1 shRNA were synthesized and overexpressed in mouse NSCs, expression of miR-146a/b was quantified by real-time PCR at 72 h after transfection. As shown in Figure 2(C) and 2(D), miR-146a/b levels were significantly elevated after transfection. Protein expression of Notch 1, the putative target gene, was performed on miR-146a/b-transfected cells using the ImageJ tools comparative method (Figures 2E and 2F). Additionally, Notch 1 shRNA was assessed in NSCs and results were in accordance with miR-146 function; Notch 1 protein expression was down-regulated (Figures 2G and 2H).

NSC proliferation was assessed according to neural sphere diameter after overexpressed small RNAs (micron, Figure 3) and results demonstrated that NSC proliferation was stagnated. The diameter of neural spheres was measured using fluorescence microscope from 1- to 8-day cultured cells in normal NSCs, miR-146-overexpressed NSCs and shRNA-overexpressed NSCs respectively. The curves of neural spheres diameter from 1- to 8-day cultured cells in normal NSCs, miR-146-overexpressed NSCs and shRNA-overexpressed NSCs respectively. This result showed a time-dependent increase in neural sphere diameter from normal NSCs. However, the diameter of neural spheres showed no significant difference in NSCs following overexpression of small RNAs. Additionally, gene expression of the Notch pathway showed a time-dependent decrease following overexpression of small RNAs. Moreover, the diameter of neural spheres continuously increased and there was a time-dependent increase in Notch pathway expression in the control group (Figure 4).

NSCs were induced to differentiate into neural cells for 10 days. Following induction, the cells contracted and became round, triangular or cone shaped with multipolar processes. The processes continued to grow with many branches forming and cone-like terminal expansions. A number of cells demonstrated very long processes, which appeared similar to long neuronal axons. After 10 days of induction, immunofluorescence and flow cytometry assay detected β-III-tubulin and GFAP expression (Figure 4).

However, the production of neuronal cells (β-III-tubulin-positive cells) from NSCs after transfection with pre-miR-146 and shRNA was lower than from normal NSCs. Moreover, transfection of miR-146 and shRNA dramatically increased the percentage of glial cells (GFAP-positive cells) and reduced the percentage of neuronal cells in vitro (Figure 5).

In mammals, a wide variety of cells use the Notch signalling system for embryonic development and maintenance of homoeostasis in adults. The role of Notch signalling in neural development has been intensely analysed using conditional inactivation of RBP-J (recombining binding protein suppressor of hairless) and it has been shown that Notch signalling is required for both maintenance of neural stem/progenitor cells and specification of glial cells [16,17]. RBP-J, also known as CBF1, is the human homologue for the Drosophila gene suppressor of hairless. Its promoter region is classically used to demonstrate Notch1 signalling [18,19]. Precocious neuronal differentiation observed in Notch pathway-deficient neuro-competent cells is also detected in mammals, such as mice with inactivated Notch 1. A conditional Notch 1 knockout study provides further support that Notch signalling inhibits the premature onset of neurogenesis [20].

miRNAs are small, non-coding RNAs that are cleaved from 70 to 100 nt hairpin pre-miRNA precursors in the cytoplasm by RNaseIII Dicer into their mature form of 19–25 nt. Single-stranded miRNAs bind messenger RNAs of potentially hundreds of genes at the 3′-UTR region with perfect or near-perfect complementarity, resulting in degradation or inhibition of the target messenger RNA [21]. miRNAs are fundamental biological molecules that have been shown to play important roles in biological development [22]. miR-146a is a member of the miR-146 miRNA family, consisting of two evolutionary conserved miRNA genes: miR-146a and miR-146b. Previously, miR-146a was shown to be induced by endotoxin (lipopolysaccharide) through two consensus NF-kB-binding sites in the promoter region [23]. This region is highly homologous between human and mouse, suggesting an evolutionarily conserved regulatory mechanism for controlling miR-146a expression. A previous study provided some evidence that miR-146a overexpression in human glioblastomas exerts an anti-tumour effect, which is probably related to the capacity of this miRNA to target the 3′-UTR region of Notch 1 mRNA [24]. miR-146a constitutes an endogenous feedback system to counteract the oncogenic potential of dysregulated signalling pathways in human glioblastomas, such as activation of EGFR (endothelial growth factor receptor) and inactivation of Pten (phosphatase and tensin homolog) in gliomas. By regulating multiple targets, including the key NSC factor Notch 1, a miR-146a-mediated innate regulatory mechanism provides a native safeguarding mechanism to restrict the formation of glioma stem-like cells and glioma growth by directly controlling expression of Notch 1 [24]. Our current results demonstrate that miR-146 plays an important role in mouse NSC differentiation and that Notch 1 is a key target of miR-146 in mouse NSCs. Each miRNA could have multiple target genes [15,25]; indeed, several target genes have been predicted and some tested for miR-146, including those that encode the transcription factors NF-kB (nuclear factor kappa-light-chain-enhancer of activated B cells), BRMS1 (breast cancer metastasis-suppressor 1) and LPS (Lipopolysaccharides), as well as components of the FGF signalling pathway [26–28]. One of the questions addressed in the present study is whether the cell differentiation effect mediated by miR-146 in NSCs is directly related to repression of Notch 1 expression. Results suggest that miR-146 regulates NSC differentiation through repression of Notch 1 expression. Although Notch 1 is an important target gene of miR-146, other targets may also play a role in miR-146 function in NSCs. Transfection of miR-146 dramatically increases the percentage of GFAP-positive cells, and reduces the percentage of β-III-tubulin-positive cells in vitro. Previous studies have shown that miR-146 overexpression represses cell proliferation in gliomas [24]. In the present study, we analysed the role of miR-146 in neuronal differentiation from NSCs. Our results demonstrated that miR-146 promotes differentiation of glial cells from NSCs and increases the percentage of GFAP-positive cells in vitro.

In the present study, we analysed the role of miR-146 in proliferation and differentiation of NSCs. The results demonstrated that miR-146 inhibited NSC proliferation and promoted differentiation of glial cells from NSCs and increased the percentage of β-III-tubulin-positive cells in vitro under contained serum medium conditions.

Wei-Zhong Xiao carried out the cell culture, participated in the cell assay and drafted the manuscript. An-Qing Lu carried out the detection of genes and protein. Xin-Wei Liu participated in drafted the manuscript. Zhe Li participated in performed the statistical analysis. Ying Zi and Zhi-Wei Wang conceived of the study, and participated in its design and coordination.

We thank prof. Wanjin Hong for valuable suggestions and Xinwei Liu for effective feedback contraction of the manuscript.

ddH₂O

double distilled water

FGF

fibroblast growth factor

GFAP

Glial fibrillary acidic protein

HEK

human embryonic kidney

NF-kB

nuclear factor kappa-light-chain-enhancer of activated B cells

NSC

neural stem cell

Pax 6

paired box protein Pax-6; RBP-J, recombining binding protein suppressor of hairless; Sox2, sex determining region Y–box 2