miR-7 and miR-214 are specifically expressed during neuroblastoma differentiation, cortical development and embryonic stem cells differentiation, and control neurite outgrowth in vitro

Biochemical and Biophysical Research Communications 394 (2010) 921–927

Contents lists available at ScienceDirect

Biochemical and Biophysical Research Communications journal homepage: www.elsevier.com/locate/ybbrc

miR-7 and miR-214 are specifically expressed during neuroblastoma differentiation, cortical development and embryonic stem cells differentiation, and control neurite outgrowth in vitro Hailan Chen a, Ruby Shalom-Feuerstein b,c,d, Joan Riley a, Shu-Dong Zhang a, Paola Tucci a,e, Massimiliano Agostini a, Daniel Aberdam b,c,d, Richard A. Knight a, Giuseppe Genchi f, Pierluigi Nicotera a,g, Gerry Melino a,e,*, Mariuca Vasa-Nicotera a,** a

Medical Research Council Toxicology Unit, Hodgkin Building, Lancaster Road, PO Box 138, Leicester LE1 9HN, UK INSERM U898, Nice, France c University of Nice-Sophia Antipolis, Nice, France d INSERTECH, Bruce Rappaport Institute of the Technion, Haifa, Israel e Biochemistry Laboratory, IDI-IRCCS, C/O Department of Experimental Medicine and Biochemical Sciences, University of Rome ‘‘Tor Vergata”, 00133 Rome, Italy f Biochemistry Laboratory, Department of Pharmaco-Biology, University of Calabria, 87036 Rende (Cs), Italy g Deutsche Zentrum Fuer Neurodegenerative Erkrankungen (DZNE), Bonn, Germany b

a r t i c l e

i n f o

Article history: Received 9 March 2010 Available online 15 March 2010 Keywords: Apoptosis Cell death microRNA Neural differentiation Central nervous system Microarray

a b s t r a c t The mammalian nervous system exerts essential control on many physiological processes in the organism and is itself controlled extensively by a variety of genetic regulatory mechanisms. microRNA (miR), an abundant class of small non-coding RNA, are emerging as important post-transcriptional regulators of gene expression in the brain. Increasing evidence indicates that miR regulate both the development and function of the nervous system. Moreover, deficiency in miR function has also been implicated in a number of neurological disorders. Expression profile analysis of miR is necessary to understand their complex role in the regulation of gene expression during the development and differentiation of cells. Here we present a comparative study of miR expression profiles in neuroblastoma, in cortical development, and in neuronal differentiation of embryonic stem (ES) cells. By microarray profiling in combination with real time PCR we show that miR-7 and miR-214 are modulated in neuronal differentiation (as compared to miR-1, -16 and -133a), and control neurite outgrowth in vitro. These findings provide an important step toward further elucidation of miR function and miR-related gene regulatory networks in the mammalian central nervous system. Ó 2010 Elsevier Inc. All rights reserved.

1. Introduction Highly orchestrated programmes of gene expression act to shape the developing nervous system. This tight regulation is mediated by a variety of transcriptional and post-transcriptional events that control the expression of individual gene products. More recently, it has become clear that protein expression can also be modulated by several classes of small RNAs, including small interfering (si), Piwi and microRNA (miR). The combination of these diAbbreviations: miR, microRNA; si, small interfering; CNS, central nervous system; ES, embryonic stem; ESR1, oestrogen receptor alpha; RA, retinoic acid; DIV, days in vitro; E, embryonic; P, postnatal day; W, weeks postnatal * Corresponding author at: Medical Research Council Toxicology Unit, Hodgkin Building, Lancaster Road, PO Box 138, Leicester LE1 9HN, UK. ** Corresponding author. E-mail addresses: [email protected], [email protected] (G. Melino), mvn1@lei cester.ac.uk (M. Vasa-Nicotera). 0006-291X/$ - see front matter Ó 2010 Elsevier Inc. All rights reserved. doi:10.1016/j.bbrc.2010.03.076

