Neuroblastoma is an embryonic tumor of the sympathetic nervous system which arises during fetal or early postnatal life from sympathetic cells derived from the neural crest [
1]. It is the most common solid extracranial malignancy of childhood and is responsible for 15% of all pediatric cancer deaths. Neuroblastoma is an extremely heterogeneous disease; tumors can spontaneously regress or differentiate, even without therapy, or display a very aggressive malignant phenotype that is poorly responsive to current intensive multimodal therapy [
2‐
4]. Despite high-dose chemotherapy, surgery and radiotherapy, in half of cases the tumor has a survival rate of less than 40% [
5]. Unsatisfactory response to classical therapies may be attributable to the clinical, biological and histological heterogeneity of “neuroblastomas”. Thus, the identification of novel selective target molecules is needed, to improve existing therapies and to develop new, specific, innovative and less aggressive therapeutic approaches [
6,
7]. Our previous study recognized one of the molecular pathways involved in neuroblastoma to be a component of the Notch signaling pathway [
8]. The Notch pathway is an highly conserved cell signaling system that regulates cell fate decisions during embryogenesis, modulates the differentiated state of mature cells and is also one of the main factors in the regulation of “cancer stem cells” [
9,
10]. Several studies have demonstrated the importance of Notch signaling in the tumor microenvironment and its involvement in many aspects of the disease: the onset of tumor, angiogenesis, the ability to invade tissues and metastasize [
11‐
16]. Depending on organ and tissue type, Notch signaling can function either as a promoter to support tumor development or as a suppressor to inhibit tumor growth. Deregulated expression of Notch proteins, ligands, and targets has been described in a multitude of solid tumors including renal, lung, pancreatic, hepatocellular and gastric carcinoma, melanoma and medulloblastoma [
17]. Notch activation is responsible for increased growth and proliferation of neuroblastoma cell lines. On the contrary, Notch pathway downregulation by gamma-secretase inhibitors causes proliferation arrest and cell differentiation [
8]. In this study we wanted to investigate in detail the role of Notch signaling in neuroblastoma and we analyzed the role of Notch pathway components, three receptors (Notch1, Notch 2 and Notch 3) and five canonical ligands (Delta-like 1, Delta-like 2, Delta-like 4, Jagged 1 and Jagged 2), in three neuroblastoma cell lines. We identified DLL1 as the Notch pathway component highly expressed in IMR-32 cells, a cell line with a high degree of
MYCN amplification. About 20% of neuroblastoma cases are characterized by
MYCN gene amplification, which has been correlated with tumor progression and is routinely used as a clinical biomarker for treatment stratification [
18,
19]. The correlation between Delta-like Notch ligand expression and development of other tumors has already been characterized. Overexpression of DLL1 was identified in choriocarcinoma [
20] and hepatocellular carcinoma [
21], while Delta-like 4 (
DLL4) expression was correlated to tumor initiation and progression of glioblastoma [
22], poor prognosis in pancreatic cancer [
23] and colon cancer [
24]. We evaluated the possibility to act on the expression of
DLL1 by using miRNAs. During the past decades the involvement of miRNAs in several human diseases, including cancer, has been intensively investigated. miRNAs are a class of small, 19–22 nucleotides, non-coding endogenous single-stranded RNAs that act as post-transcriptional regulators of specific messenger transcripts (mRNAs), resulting in targeted degradation or suppression of gene expression [
25,
26]. More than 4469 miRNAs have been identified in Homo sapiens, of which 1881 are precursors and 2588 are mature (miRBase, Release 21: June 2014) and most of these miRNAs are highly conserved across species. It has been reported that miRNAs are able to control more than 60% of human protein-coding genes [
27,
28]. In physiologic conditions miRNAs are key regulators involved in biological processes such as development, proliferation, differentiation, migration, neuroplasticity, survival and death. miRNAs dysregulation contributes to the onset of different pathologies such as heart disease, diabetes, mental disorders and cancer. Because 50% of miRNAs genes are located at genomic sites associated with cancer-specific chromosomal rearrangements and because of the proximity of their genes to chromosomal breakpoints, miRNAs have been associated with tumorigenesis. In some cancer types miRNAs appear to be upregulated and are thus thought to act as oncogenes, while they are downregulated in other types of cancers, which may be indicative of a tumor suppressor function. miRNAs expression is dynamic: many miRNAs are deregulated in early stages of tumor development and upregulated during cancer progression, which underscores the importance of the cellular microenvironment [
29]. miRNAs can be used as biomarkers to discriminate cancer from normal tissue, to diagnose the onset of a tumor, to indicate the degree of dissemination and to monitor the response to drug treatments, or as therapeutic targets in the design of a real “miRNA-based therapy” [
28]. In silico analyses suggest that
DLL1 is one of the targets of the miRNA-34 family; miRNA-34a maps to the distal region of chromosome 1p which is commonly deregulated or deleted in neuroblastoma (
www.mirbase.org). miRNA-34a can antagonize many different oncogenic processes by regulating genes that function in various cellular pathways. The anti-oncogenic activity of miRNA-34a has been demonstrated in cancer cells of the lung [
30,
31], pancreas [
32,
33], brain [
34,
35], ovary [
36], prostate [
37] as well as in lymphoma and leukemia [
38]. miRNA-34a inhibits the propagation properties of tumor-initiating cells derived from medulloblastoma [
39] and it is downregulated in glioblastoma tissues, where its overexpression could suppress cell proliferation and induce apoptosis, indicating that this miRNA may act as tumor suppressor also in this type of tumor [
40]. miRNA-34b is significantly downregulated in prostate cancer and its reconstitution induced anti-proliferative and antimigratory effects and suppressed tumor growth in an in vivo xenograft nude mouse model, suggesting the tumor suppressor function of this miRNA [
41]. Also, in breast cancer, miRNA-34b acts as an oncosuppressor regulating the complex estrogenic pathway, which could lead to the development of new therapeutic strategies [
42]. The miRNA-34 family was the most extensively studied miRNAs in neuroblastoma and Welch and colleagues were the first to report that miRNA-34a was generally expressed at lower levels in unfavorable primary neuroblastomas and cell lines compared to normal adrenal tissue. miRNA-34a induced cell cycle arrest, apoptosis, and significantly reduced tumor growth in an in vivo orthotopic murine model of neuroblastoma [
35]. Our data indicate that, within the miRNA-34 family, miRNA-34b induced significant downregulation of DLL1 mRNA expression levels, cell differentiation and arrested cell proliferation in IMR-32 neuroblastoma cells. This study identified Notch ligand DLL1 as a new and specific molecular target in childhood neuroblastoma, suggesting that miRNAs could be a novel therapeutic tool to develop an effective strategy to attack “DLL1 positive” neuroblastoma.