Neurochem Res DOI 10.1007/s11064-014-1247-9

OVERVIEW

MicroRNAs in Neuroblastoma: Small-Sized Players with a Large Impact Feng Zhi • Rong Wang • Qiang Wang • Lian Xue • Danni Deng • Suinuan Wang Yilin Yang

•

Received: 13 November 2013 / Revised: 22 December 2013 / Accepted: 21 January 2014 Ó Springer Science+Business Media New York 2014

Abstract Neuroblastoma, a malignant embryonal tumor of the sympathetic nervous system, is the most common solid extracranial malignancy of childhood and accounts for 15 % of all childhood cancer deaths. The biological behavior of neuroblastoma is extensively heterogeneous, ranging from spontaneous regression to rapid progression despite multimodal aggressive therapy. Although the molecular basis of neuroblastoma has received considerable attention over the past decade, elucidating the mechanisms for the aggressive progression of neuroblastoma is needed for improving the efficacy of treatment. miRNAs (microRNAs) are small non-coding RNA molecules generally 19–22 nucleotides in length. miRNAs regulate 60 % of human gene expression at the post-transcriptional level by targeting regions of sequence complementarity on the 30 -untranslated regions (30 -UTRs) of specific mRNAs. miRNAs can either cause degradation of mRNAs or can inhibit their translation and therefore play major roles in normal growth and development. miRNA dysregulation has oncogenic or tumor-suppressive functions in virtually all forms of cancer, including neuroblastoma. The present review highlights the current insights on dysregulated miRNAs in neuroblastoma and on their roles in the

Feng Zhi and Rong Wang have contributed equally to this work. F. Zhi  R. Wang  L. Xue  D. Deng  Y. Yang (&) Modern Medical Research Center, Third Affiliated Hospital of Soochow University, 185#, Juqian Road, Changzhou 213003, Jiangsu, China e-mail: [email protected] Q. Wang  S. Wang Department of Neurosurgery, Third Affiliated Hospital of Soochow University, 185#, Juqian Road, Changzhou 213003, Jiangsu, China

diagnosis, prognosis, and treatment of this malignancy. As a rapidly evolving field of basic and biomedical sciences, miRNA research holds a great potential to impact on the management of neuroblastoma. Keywords MicroRNA  Neuroblastoma  Differentiation  Tumorigenesis  Metastasis  Diagnosis

Introduction Neuroblastoma, the most common extracranial childhood tumor, originates from precursor cells of the sympathetic nervous system and accounts for [15 % of all childhood cancer deaths [1]. The heterogeneous clinical behavior, ranging from spontaneous regression to rapid progression, is attributable to both biological and genetic characteristics of the tumor [2]. Neuroblastomas with favorable clinical traits (Stages 1, 2 and 4S) often undergo complete regression or differentiation without therapy, while high-stage, highly aggressive neuroblastoma often ends fatally despite recent therapeutic improvements [3]. Survival rates of patients with unfavorable neuroblastoma can only be improved by a better understanding of the molecular pathogenesis of neuroblastoma. This better understanding may also ameliorate patient stratification and prognostic prediction and spur development of superior targeted therapies. miRNAs (microRNAs) are a class of non-coding, 19–22 nucleotide, single-stranded RNAs transcribed in a developmental- and tissue-specific manner during tumorigenesis [4]. More than 4,500 mature miRNAs have been identified in humans (http://www.mirbase.org), and most of these miRNAs are highly conserved across species. The expression of more than 60 % of human protein-coding genes is controlled by miRNAs [4]. Several miRNAs can target the same mRNA

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and, conversely, each miRNA can target several mRNAs, thus leading to additional layers of post-transcriptional control of gene expression. Dysregulation of miRNAs was discovered to play an important role in a wide variety of human cancers by regulating the expression of various oncogenes and tumor suppressors, thereby supporting the proposal of using miRNAs as novel effective targets for anticancer therapies [5–7]. This review will highlight the major findings from the existing literature on miRNAs and discuss their significance in the tumorigenesis of neuroblastoma. In addition, it will discuss the potential use of miRNAs as diagnostic and prognostic biomarkers, as well as present targets for future therapeutics.

