Non-Coding RNAs Participate in the Pathogenesis of Neuroblastoma

Neuroblastoma is one of the utmost frequent neoplasms during the first year of life. This pediatric cancer is believed to be originated during the embryonic life from the neural crest cells. Previous studies have detected several types of chromosomal aberrations in this tumor. More recent studies have emphasized on expression profiling of neuroblastoma samples to identify the dysregulated genes in this type of cancer. Non-coding RNAs are among the mostly dysregulated genes in this type of cancer. Such dysregulation has been associated with a number of chromosomal aberrations that are frequently detected in neuroblastoma. In this study, we explain the role of non-coding transcripts in the malignant transformation in neuroblastoma and their role as biomarkers for this pediatric cancer.


INTRODUCTION
Neuroblastoma is a neoplasm originated from the neural crest of the sympathetic part of autonomic system (1) during the embryonic life (2). This malignancy is among the most common childhood cancers particularly during the first year of life (3). Neuroblastoma has a heterogeneous course in terms of both pathobiology and clinical manifestations. Several therapeutic options such as surgical removal of the tumor, chemotherapy, radiotherapy, and bone marrow transplantation are being applied for neuroblastoma (4). Spontaneous regression might also happen in the course of neuroblastoma (5). This tumor is associated with several genetic and chromosomal abnormalities that affect its clinical course and prognosis namely MYCN amplification, loss of distal portion of chromosome (chr) 1p and gain of 17q (6). Other chromosomal abnormalities detected in neuroblastoma are loss of 11q, 3p, 4p, 9p, 14q, and gain of 1q, 7q, 2p, and 11p (7)(8)(9). In addition to these chromosomal aberrations, dysregulation of several genes including non-coding RNAs (ncRNAs) are linked with this cancer (10). These kinds of transcripts have regulatory impact on other genes, hence constructing an epigenetic layer of gene regulation. They are classified based on their sizes to long non-coding RNAs (lncRNAs) and microRNAs (miRNAs) with the former having more than 200 nucleotides and the latter being about 22 nucleotides (11). Based on the speculation stated by the ENCODE consortium regarding the recognition of "biochemical functions for 80% of the genome" (12), ncRNAs have attained much attention during the recent decade particularly in the field of cancer research. In the current study, we explain the role of lncRNAs and miRNAs in the evolution of neuroblastoma and their role as biomarkers for this pediatric cancer.

Dysregulated miRNAs in Neuroblastoma
Chen and Stallings have measured expression of 157 miRNAs in neuroblastoma samples. They have displayed differential pattern of 32 miRNAs between tumor with favorable prognosis and those with poor prognosis. Notably, several of these miRNAs were down-regulated in neuroblastoma samples harboring MYCN amplification, which was associated with unfavorable outcome. Cell line studies have shown the role of retinoic acid in the modulation of expression of miRNAs in a MYCN-amplified cell line. Among the dysregulated miRNAs has been miR-184 which participates in the regulation of apoptosis. MYCN might exert its tumorigenic effects via modulating expression of miRNAs that participate in neural cell differentiation or apoptotic processes (13).
Among the firstly discovered tumor suppressor miRNAs in neuroblastoma was miR-34a (14), which is transcribed from a frequently deleted region in neuroblastoma i.e. 1p36. 23. This miRNA was particularly down-regulated in neuroblastoma samples with 1p deletion (14). Since miR-34a inhibits expression of the E2F3 transcription factor, its down-regulation facilitates cell cycle progression (14). Subsequent studies have also verified the tumor suppressive impact of miR-34a in the neuroblastoma cells and its inhibitory effects on the expression of BCL2 and MYCN (15,16). miR-34a also binds with the 3' UTR of ATG5 and CD44 transcripts and decreases their expressions. Down-regulation of miR-34a in neuroblastoma cells results in the over-expression of ATG5 and CD44 (17,18). CD44 is a cell surface receptor which can bind with hyaluronan and induce expression of genes that promote progression of cancer (19). ATG5 can dissociate V1V0-ATPase, increase pH in multivesicular bodies and enhance secretion of exosomes to facilitate cancer metastasis (20). Thus, miR-34a affects the progression of neuroblastoma through different mechanisms. Figure 1 shows some aspects of participation of miR-34a in the pathogenesis of neuroblastoma.
miR-542-5p is another tumor suppressor miRNA whose down-regulation in neuroblastoma has conferred poor clinical outcome. Notably, forced up-regulation of miR-542-5p has resulted in attenuation of neuroblastoma invasive properties and tumor growth bot in vitro and in vivo (21). Moreover, expression of miR-490-5p has been diminished in neuroblastoma tissues and cells. Forced overexpression of miR-490-5p has diminished cell proliferation migration and invasiveness, prompted G0/G1 arrest in cells and induced cell apoptosis. MYEOV has been confirmed to be the target of miR-490-5p through which miR-490-5p blocks neuroblastoma progression (22). Table 1 recapitulates the results -miR-34a down-regulation increases cell proliferation, migration, and invasion. - 18 NB primary and their metastatic tissues SH-SY5Y, IMR-32 CD44 -miR-34a repression results in enhanced metastasis, proliferation, and invasion rates in NB cells.
- -   -miR-17 down-regulates N-myc mRNA and protein levels, while ELAVL4 up-regulates N-myc and is a competitive factor for miR-17.
-  Over-expression of miR-17-5p has also been verified in primary neuroblastoma patients especially those with MYCN amplification and poor clinical outcome (68). miR-640, miR-543, miR-624-3p, and miR-196-b are among up-regulated miRNAs in neuroblastoma. Notably, these miRNAs target ING5 transcript. miRNA-ING5-histone acetylation axis has been recognized as the main route through which two anticancer drugs namely a histone deacetylase inhibitor and a proteasome inhibitor block progression of neuroblastoma (69). miR-1303 is another over-expressed miRNA in neuroblastoma.
Up-regulation of this miRNA enhanced proliferation of neuroblastoma cells through targeting GSK3b and SFRP1. miR-1303 also increased levels of MYC and CyclinD1, and diminished p21 and p27 levels (70). Table 2 lists up-regulated miRNAs in neuroblastoma.
Aberrant expression of miRNAs in neuroblastoma samples can be used as biomarkers for prediction of the course of malignancy. For instance, down-regulation of miR-490-5p has been correlated with INSS stage, lymph node involvement, and poor clinical outcome of patients with neuroblastoma (22). Similarly, decreased expression of miR-186, let-7, miR-497 and miR-432-5p predicts lower survival rates (31,34,56,63). Table 3 reviews the results of studies which evaluated this aspect of miRNAs.