verse regulatory processes on protein expression and activity confer great plasticity to the cellular responses to changes in its local environment. miRs are a recently discovered class of short non-coding RNA genes that act post-transcriptionally as negative regulators of gene expression [1]. A large body of research shows that animal miRs play fundamental roles in many biological processes, including cell growth and apoptosis, hematopoietic lineage differentiation, insulin secretion, brain morphogenesis, and muscle cell proliferation and differentiation [2,3]. During development, many miRs are expressed in neurons or show distinct expression patterns within the developing central nervous system (CNS), suggesting their importance in brain development and function. However, functional studies of miRs in the vertebrate nervous system are still very limited. A number of studies have begun to address the role of miR in neuronal differentiation. For example, a microarray comparison of the miR

922

H. Chen et al. / Biochemical and Biophysical Research Communications 394 (2010) 921–927

profile in embryonic, postnatal, and adult brain revealed differential changes in nine miRs, including miR-9 and -124 [4,5]. In vitro, the levels of both miRs increased sharply during the transition from neuronal precursors to neurons in differentiating embryonic stem (ES) cells [6]. Overexpression of both miRs shifted the differentiation of the precursors toward a neuronal fate, while inhibition had the opposite effect [7]. Another microarray study, comparing miR expression profiles in rat neuronal progenitors between E11 and E13 also emphasised the importance of miR-9 and -124 among the 21 miRs whose expression was increased and the 11 where reduced expression was found [8]. Moreover, ectopic expression of miR-124 also increased the degree of retinoid-induced differentiation in a neuroblastoma cell line [9]. In neuroblastoma cells with enhanced expression of N-MYC, there is correspondingly increased expression of miR-18a and -19a, which repress oestrogen receptor alpha (ESR1), and overexpression of ESR1 results in growth arrest and neuronal differentiation [10], suggesting that a MYC/miR/ESR1 axis is important in development of the sympathetic nervous system. Moreover, links between miRs dysfunction and neurological diseases become more and more apparent [11]. Despite the accumulating evidence that miR play important roles in brain development and disorders, our knowledge of miR function in the vertebrate nervous system is still quite limited. We have established a classic model of neuronal differentiation and apoptosis treating neuroblastoma cells with retinoic acid (RA) [12–16]. By combining microarray expression profiling with miRspecific real time PCR, we have compared the expression profiles of miR in neuroblastoma cells induced to differentiate with RA, in the development of mouse brain cortex, and in neuronal differentiation from mouse ES cells. While there are some differences in the pattern of miR expression in the different models, we did not identify a prominent expression for miR-9 and -124 in any system, but did find additional miRs, namely miR-7 and miR-214, implicated in neuronal differentiation and in the control of neurite outgrowth in vitro, which will serve as an important basis for detailed studies of individual miR, their target genes, and the miR-related regulatory networks in the mammalian central nervous system.

2. Materials and methods 2.1. Human neuroblastoma cell line SH-SY5Y cells (ATCC, UK) were maintained in non-differentiating medium (DMEM, 10% FCS, 1% penicillin/streptomycin). Twenty-four hours of post-seeding, the non-differentiating medium was replaced with differentiating medium (DMEM, 1% FCS, 1% penicillin/streptomycin, 1% Glutamine, 10 lM RA) and cells incubated for further 48 h. 2.2. Primary culture of mouse cortical neurons Mouse cortical neuronal cultures were prepared from E16–E17 mouse embryos and cultured on poly-D-lysine-coated cell culture dishes in a defined serum-free medium (Neurobasal, 2% B-27 supplement, 2 mM glutamine, 1% penicillin/streptomycin). Cytosine arabinoside (10 lM) was added at day 7 after plating. The cells were then collected in Trizol (Invitrogen) every 2 days until day in vitro (DIV) 12. 2.3. Mouse embryonic stem (ES) cells Mouse ES cell lines CGR8 and CGR8/Sox1-GFP (a gift of Smith) were routinely cultured in flasks coated with 0.1% gelatin in ES medium (GMEM, 10% FCIII, 1% nonessential amino acids, 1 mM