miRNA Biogenesis Pathway Since the first miRNA lin-4 was discovered by Lee et al. [8], the number of known human miRNAs has grown to over 4,500 and is still growing [9]. The canonical miRNA maturation process includes the production of the primary miRNA transcript, the pri-miRNA, by RNA polymerase II or III and the cleavage of the pri-miRNA by the microprocessor complex Drosha/DGCR8 in the nucleus. The resulting precursor hairpin, the pre-miRNA, is exported from the nucleus by Exportin-5-Ran-GTP [10, 11]. In the cytoplasm, the RNase Dicer, in complex with the doublestranded RNA-binding protein TRBP, cleaves the premiRNA hairpin to its mature length [12]. The functional strand of the mature miRNA is loaded together with the Argonaute (Ago2) proteins into the RISC (RNA-induced silencing complex). There, the mature miRNA guides RISC to regulate target mRNAs by either direct cleavage of the target mRNA or inhibition of target protein synthesis, depending on the degree of sequence complementarity between the miRNAs and their target 30 -UTRs (30 untranslated regions) [13]. In addition to the canonical miRNA biogenesis pathway that is Drosha/DGCR8dependent and Dicer-dependent, there also exist alternative miRNA biogenesis pathways, such as the mirtron pathway, which is Drosha/DGCR8-independent and Dicer-dependent, and the miR-451 biogenesis pathway, which is Drosha/DGCR8-dependent and Dicer-independent [14].

miRNA Dysregulation in Neuroblastoma A number of expression profiling studies have demonstrated dysregulation of miRNAs in neuroblastoma. Many researchers choose to use high-throughput technologies to explore aberrantly expressed miRNAs in neuroblastoma. Chen et al. [15] conducted one of the first miRNA profiling studies of neuroblastoma samples. They examined the

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expression of 157 miRNAs in primary neuroblastomas and indicated that 32 miRNAs were differentially expressed in favorable and unfavorable tumor subtypes. Furthermore, they found that miRNA expression levels substantially changed in a MYCN amplified cell line following exposure to RA (retinoic acid), a compound that reduces MYCN expression and induces neuronal differentiation. These findings suggested the potential role of miRNAs in neuroblastoma pathogenesis. Schulte’s group [16] identified that 7 miRNAs were significantly upregulated in MNA (MYCN-amplified) neuroblastomas compared with nonMNA neuroblastomas. Bray et al. [17] examined the miRNA expression levels between MNA and non-MNA neuroblastomas and found a total of 37 miRNAs that were significantly differentially expressed: 14 miRNAs were upregulated and 23 miRNAs were downregulated. 50 unique miRNAs identified by Mestdagh et al. [18] were differentially expressed between low-risk or high-risk MNA and MYCN single-copy tumors. Among them, miR92a and miR-15b expression levels were linked with increased MYCN/c-MYC signaling, while miR-128a and miR-628-3p expression levels were inversely linked with increased MYCN/c-MYC signaling. De Peter et al. [19] compared the miRNA expression profile in low-risk and high-risk neuroblastoma patients. They established a 25-miRNA signature, 14 were highly expressed in highrisk patients, while 11 were highly expressed in low-risk patients. In Gattolliat’s work [20], they found that 17 miRNAs were differentially expressed between low-risk and high-risk neuroblastomas, and most of them belonged to the imprinted human 14q32.31 miRNA cluster. miR487b and miR-410 were the most significantly downregulated miRNAs in the high-risk group. Lin et al. [21] observed global downregulation of miRNA expression in advanced neuroblastomas and identified 27 miRNAs differentially expressed between low-risk and high-risk patients. Guo et al. [22] found 54 miRNAs that were significantly altered in metastatic neuroblastoma compared to primary neuroblastoma. Through a novel integrated analysis, Das’s group [23] identified 67 epigenetically silenced miRNAs in primary neuroblastoma tumors and cell lines. This set of epigenetically regulated miRNAs targeted a multitude of genes that were overexpressed in tumors from patients with poor survival with extensive redundancy. Some researchers focus on individual miRNAs that may play important roles in neuroblastoma. The two most commonly reported miRNAs are miR-34a and the miR-1792 cluster. miR-34a is one of the most extensively studied miRNAs in neuroblastoma. Welch et al. [24] first reported that miR-34a was generally expressed at lower levels in unfavorable primary neuroblastomas and cell lines relative to normal adrenal tissue. Subsequently, Wei et al. [25] confirmed that neuroblastomas with 1p36 loss expressed