Dysregulated lncRNAs in Neuroblastoma
LncRNAs can regulate expression of genes via different mechanisms including alterations in chromatin configuration, modulation of transcription, splicing, mRNA stability and bioavailability as well as post-translational modifications (83). Therefore, they contribute in the pathogenesis of human cancers. Prajapati et al. have analyzed RNA-seq data of a number of neuroblastoma samples to recognize their differential expression in among primary neuroblastoma, relapsed ones and metastasized tumors. They reported up-regulation of RFPL1S,   - The up-regulation of miR-3613-3p increases viability but reduces the apoptosis of NB cells.
- Over-expression of this transcript has been associated with poor survival of patients. This lncRNA has been mapped to the region of 17q which is amplified in neuroblastoma and exerts oncogenic effects in this type of cancer (89). Tables 4 and 5 enlist over-expression and decreased expression lncRNAs in neuroblastoma, respectively. Dysregulation of several lncRNAs in neuroblastoma samples has been correlated with survival of patients. For instance, high levels of DLX6-AS1, lncNB1, LINC01296, SNHG16 and RMRP expression have been linked with poor prognosis and lower survival (90,92,94,95,104). Table 6 summarizes the results of studies which assessed correlation between expression levels of lncRNAs and survival of patients with neuroblastoma.

Expression and Function of circRNAs in Neuroblastoma
Circular RNAs (circRNAs) constitute a group of ncRNAs which are produced from exons or introns through construction of covalently-closed circles (134). Recent studies have shown dysregulation of this type of ncRNAs in cancers. For instance, circDGKB has been shown to be over-expressed in neuroblastoma tissues versus normal dorsal root ganglia. Notably, overexpression of this circRNA has been an indicator of poor survival of these patients. Mechanistically, circDGKB enhances cell proliferation, migration and invasion of neuroblastoma cells while inhibiting cell apoptosis. Moreover, up-regulation of circDGKB reduced expression level of miR-873 and increased GLI1 expression (135). Table 7 recapitulates the results of studies which assessed function of circRNAs in neuroblastoma.

Polymorphisms Within ncRNAs and Risk of Neuroblastoma
Single nucleotide polymorphisms (SNPs) within lncRNAs or miRNAs can modulate expression or activity of these transcripts, thus being implicated in the development of neuroblastoma. The        role of a number of SNPs within lncRNAs such as LINC00673, H19, MEG3 and HOTAIR has been evaluated in this regard (137)(138)(139)(140). Moreover, the rs4938723 within miR-34b/c has been associated with risk of this kind of cancer (141). Notably, some studies have appraised these associations in certain subgroups of patients. For instance, the association between rs4938723 TC and CC genotypes is prominent in all age-based subgroups, both sexes, retroperitoneal tumors as well as tumors originated from other sites, and all clinical stages (141). Such detailed analyses have not been done for all assessed SNPs. Table 8 summarizes the results of studies which assessed contribution of SNPs within ncRNAs in conferring the risk of neuroblastoma.