sodium pyruvate, 0.1 mM b-mercaptoethanol and 103 U/ml LIF (Leukemia Inhibitory Factor)). For neural differentiation, confluent NIH-3T3 cells were fixed with 3% formaldehyde, washed with PBS and incubated with glycine to saturate free formaldehyde sites. For ES cells differentiation, the cells were cultured on fixed NIH-3T3 cells in differentiation medium (similar to ES medium but without LIF and FCIII, and with 10% knock out serum). 2.4. Microarray printing, labelling, and hybridization We use in-house made two-colour cDNA microarrays to measure the expression levels of miR. Briefly, 3.0 lg of total RNA was labelled using FlashTag™ kits according to the manufacturers instructions (Genisphere). Reference RNA (or control RNA, e.g., RNA from native SH-SY5Y cells, or DIV 0 for mouse cortical neurons, or differentiation day 0 for mouse ES cells), labelled with OysterÒ-550 was hybridized against RNA labelled with OysterÒ-650 from the experiment RNA (e.g., RNA from RA-differentiated SHSY5Y cells, or different DIV for mouse cortical neurons, or differentiation days for mouse ES cells), and reverse (Reference-OysterÒ650, experiment-OysterÒ-550) labelling reactions. Hybridizations were performed overnight at 52 °C on microarrays printed in-house using the miRCURY LNA™ ready-to-spot probe set version 208010V8.1 (Exiqon). Following hybridization, the microarrays were washed at RT in 2 SSC containing 0.2% (w/v) SDS for 5 min, 1 SSC for 5 min, 0.2 SSC for 5 min, and dried by centrifugation. 2.5. Microarray data analysis Microarray slides were scanned using an Axon 4200A scanner to acquire the microarray images which were subsequently processed with GenePix Pro software (Molecular Devices) to generate the expression data in GPR (Genepix Result File) format, all according to manufacture’s instructions. The median fluorescent intensity of a feature spot on a microarray slide was used to represent the expression level of the corresponding gene. Each microarray was then normalized by globally shifting the mode of log-ratio values to 0, implicitly making the assumption that on each microarray slide most genes are not differentially expressed between the two samples. The normalized data were subsequently analyzed using the statistical method described by Zhang and Gant [17] for gene expression experiments involving both forward and reverse labelled microarrays. In the miR microarray data, the threshold p-value was set such that on average only two of the genes (miRs) declared significant is expected to be false, then genes (miRs) with p-values lower than the above set threshold are declared as statistically significant. The changes >1.6-fold and p < 0.05 of up-regulated miRs and the changes <0.7-fold and p < 0.05 of down-regulated miRs were used for further analysis. Significant changes were verified by real time PCR as described below. 2.6. Real time PCR Total RNA from cells was isolated using Trizol (Invitrogen) according to the manufacturer’s instructions. Then RNA was reverse transcribed using TaqMan MicroRNA Reverse Transcription kit and qRT-PCR was performed with TaqMan universal master mix (Applied Biosystem) and specific primers for miRs. SnoRNA202 (mouse) or RNU6B (human) were used as internal control (Applied Biosystem). The expression of each miR was defined from the threshold cycle (Ct), and relative expression levels were calculated using the 2 DDC t method after normalization with reference to expression of internal control.