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lower levels of miR-34a than those with normal copies of 1p36. Furthermore, more and more groups confirmed the role of miR-34a in neuroblastoma. Cole et al. [26] reported that the expression of miR-34a was lower in primary tumors with a hemizygous deletion of the miR-34a locus compared with those without a 1p36 deletion, but no mutations were discovered by resequencing the miR-34a locus in 30 neuroblastoma cell lines. Feinberg et al. [27] observed that the reduced expression levels of miR-34a in neuroblastomas harboring 1p36 loss were not caused by mutations in the TP53-binding site. The miR-17-92 cluster, also called Oncomir-1, is among the first miRNAs to be validated as showing oncogenic potential. It is also involved in the neuroblastoma pathogenesis. Schulte et al. [28] reported that the miR-17-92 cluster and the miR-181 family were overexpressed in unfavorable neuroblastomas and that miR-542-5p and miR628 were expressed in favorable neuroblastomas and virtually absent in unfavorable neuroblastomas. Mestdagh et al. [29] found that the miR-17-92 cluster activity was the highest in MNA tumors, followed by MYCN single copy high-risk tumors and MYCN single copy low-risk tumors. Fontana et al. [30] found that in primary neuroblastoma, the majority of cases showed a rise of miR-17-5p level, which was particularly severe in patients with MYCN amplification. Laneve et al. [31] found that 3 neuronal miRNAs, miR-9, miR-125a, and miR-125b, were downregulated in primary neuroblastoma tumors. There are also many other individual miRNAs that are differentially expressed in neuroblastoma. miR-27b was downregulated in neuroblastoma tissues, compared to adjacent non-cancer tissue [32]. miR-29a, miR-29b, and miR-29c were downregulated in neuroblastomas compared with normal tissues [33]. The high expression of miR-128 was coincided with favorable features, such as early age at diagnosis, favorable Shimada category, prognostically favorable stages and MYCN single-copy status [34]. miR137 was downregulated in primary neuroblastomas. It was also expressed at lower levels in patients with MYCN amplification or high NTRK1 (neurotrophic tyrosine kinase receptor type 1) expression than in patients with MYCN single copy or low NTRK1 expression [35]. miR-145 expression was downregulated in either human neuroblastoma tissues or in cultured neuroblastoma cell lines compared with normal dorsal ganglia [36]. miR-149-3p was significantly lower in tumors of high-risk groups than in tumors of low- and intermediate-risk groups [37]. miR-152 and miR-338 were overexpressed in many neuroblastoma samples and in all neuroblastoma cell lines, compared to adrenal gland, whereas miR-200b was slightly downregulated [38]. miR-204 was significantly lower in patients with known high-risk prognostic factors, including MYCN amplification, 11q- deletion, and INSS (international

neuroblastoma staging system) stage 3 or 4 [39]. miR-3805p was substantially overexpressed in primary neuroblastoma samples relative to human brain. Expression of miR380-5p did not correlate with individual age or tumor stage; however, tumors with MYCN amplification had significantly lower expression of miR-380-5p [40]. miR-885-5p was downregulated in primary neuroblastomas with segmental 3p loss [41].