DISCUSSION
Recent studies have demonstrated abnormal expression of lncRNAs, miRNAs and circRNAs in neuroblastoma. Besides, some SNPs within lncRNAs and miRNAs confer risk of neuroblastoma. In vitro studies have shown the functional interactions between a number of these ncRNAs and MYCN, the oncogene that has essential roles in the pathogenesis of this type of cancer. Moreover, certain miRNAs have been shown to target tyrosine kinase receptors. For instance, hsa-miR-376c is predicted to target ALK tyrosine kinase receptor. Notably, this miRNA has been up-regulated in neuroblastoma samples of long-survivors (146). Expressions of a number of other ncRNAs have been shown to stratify neuroblastoma patients based on their risk of recurrence and clinical outcome. The observed dysregulation of ncRNAs in neuroblastoma can be explained by their association with the frequent chromosomal abnormalities in this kind of cancer. Amplification of genomic loci corresponding to these transcripts is a possible route for their up-regulation (86). Moreover, epigenetic factors participate in the regulation of ncRNAs expression in neuroblastoma, as several lines of evidence points to the role of retinoic acid and its derivatives in the reversal of such dysregulation. Consistent with these observations, ATRA has been lately shown to induce differentiation of a number of neuroblastoma cell lines or activate apoptosis in these cells (147).
As a number of ncRNAs regulate tumorigenic process downstream of MYCN, dysregulation of these transcripts might represent an alternative mechanism of MYCN upregulation/amplification in neuroblastoma. In vivo studies have demonstrated the efficacy of miRNA antagonism in suppression of proliferation of MYCN-amplified neuroblastoma cells in animal models (68). However, these results have not been replicated in clinical settings. Administration of miRNA mimics in clinical settings has encountered some problems most of the being related with the distribution of these transcripts in the body and enrichment in the target organs. Encapsulation of these small transcripts in nanoparticle vesicles is expected to enhance their stability and their presence in the circulation, permitting further time for their amassment in tumor tissues (148).
Multidrug resistance is a problem in the treatment of patients with neuroblastoma. Such phenotype has been associated with a number of genetic abnormalities such as over-expression of MYCN oncogene, hyper-activation of tyrosine kinase receptors (BDNF-TrkB) or reduced expression and activity of tumor suppressor genes including p53 (148). Therefore, ncRNAs that modulate expression of these elements or function in the downstream of these molecules can also be involved in the multidrug resistance of these cells. Therefore, modulation of expression of these transcripts represents a novel modality to combat multidrug resistance in neuroblastoma.   ERK/AKT NEAT1 up-regulation lowers cell proliferation, migration, and invasion rates.
- (133) Expression profile of ncRNAs has been correlated with patients' survival. The underlying mechanism of this observation has been clarified in some cases. For instance, hsa-miR-383, hsa-miR-548d-5p, hsa-miR-939 and hsa-miR-877* miRNAs which have been down-regulated in neuroblastoma samples from long-survivors (146) target a number of genes being involved in the neuronal differentiation (149).  High levels of lncNB1 correlate with poor prognosis. - Genomics Analysis and Visualization Platform for NB dataset: Two groups of high (=21) and low (=67) for LINC01296 expression High LINC01296 expression is associated with poor outcome. - GSE62564 dataset: two groups of low and high expression for SNHG16 expression, each containing 249 patients High levels of SNHG16 expression correlates with lower EFS and OS. - High (=34) and low (=10) expressing groups of RMRP lncRNA Higher RMRP expression relates to poor prognosis and survival. - GAU1 expression in two groups from TCGA dataset: high (=44) and low (=44) High GAU1 expression correlates with lower OS. - CAI2 expression in NB patients: high=19, low=43 CAI2 expression negatively correlates with OS and EFS. - NBAT-1 expression in 2 cohorts: 1) 50 high and 43 low, 2) 314 high and 184 low NBAT expression significantly correlates with OS and EFS.
NBAT-1 is an independent prognostic marker in predicting EFS. - DUXAP8 expression in two groups: group 1: 1 + 2+4S stage (n = 18) and group 2: 3 + 4 stage (n = 27) The survival rate is low in high expression of the DUXAP8 group compared with lower expression of the DUXAP8 group.
-   neuroblastoma is lack of longitudinal assessment of expression of these transcripts to unravel temporal changes during the course of disease. Conduction of this type of studies would facilitate approval of the diagnostic and prognostic power of ncRNAs.