H. Chen et al. / Biochemical and Biophysical Research Communications 394 (2010) 921–927

2.7. High-throughput neurite outgrowth assays SH-SY5Y cells were maintained in non-differentiating medium. Twenty-four hours of post-seeding, non-differentiating medium was replaced with differentiating medium, containing 10 lM RA, and the cells incubated for further 48 h. The cells were then transfected with 10 nM Pre-miR™ miRNA Precursors, or 75 nM AntimiR™ miRNA Inhibitors, or FAM-labeled Negative Control (Ambion), using the SiPORT neoFX transfection agent (Ambion) according to the manufacturer’s instruction. After 48 h the cells were fixed with 3% paraformaldehyde in PBS followed by treatment with 0.1% Triton X-100 for 5 min and 50% normal goat serum (NGS) in PBS for 1 h at RT. Neuronal-specific anti-b III tubulin antibody was used to stain the neurite processes (1:5000). Bound antibody and nucleus were visualized using Alexa 546-conjugated secondary antibody (1:500) and Hoechst 33342 (1:1000), respectively. 2.8. Automated image analysis Cells were imaged using a Cellomics Kinetic Scan Reader high content microscope system and analysed using neurite outgrowth for the outgrowth assay. Twenty images per well were taken at 10 magnification in a fully automated and blind manner. The total number of cell counts and the average of neurite length per neuron in each experiment in duplicate were determined for neurite outgrowth assay. 3. Results 3.1. miR-7 and miR-214 are modulated during neuroblastoma cells differentiation In order to identify the miRs involved in the differentiation of human SH-SY5Y cells, we analysed the expression profile of miR in the cells induced to differentiate with RA (10 lM) using microarrays with 700 oligonucleotide probes complementary to mature forms of miRs of human origin, based on version 10.1 of the Sanger miRBase (http://microrna.sanger.ac.uk/sequences). Data from the microarray (analyzed as described in Section 2) showed that 73 miRs were modulated during differentiation induced by RA (Table S1). To validate the microarray platform, we confirmed the expression of 12 miRs which were most strongly (statistical values are shown in the table) up- or down-regulated (Table 1) by qRT-PCR, using the same RNA samples that were used for the microarrays. Real time PCR confirmed the modulation of several miRs, including down-regulation of miR-7 (Fig. 1A), and up-regulation of miR-214 (Fig. 1B) after differentiation of the cells with RA for 48 h.

Table 1 miR regulated during RA-induced neuroblastoma differentiation. Name

Fold

p-Value

Sanger mature miR-sequence

Up-regulated hsa-miR-132 hsa-miR-16 hsa-miR-27b hsa-mir-27a hsa-miR-214 hsa-miR-197

2.67 2.62 2.45 1.99 1.80 1.65

0.0138848 0.0300957 0.0236875 0.0048806 0.0071070 0.0001151

59-uaacagucuacagccauggucg-80 14-uagcagcacguaaauauuggcg-35 51-uucacaguggcuaaguucugc-81 51-uucacaguggcuaaguuccgc-71 71-acagcaggcacagacaggcagu-92 48-uucaccaccuucuccacccagc-69

Down-regulated hsa-miR-133a hsa-miR-508-3p hsa-miR-7 hsa-miR-1 hsa-mir-205 hsa-mir-20b

0.42 0.56 0.56 0.57 0.66 0.69

0.0001546 0.0005188 0.0500789 0.0076927 0.0098568 0.0001416

53-uuugguccccuucaaccagcug-74 61-ugauuguagccuuuuggaguaga-83 24-uggaagacuagugauuuuguugu-46 53-uggaauguaaagaaguauguau-74 34-uccuucauuccaccggagucug-55 6-caaagugcucauagugcagguag-28