Role of miRNAs in Neuroblastoma Differentiation Differentiation is a complex multi-step cell specialization process that begins with the installation of a cell lineagespecific genetic program, including both gene expression and gene silencing. Uncontrolled proliferation and disturbed differentiation of neural crest cells are thought to be the reason underlying neuroblastoma development. Many miRNAs have been shown to be involved in neuroblastoma differentiation, especially when induced by RA. The miRNAs that are correlated with neuroblastoma differentiation are summarized in Table 1 and Fig. 1. Laneve et al. [31] were the first group to report that miRNAs correlated with neuroblastoma differentiation. They identified 14 miRNAs that were upregulated after RA treatment, including 2 brain-specific (miR-9 and miR-124) and 2 brain-enriched (miR-125a and miR-125b) miRNAs. Two years later, Beveridge et al. [42] identified 43 miRNAs with altered expression following RA treatment: 32 miRNAs displayed significantly decreased expression and 11 miRNAs showed significantly increased expression as a result of differentiation. The entire miR-17 family was over-represented among the downregulated miRNAs. Downregulation of miR-17 family expression in response to RA resulted in neuronal differentiation by regulating BCL2 (B cell lymphoma 2), MEF2D (myocyte enhancer factor-2D), and zipper protein kinase (MAP3K12; aka ZPK/MUK/DLK). Le et al. [43] reported that 6 miRNAs were significantly upregulated during differentiation induced by RA. The ectopic expression of either miR-124 or miR-125b could increase the percentage of differentiated neuroblastoma cells with neurite outgrowth by repressing multiple targets. Chen et al. [44] analyzed the miRNA expression profile after differentiation induced by RA using a microarray and real-time PCR and found that miR-7 was downregulated and miR-214 was upregulated during neuronal differentiation and neurite outgrowth in vitro. Overexpression of miR-214 could increase neurite length, while overexpression of miR-7 had the opposite affect. Meseguer et al. [45] showed that 42 different miRNAs were significantly changed after RA treatment in neuroblastoma cells: 26 were upregulated and 16 were downregulated. Suppression of miR-10a and miR-10b

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Neurochem Res Table 1 miRNAs aberrantly expressed in neuroblastoma and their regulatory roles in tumorigenesis Dysregualted miRNA

Trend

miR-7

Up

miR-9

Down

miR-10a, 10b

Down

Target

Putative function

References

Inhibit neuronal differentiation

[44]

ID2

Inhibit cell proliferation

[31, 56]

MMP-14

Suppress cell invasion, metastasis, and angiogenesis

[58]

NCOR2

Inhibit neuronal differentiation and cell migratory and metastatic abilities

[45, 46]

miR-15a

Down

RECK

Decrease cell migration

[59]

miR-17–92 cluster

Up

BCL2, MEF2D, MAP3K12

Inhibit neuronal differentiation

[42]

p21, BIM, TGFBR2

Increase cell proliferation, inhibit cell apoptosis, decrease cell adhesion

[29, 30]

Inhibit neuronal differentiation, increase cell proliferation

[49]

miR-18a

Up

miR-27b

Down

PPARc

Inhibit cell proliferation

[32]

miR-34a

Down

SYT1, STX1A, E2F3, MYCN

Promote neural differentiation, inhibit cell proliferation, induce cell cycle arrest, promote cell apoptosis

[24, 25, 47] [26, 53]

miR-101, let-7

Down

MYCN

Inhibit cell proliferation

[57]

miR-103

Down

ID2

Inhibit cell proliferation

[31, 56]

CDK5R1

Decrease cell migration

[60]

miR-107

Down

CDK5R1

Decrease cell migration

[60]

miR-124

Down

PTBP1

Promote neural differentiation

[43, 48]

miR-125a

Down

t-crkC

Inhibit cell proliferation

[31]

miR-125b

Down

Promote neural differentiation, inhibit cell proliferation

[31, 43]

miR-128

Up

NTRK3

Promote cell proliferation

[51]

Down

Reelin, DCX

Reduced cell motility and invasiveness

[34]

miR-137

Down

KDM1A

Inhibit cell proliferation

[35]

miR-145

Down

miR-149-3p

Down

miR-152 miR-184

Inhibit cell proliferation, invasion and metastasis

[36]

Akt1, E2F1

Inhibit cell proliferation, induce cell apoptosis

[37]

Down

DNMT1

Inhibit cell invasiveness and anchorage independent growth

[38, 61]

Down

AKT2

Inhibit cell proliferation, induce cell cycle arrest and apoptosis

[15, 54]

miR-200b

Down

Inhibit cell invasiveness

[38]

miR-214

Down

Promote neural differentiation

[44]

miR-335

Down

Inhibit cell invasive and migratory abilities

[62]

miR-340

Down

Inhibit cell proliferation

[23] [40]

miR-380-5p

Up

Promote cell proliferation, inhibit cell apoptosis

miR-542-5p

Down

Inhibit cell invasive and migratory abilities

[63]

miR-558

Up

Promote cell proliferation

[52]

miR-885-5p

Down

Inhibit cell proliferation, induce cell cycle arrest and apoptosis

[41]

CDK2, MCM5

endogenous expression could impair RA-induced morphological differentiation without altering proliferation and increase the migratory and metastatic abilities of neuroblastoma cells. Foley et al. [46] confirmed these results when they profiled 364 miRNAs following RA treatment of neuroblastoma cell lines and found both miR-10a and miR10b to be highly overexpressed. Ectopic overexpression of

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these miRNAs led to a major reprogramming of the transcriptome and a differentiated phenotype that was similar to that induced by RA. These results indicated that miR10a and miR-10b were potent inducers of neuroblastoma cell differentiation. Further studies showed that the differentiation was induced through targeting NCOR2 (nuclear receptor corepressor 2), which in turn induced a

Neurochem Res Fig. 1 miRNAs regulate neuroblastoma differentiation by targeting different genes

cascade of primary and secondary transcriptional alterations, including the downregulation of MYCN. Many individual miRNAs are also involved in neuroblastoma differentiation. miR-34a was increased during RA induced differentiation of neuroblastoma cell line [24], while RA induced neuroblastoma differentiation was inhibited by knockdown of miR-34a through regulation of synaptic protein targets, including SYT1 (synaptotagmin1) and STX1A (syntaxin-1A) [47]. miR-124 could promote neuronal differentiation by triggering brain-specific alternative pre-mRNA splicing through targeting PTBP1 (polypyrimidine tract binding protein 1) [48]. miR-340 was upregulated by demethylation of an upstream genomic region that occurs during RA induced neuroblastoma cell differentiation [23]. The inhibition of miR-18a in neuroblastoma cells led to the outgrowth of varicosity-containing neurites and the induction of sympathetic neuron differentiation markers [49]. MYCN knockdown could also induce neuroblastoma differentiation. Buechner et al. [50] revealed that 23 miRNAs were differentially expressed during the MYCN knockdown-mediated neuronal differentiation of MNA neuroblastoma cells. The expression changes were bidirectional, with 11 and 12 miRNAs being up- and downregulated, respectively. miR-21 was strongly upregulated upon MYCN knockdown and the subsequent differentiation. However, overexpression of miR-21 did not prevent the differentiation of neuroblastoma cells.

Role of miRNAs in Neuroblastoma Tumorigenesis miRNAs can function as oncogenes during neuroblastoma tumorigenesis, as summarized in Table 1 and Fig. 2. The miR-17-92 cluster, which has been shown to be aberrantly expressed in neuroblastoma, plays important roles in neuroblastoma development. Fontana et al. [30] demonstrated that overexpression of the miR-17-92 cluster in MYCNnon-amplified neuroblastoma cells strongly augments their

in vitro and in vivo tumorigenesis. However, in vitro or in vivo treatment with antagomir-17 abolishes the growth of MYCN amplified and therapy resistant neuroblastoma cells through p21 and BIM (Bcl-2 interacting mediator of cell death) upmodulation, leading to cell cycling blockade and activation of apoptosis, respectively. Mestdagh et al. [29] further demonstrated that miR-17-92 cluster could increase cellular proliferation and significantly decrease intercellular cell adhesion by targeting TGFBR2 (transforming growth factor beta receptor 2). These results suggest a functional collaboration between the miR-17-92 cluster and the development of neuroblastoma. There are also many other oncogenic miRNAs whose overexpression would increase proliferation and whose inhibition would cause cell death or apoptosis. Inhibition of miR-18a could lead to severe growth retardation in neuroblastoma cells [49]. miR-128 overexpression significantly increased cell number by targeting NTRK3 (neurotrophic tyrosine kinase receptor type 3) [51]. miR-380-5p overexpression could cooperate with activated HRAS oncoprotein to transform primary cells, block oncogene-induced senescence and form tumors in mice, while inhibition of endogenous miR380-5p in neuroblastoma cells resulted in induction of p53 and extensive apoptotic cell death [40]. miR-558 could markedly increase colony formation, proliferation, and tumor growth in vitro and in vivo [52]. Conversely, miRNAs can influence carcinogenesis by acting as tumor suppressors and thus can play a significant role in disease progression, as summarized in Table 1 and Fig. 2. miR-34a is strongly downregulated in neuroblastoma as discussed before. Welch et al. [24] showed that reintroduction of miR-34a caused a dramatic reduction in cell proliferation through the induction of a caspasedependent apoptotic pathway by directly targeting E2F3 (E2F transcription factor 3). Wei et al. [25] found that miR34a caused significant suppression of cell growth through increased apoptosis and decreased DNA synthesis in neuroblastoma cell lines by targeting MYCN. Cole et al. [26]