923

3.2. Effect of miR-7 and miR-214 on neurite outgrowth during neuroblastoma cells differentiation To investigate whether RA-induced modulation of these two miRs has functional significance, we measured the neurite length of SH-SY5Y cells transfected with either pre-miR precursors, or anti-miR inhibitors, or with negative controls. We took advantage of the high throughput technology to automatically visualize and measure neurite outgrowth. We tested all miRs shown in Table 1, but only two showed a coherent behaviour, that is opposite effect of pre-miR versus anti-miR. In fact miR-1, -16 and -133a were not able to show a direct coherent effect on neurite outgrowth. We found that neurite length significantly increased when miR-214 (Fig. 1C and D) (whose expression was increased following RA, Fig. 1B) was over-expressed. However, when the expression of the miR-214 was inhibited, neurite length remained statistically unchanged (Fig. 1D). Conversely (Fig. 1C and D) we found a reduction in the neurite outgrowth by transfecting the cells with the precursor of miR-7, and an increase of the neurite outgrowth when the SH-SY5Y were transfected with an inhibitor of the miR-7. Fig. 1D shows the statistical validation of results obtained by high throughput analysis. These results indicate that not only miR-7 and miR-214 were modulated during neuroblastoma differentiation, but also that they had a relevant role during this phase, as they are able to modulate per se the outgrowth of neurites. Therefore, we decided to investigate the involvement of these two miRs in other more physiological ex vivo cellular models, namely mouse cortical neurons in vitro and cerebellar cortical neurons during late developmental stages. 3.3. Expression of miR-7 and miR-214 during differentiation of mouse cortical neurons ex vivo miR expression levels were also evaluated in spontaneously differentiating primary mouse cortical neurons in vitro every 2 days until DIV 12. The miR-7 levels, whose expression after microarray and real time PCR was found down-regulated during neuroblastoma differentiation, increased progressively during differentiation of mouse primary cortical neurons in vitro (Fig. 2A). In keeping with the expression pattern in RA-differentiated neuroblastoma cells, the levels of miR-214 increased (Fig. 3A). 3.4. miR expression during development of mouse cerebral cortex Figs. 2B and 3B shows the changes in expression levels of miRs7 and -214 during the development of mouse brain cortex from E13 to 6 weeks postnatal. The data are expressed relative to their respective values at E13. During embryonic and postnatal development the levels of miR-214 dropped significantly (Fig. 3B), while expression of miR-7 did not change during the embryonic development but progressively increased during postnatal cortical development (Fig. 2B). 3.5. miR expression during neuronal differentiation of mouse ES cells In vitro ES cells can be induced to differentiate, recapitulating the physiological in vivo differentiation. Because of the high relevance of this model, we decided to evaluate in this model the regulation of miR-7 and miR-214. In order to study the possible role of miRs in neural commitment, mouse ES cells were cultured on fixed feeder PA6 fibroblast cells in the absence of serum. To quantify the efficacy of neural differentiation, we used ES cells with stable GFP gene expression under the control of the sox-1 promoter. Cells were collected at the

924

H. Chen et al. / Biochemical and Biophysical Research Communications 394 (2010) 921–927

Fig. 1. Real time PCR and high-throughput screen of miR regulated during neuroblastoma cells differentiation. Cells were treated for 48 h with 10 lM RA and endogenous levels of miR-7 (A) and miR-214 (B) were assayed by qRT-PCR in order to validate the results obtained from the microarray; the result were normalized to RNU6b. Data represent mean ± SD of three different experiments analyzed in triplicate. (C) Example of neurite outgrowth evaluation by immunostaining, as described in Section 2, of SHSY5Y cells transfected with pre-miR, or anti-miR, or a negative control. (D) SH-SY5Y were transfected with pre-miRs, or with anti-miRs, or a negative control, and neurite growth was measured as described in Section 2. The results were expressed as average of the neurite length per outgrowth neuron. Experiment was performed in triplicate, and 25 or 40 images acquired per well. ***p < 0.001; **p < 0.01; *p < 0.05; t-test.

indicated times and neural differentiation was evaluated by FACS analysis, immunofluorescence microscopy and real time PCR (data not shown). ES cells were differentiated into large colonies of neural cells. Within 7 days of culture, 60% and 80% of the cells expressed the putative neural precursor markers, CD57 and sox-1, respectively. Enhancement in the expression levels of the bIII-

tubulin and neurofilament, was demonstrated by immunofluorescence microscopy and real time PCR (data not shown), which together showed efficient neuronal differentiation. miR-214 showed an early increase in expression at day 1, which subsequently declined on days 4 and 7 (Fig. 3C). In contrast, expression of miR-7 decrease although with an irregular fashion (Fig. 2C).