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Neurochem Res Fig. 2 miRNAs regulate neuroblastoma tumorigenesis by targeting different genes

reported that the likely mechanism of miR-34a growth inhibition is through cell cycle arrest followed by apoptosis. Tivnan et al. [53] reported that over expression of miR34a in neuroblastoma cell lines led to a significant decrease in cell number, induced cell cycle arrest and subsequent apoptosis activation in vitro, and reduced tumor growth in vivo. Although miR-34a exerts its tumor-suppressive function through different target genes, these results highlight the potential use of miR-34a in neuroblastoma treatment. miR-184 is another downregulated miRNA in neuroblastoma. Chen et al. [15] reported that miR-184 overexpression induced neuroblastoma cell cycle arrest and apoptosis. Tivnan et al. [54] reported that ectopic overexpression of miR-184 was anti-proliferative in vitro and in vivo. Foley et al. [55] demonstrated that the function of miR-184 to inhibit neuroblastoma cell survival and promote apoptosis was through targeting AKT2 (v-akt murine thymoma viral oncogene homolog 2). Many other miRNAs have been demonstrated to have tumor suppressive functions in neuroblastoma. Overexpression of miR-9 and miR103 could reduce neuroblastoma cell proliferation by targeting the differentiation inhibitor ID2 (inhibitor of DNA binding 2) [56]. 3 neuronal miRNAs (9, 125a, and 125b) could control neuroblastoma cell proliferation in an additive manner by repressing the t-crkC (truncated isoform of the neurotrophin receptor tropomyosin-related kinase C), whose downregulation was critical for cell growth [31]. miR-27b overexpression could block cell growth in vitro and tumor growth in vivo by inhibiting PPARc (peroxisome proliferator-activated receptor c), which triggers an increased inflammatory response [32]. miR-101 and let-7 could target the proto-oncogene MYCN and inhibit cell proliferation in MYCN-amplified neuroblastoma [57]. Reexpressing miR-137 in neuroblastoma cell lines could increase cell apoptosis, decrease cell viability and inhibit cell proliferation by directly targeting KDM1A (lysine (K)specific demethylase 1A) [35]. Overexpression of miR-145

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could significantly inhibit the cell growth of neuroblastoma in vitro [36]. miR-149-3p overexpression could decrease cell growth and enhance apoptosis through repressing the expression of AKT1 (v-akt murine thymoma viral oncogene homolog 1) and E2F1 (E2F transcription factor 1) [37]. miR-340 could induce cell apoptosis in a cell context dependent manner, indicating a tumor suppressive function for this miRNA [23]. Enforced miR-885-5p expression in neuroblastoma cell lines could inhibit proliferation, trigger cell cycle arrest, and induce apoptosis by targeting CDK2 (cyclin-dependent kinase 2) and MCM5 (minichromosome maintenance complex component 5) [41]. In summary, some miRNAs have been shown to participate in neuroblastoma tumorigenesis and much more efforts still have to be made to reveal the roles of deregulated miRNAs in neuroblastoma carcinogenesis and progression.

Role of miRNAs in Neuroblastoma Metastasis Metastases are present in approximately 50 % of neuroblastoma patients at diagnosis and represent the most important clinical prognostic factors in neuroblastoma besides age. The exact mechanisms leading to metastases in neuroblastoma are unclear. The miRNAs correlated with neuroblastoma metastasis are summarized in Table 1 and Fig. 3. Overexpression of miR-9 suppressed the invasion, metastasis, and angiogenesis of neuroblastoma cells in vitro and in vivo by targeting MMP-14 (matrix metallopeptidase 14) [58]. Knockdown of miR-10a and -10b impaired the RA induced reduction of migratory and metastatic abilities of neuroblastoma cells [45]. Suppression of miR-15a could significantly decrease neuroblastoma cell migration by targeting RECK (reversioninducing-cysteine-rich protein with kazal motifs) [59]. miR-103 and miR-107 overexpression could cause a