H. Chen et al. / Biochemical and Biophysical Research Communications 394 (2010) 921–927

Fig. 2. miR-7 regulation during neuronal differentiation. (A) Expression levels of miR-7 during mouse cortical neuron development. Steady state expression levels were evaluated by qRT-PCR in primary mouse cortical neuron in culture every 2 days until DIV 12 (DIV, day in vitro). (B) Expression of miR-7, by qRT-PCR, during the development of mouse brain cortex for a period from E13 to adult (E, embryonic; P, postnatal day; W, weeks postnatal) (N = 6 at E13, E16, E18 and P1; N = 3 for P11, 3W and N = 3 at 6W). (C) Regulation of miR-7 during the ES cells neuronal differentiation after 1, 4, and 7 days. The results were normalized to SnoRNA202. Data represent mean ± SD of three different experiments analyzed in triplicate.

925

Fig. 3. miR-214 regulation during neuronal differentiation. (A) Expression levels of miR-214 during mouse cortical neuron development. Steady state expression levels were evaluated by qRT-PCR in primary mouse cortical neuron in culture every 2 days until DIV 12. (B) Expression of miR-214, by qRT-PCR, during the development of mouse brain cortex for a period from E13 to adult. Symbols as in Fig. 2. (C) Regulation of miR-214 during the ES cells neuronal differentiation after 1, 4, and 7 days. The result were normalized to SnoRNA202. Data represent mean ± SD of three different experiments analyzed in triplicate.

4. Discussion 3.6. Regulation of other miRs Even though other miRs were not sufficient per se to cause neurite outgrowth as discussed above (Table 1 and Fig. 1), we evaluated the regulation of other miRs using our cellular models. miR-1, as reported in Fig. 4, showed a consisted up-regulation in all differentiation models tested. This indicates a relevant, although not necessary not sufficient, role of miR-1 during neuronal differentiation. miR-133a (Fig. S1) showed a consistent induction in all models, despite that it was not significantly regulated in RA-treated neuroblastoma cells. miR-16 was less consistent (Fig. S2).

The nervous system undergoes extensive changes in patterning, remodelling, and cell specification during development. In mature mammals, it consists of networks of cells that reach every organ and part of the body to conduct impulses back and forth to control essential physiological responses to internal and external stimuli in a timely fashion. To accomplish its tasks, the nervous system uses a large number of cells with different properties to form exceedingly complex structures and depends on an array of elaborate gene regulatory mechanisms for its development and function. miRs are involved in a variety of physiological and pathological processes in multicellular organisms [1–3], ranging from patterning (for example in the epidermis [18,19]) to cancer development.

926

H. Chen et al. / Biochemical and Biophysical Research Communications 394 (2010) 921–927

Fig. 4. miR-1 regulation during neural differentiation. Expression level of miR-1 during in vitro differentiation of neuroblastoma cells treated with RA (A), and during mouse cortical neuronal development (B). (C) Expression during the development of brain cortex in mouse from E13 to adult stages. (D) Regulation of miR-1 during the ES cells neuronal differentiation. All technical details and symbols are as described in Fig. 2.