Neurochem Res Fig. 3 miRNAs regulate neuroblastoma metastasis by targeting different genes

reduction in neuroblastoma migration ability by modulating CDK5R1 (cyclin-dependent kinase 5 regulatory subunit 1) expression [60]. miR-128 upregulation could reduce neuroblastoma cell motility and invasiveness by inhibiting Reelin and DCX (doublecortin) expression [34]. miR-145 was downregulated in neuroblastoma and that ectopic expression of miR-145 could efficiently inhibit cell invasion and metastasis both in vitro and in vivo [36]. miR-152 could negatively impact cell invasiveness and anchorage independent growth through targeting DNMT1 (DNA (cytosine-5-)-methyltransferase 1) [61]. The enforced expression of miR-200b and knockdown of miR-152 resulted in a significant decrease of the invasion activity of neuroblastoma cells [38]. miR-335 could suppress the invasive and migratory potential of neuroblastoma cells by directly targeting multiple genes from the non-canonical TGF-b signaling pathway [62]. The ectopic overexpression of miR-542-5p decreased the invasive potential of neuroblastoma cell lines in vitro as well as primary tumor growth and metastases in an orthotopic mouse xenograft model [63]. Collectively, we have begun to identify differentially expressed miRNAs in the metastasis of neuroblastoma; however, due to the limited number and limited scope of the function of these miRNAs, more research is necessary to understand the metastatic process in this disease.

Role of miRNAs in Neuroblastoma Diagnosis and Prognosis The hallmark of neuroblastoma is its clinical and biological heterogeneity, with the likelihood of cure varying widely according to age at diagnosis, extent of disease, and tumor biology. A number of genetic and genomic changes have been identified in neuroblastoma tumors that are relevant to clinical progression. However, no single genetic change has been found to be common to all neuroblastoma tumors,

suggesting a complex underlying genetics of neuroblastoma and that aberrant expression or regulation of multiple genes may work together to initiate the malignant transformation of undifferentiated neuroblasts. miRNAs has opened up new areas for the exploration of potential diagnostic and prognostic biomarkers. A miRNA signature, which is composed of several miRNAs, may ultimately be more successful at prediction than the other potential biomarkers discovered to date in neuroblastoma. Bray et al. [17] identified the first miRNA signature for neuroblastoma diagnosis and prognosis. They established a 15-miRNA signature that was significantly associated with poor overall patient survival with 72.7 % sensitivity and 86.5 % specificity. Among these miRNAs, lower expression of miR-542-5p was highly associated with poor patient EFS (event-free survival) and OS (overall survival). Schulte et al. [64] supported the conclusion made by Bray. They demonstrated that miRNA signatures could be used as biomarkers to stratify neuroblastoma patients according to clinical course and that these signatures were superior to the predictive power of individual miRNA expression. The expression of miR-542-5p was predictive of survival. Lin et al. [21] identified 27 miRNAs that can clearly distinguish low- from high-risk patients. De Peter et al. [19] established a prognostic 25-miRNA signature that could significantly discriminate patients with respect to progression-free survival and overall survival, both in fresh frozen tumor samples as well as in archived FFPE samples. Individual miRNAs are often predictive of survival. miR-15a expression positively correlated with neuroblastoma clinical pathological stage [59]. miR-17-92 cluster activation was a marker for poor survival in neuroblastoma patients [29]. High expression of miR-128 is associated with favorable features, such as favorable Shimada category or very young age at diagnosis [34]. miR-137 was downregulated in primary neuroblastomas and correlated with poor patient prognosis [35]. miR-204 was downregulated in primary neuroblastoma and was predictive of

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patient event-free survival and overall survival, independent of established known risk factors [39]. miR-380-5p was highly expressed in neuroblastoma and that high expression correlated with poor outcome in MYCN amplified neuroblastoma [40]. miR-487b expression was associated with overall survival and disease-free survival in neuroblastoma patients, independent of clinical covariates. Moreover, miR-487b and miR-410 expression was significantly associated with disease-free survival in nonMYCN-amplified favorable neuroblastoma. Expression of miR-487b and miR-410 shows predictive value beyond the classical high-/low-risk stratification and is a biomarker of relapse in favorable neuroblastoma [20]. Over the past several years, intensive studies have demonstrated that miRNAs are not only found intracellularly, but are also detected outside of cells, including in various body fluids (i.e., serum, plasma, saliva, urine, and milk). Alterations in the expression level and composition of these extracellular circulating miRNAs have been shown to tightly correlate with various diseases, including cancers [65–69]. These results suggest the use of circulating miRNAs as a non-invasive biomarker to assess and monitor the clinical status of neuroblastoma patients. However, there are no published reports on the identification of circulating miRNAs in neuroblastoma. Potential Role of miRNAs as Neuroblastoma Therapeutics In view of the functional involvement of miRNAs in the development and progression of cancer, attempts are underway to develop therapeutic strategies using miRNAs. One strategy is to block upregulated oncogenic miRNAs by antisense oligonucleotides. Tivnan et al. [70] targeted delivery of miR-34a using anti-disialoganglioside GD2coated silica nanoparticles in tumor-bearing mice. This approach resulted in significantly decreased tumor growth, increased apoptosis, and a reduction in vascularization. Another method is to rescue downregulation of tumor suppressor miRNAs using miRNA mimics. Zhang et al. [58] reported that overexpression of miR-9 inhibited the invasion, metastasis, and angiogenesis of neuroblastoma in vivo. Lee et al. [32] demonstrated that miR-27b overexpression could block tumor growth both in vitro and in vivo by inhibiting PPARc. Tivnan et al. [53] reported that over expression of miR-34a reduced tumor growth in vivo. Zhang et al. [36] reported that miR-145 could significantly inhibit the growth, invasion, metastasis, and angiogenesis of neuroblastoma cells in vitro and in vivo through direct targeting of HIF-2a, suggesting the potential value of miR-145 as a novel therapeutic target for treating neuroblastoma. Tivnan et al. [54] reported that

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overexpression of miR-184 led to a significant reduction in tumor growth relative to negative control-treated cohorts in a xenograft model of neuroblastoma. Swarbrick et al. [40] reported that in vivo delivery of a miR-380-5p antagonist decreased tumor size in an orthotopic mouse model of neuroblastoma. Traditional chemotherapy in combination with miRNAbased treatments may be a new strategy for the clinical management of neuroblastoma. Chakrabarti et al. [71] found that overexpression of miR-93 and miR-7-1, respectively, decreased and increased the efficacy of the combination of 4-HPR (N-(4-hydroxyphenyl) retinamide) and EGCG ((-)-epigallocatechin-3-gallate) to inhibit the growth of neuroblastoma cells. Furthermore, Chakrabarti et al. [72] found that overexpression of miR-7-1 could increase the efficacy of green tea polyphenols to induce apoptosis in neuroblastoma cells. Finally, Ryan et al. [39] reported that ectopic miR-204 expression significantly increased sensitivity to cisplatin and etoposide in vitro. These results reveal a potential for miRNAs to be used as targets for neuroblastoma therapy. However, as our understanding of the role of miRNAs in neuroblastoma is still very limited, whether miRNAs can be used directly for the treatment of neuroblastoma patients remains unknown. Furthermore, extensive preclinical studies for safety and toxicity are necessary before a miRNA-based treatment can be considered in humans.

Conclusion and Perspectives miRNAs are an astonishing new class of gene regulators, and it has been demonstrated that these molecules play a crucial role in neuroblastoma development and progression. These discoveries not only provide new insights into the molecular pathogenesis of neuroblastoma but also raise hopes for the application of miRNAs in neuroblastoma diagnosis, prognostication, and therapy. Unfortunately, translation of these preliminary research data into clinical application is not feasible at this stage. However, these findings provide a very promising basis for future studies to determine the effects of miRNA in neuroblastoma treatment. In summary, the presented data support the enormous clinical potential of miRNAs in neuroblastoma and mandate further intensive investigations in this field. Acknowledgments This work was supported by National Natural Science Foundation of China: 31071046, 81302197; Changzhou Social Development Project: CS20092015, CS20102010; Changzhou Health Bureau Project: ZD200903, ZD201007; Changzhou Science Technology Bureau Guiding Project: CY20119004. Conflict of interest of interest.

The authors declare that they have no conflict

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