Since the central nervous system is a rich source of miRs that often display a brain specific expression pattern, and since a single miR is able to target up to a few hundreds of different mRNAs, it is hardly surprising that the number of roles assigned to miRs during all stages of central nervous system development and function is rapidly expanding. Moreover, the number of miR genes found to be expressed in the nervous system seems to be larger than that in many other organs, perhaps partly reflecting the fact that the nervous system contains many types and subtypes of cells. Toward understanding the complexity of miRs expression, in this study we have compared the expression profiles of miRs involved in neuronal differentiation in three models: differentiation of neuroblastoma cells induced with RA, time kinetics of expression in the developing mouse cerebral cortex, and neuronal differentiation from ES cells. We have focused on two miRs (miR-7 and miR-214) in all three systems out of a total of 73 whose expression was changed by microarray analysis during neuroblastoma differentiation. Even more interesting, these two miRs were able to regulate neurite outgrowth per se, suggesting a pivotal role in this process. These experiments have revealed that although during neuronal differentiation and neurodevelopment distinct areas of the central nervous system express similar miRs, the direction and degree of change relative miR levels vary significantly in different regions or in different development stages. When treated with RA the SH-SY5Y cells, neuronal MYCN-driven cancer cells, will terminally differentiate into neuron-like cells. Accompanied with the classical morphological changes of neurite outgrowth, expression of miR-214 is significantly induced over

time, as we confirmed both by microarray analysis and real time PCR, suggesting that this miR may play a role in differentiation or cell fate determination, in addition to its potential functions in adults [20–22], as we saw an increase in the development of the mouse cortical neurons and of brain cortex. Although expression of anti-miR-214 had no significant effect on neurite outgrowth of neuroblastoma cells, regulation of miR-214 not only is relevant for cell lineage-specific differentiation (i.e., SH-SY5Y), but also may influences ES cell commitment. Pluripotent ES cells, which express very low levels of miR-214, readily activate miR-214 transcription when induced to differentiate. Indeed, miR-214 accumulation is evident at the very initial stages of cell differentiation of the ES cells. Thus, after RA treatment, miR-7 was found reduced both on the array and by real time PCR, as well as in ES cells during the neuronal differentiation, which suggests that it has a more general influence on the process of differentiation and development. In contrast, miR-7 expression increased during mouse cortical neuron development and differentiation. Others [8] have also shown increases in miR-7 expression between days E11 and E13 of mouse cortical development. These apparent differences in miR-7 kinetics may, at least in part, reflect the fact that SH-SY5Y cells are dopaminergic, whereas neurons in the developing brain will contain a variety of neurotransmitters. Moreover, overexpression of miR-7, by pre-miR-7 precursor, reduced neurite outgrowth when the differentiation occurred in SH-SY5Y cells, whereas expression of antimiR-7 enhanced the size of the neural net. Although others have shown a small increase in miR-7 expression 12 days after addition

H. Chen et al. / Biochemical and Biophysical Research Communications 394 (2010) 921–927

of RA to SH-SY5Y cells, changes in expression of miR were more pronounced and occurred earlier in the differentiation process [9]. Nevertheless, we believe that our findings provide a significant evidence for a down-regulation and a functional involvement of miR-7 at an early stage (48 h) of neuroblastoma differentiation. All together these results suggest that miR expression profile can serve as a marker of neuronal development and that specific miR may contribute to the developmental process. Acknowledgments This work has been supported by the Medical Research Council, UK; ‘‘Alleanza contro il Cancro” (ACC), MIUR/PRIN (RBIP06LCA9_0023), AIRC (2008-2010_33-08), ISS ‘‘Program Italia-USA” N526D5, Italian Human ProteomeNet RBRN07BMCT_007, Telethon (GGPO4110) and RF 06 73UO3, RF07EC57UO2 G.M. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at doi:10.1016/j.bbrc.2010.03.076. References [1] A. Ventura, T. Jacks, microRNAs and cancer: short RNAs go a long way, Cell 136 (2009) 586–591. [2] D. Bartel, microRNAs: target recognition and regulatory functions, Cell 136 (2009) 215–233. [3] E. Bernstein, S. Kim, M. Carmell, et al., Dicer is essential for mouse development, Nat. Genet. 35 (2003) 215–217. [4] A. Krichevsky, K. King, C. Donahue, et al., A microRNA array reveals extensive regulation of microRNAs during brain development, RNA 9 (2003) 1274–1281. [5] K. Kosic, A. Krichevsky, The elegance of microRNAs: a neuronal perspective, Neuron 47 (2005) 779–782. [6] J. Silber, D. Lim, C. Petritsch, et al., miR-124 and miR-137 inhibit proliferation of glioblastoma multiforme cells and induce differentiation of brain tumour stem cells, BMC Med. 6 (2008) 14.

927

[7] A.M. Krichevsky, K.C. Sonntag, O. Isacson, K.S. Kosik, Specific micro-RNAs modulate embryonic stem cell-derived neurogenesis, Stem Cells 24 (2006) 857–864. [8] J. Nielsen, P. Lau, D. Maric, et al., Integrating microRNA and mRNA expression profiles of neuronal progenitors to identify regulatory networks underlying the onset of cortical neurogenesis, BMC Neurosci. 10 (2009) 98. [9] M. Le, H. Xie, B. Zhou, et al., microRNA-125b promoted neuronal differentiation in human cells by repressing multiple targets, Mol. Cell. Biol. 29 (2009) 5290– 5305. [10] J. Loven, N. Zinin, T. Wahlstrom, et al., MYCN-regulated microRNAs repress estrogenreceptor-{alpha} (ESR1) expression and neuronal differentiation in human neuroblastoma, Proc. Natl. Acad. Sci. USA (2010). epub ahead of print. [11] S. Hebert, B. De Strooper, Alterations of the microRNA network cause neurodegenerative disease, Trends Neurosci. 32 (2009) 199–206. [12] G. Melino, C.J. Thiele, R.A. Knight, M. Piacentini, Retinoids and the control of growth/death decisions in human neuroblastoma cell lines, J. Neurooncol. 31 (1997) 65–83. [13] G. Melino, M. Draoui, M. Piacentini, et al., Retinoic acid receptors mediate tissue-transglutaminase induction in human neuroblastoma cells undergoing apoptosis, Exp. Cell Res. 255 (1997) 55–61. [14] S. Bernardini, G. Melino, F. Saura, et al., Expression of co-factors (SMRT and Trip-1) for retinoid acid receptors in human neuroectodermal cell lines, Biochem. Biophys. Res. Commun. 234 (1997) 278–282. [15] P.E. Lovat, M. Annicchiarico-Petruzzelli, M. Corazzari, et al., Differential effects of retinoic acid isomers on the expression of nuclear receptor co-regulators in neuroblastoma, FEBS Lett. 445 (1999) 415–419. [16] P.E. Lovat, S. Oliverio, M. Corazzari, et al., Bak: a downstream mediator of fenretinide-induced apoptosis of SH-SY5Y neuroblastoma cells, Cancer Res. 63 (2003) 7310–7313. [17] S. Zhang, T. Gant, A statistical framework for the design of microarray experiments and effective detection of differential gene expression, Bioinformatics 20 (2004) 2821–2828. [18] A.M. Lena, R. Shalom-Feuerstein, P. Rivetti di Val Cervo, et al., miR-203 represses ’stemness’ by repressing DeltaNp63, Cell Death Differ. 15 (2008) 1187–1195. [19] D. Aberdam, E. Candi, R.A. Knight, G. Melino, miRNAs, ’stemness’ and skin, Trends Biochem. Sci. 33 (2008) 583–591. [20] S. Decembrini, D. Bressan, R. Vignali, et al., microRNAs couple cell fate and developmental timing in retina, Proc. Natl. Acad. Sci. USA 15 (2009) 21179– 21184. [21] A.S. Flynt, N. Li, E.J. Thatcher, et al., Zebrafish miR-214 modulates Hedgehog signaling to specify muscle cell fate, Nat. Genet. 39 (2007) 259–263. [22] A.H. Juan, R.M. Kumar, J.G. Marx, et al., mir-214-dependent regulation of the polycomb protein Ezh2 in skeletal muscle and embryonic stem cells, Mol. Cell 36 (2009) 61–74.