Temporal lobe epilepsy is a common, chronic neurologic disorder characterized by recurrent spontaneous seizures. MicroRNAs (miRNAs) are small, non-coding RNAs that regulate post-transcriptional expression of protein-coding mRNAs, which may have important roles in the pathogenesis of neurologic disorders. In models of prolonged, injurious seizures (status epilepticus) and in experimental and human epilepsy, we found up-regulation of miR-134, a brain-specific, activity-regulated miRNA implicated in the control of dendritic spine morphology. Silencing of miR-134 expression in vivo using antagomirs reduced hippocampal CA3 pyramidal neuron dendrite spine density by 21%, and rendered mice refractory to seizures and hippocampal injury caused by status epilepticus. Depletion of miR-134 after status epilepticus reduced the later occurrence of spontaneous seizures by over 90% and mitigated attendant pathologic features of temporal lobe epilepsy. Thus, silencing miR-134 exerts prolonged seizure suppressant and neuroprotective actions; whether these represent anticonvulsant or truly antiepileptogenic effects requires additional experimentation.

Many neuroblastoma cell lines can be induced to differentiate into a mature neuronal cell type with retinoic acid and other compounds, providing an important model system for elucidating signalling pathways involved in this highly complex process. Recently, it has become apparent that miRNAs, which act as regulators of gene expression at a post-transcriptional level, are differentially expressed in differentiating cells and play important roles governing many aspects of this process. This includes the down-regulation of DNA methytransferases that cause the de-methylation and transcriptional activation of numerous protein coding gene sequences. The purpose of this article is to review involvement of miRNAs and DNA methylation alterations in the process of neuroblastoma cell differentiation. A thorough understanding of miRNA and genetic pathways regulating neuroblastoma cell differentiation potentially could lead to targeted therapies for this disease.

Several studies have implicated the dysregulation of microRNAs in neuroblastoma pathogenesis, an often fatal paediatric cancer arising from precursor cells of the sympathetic nervous system. Our group and others have demonstrated that lower expression of miR-542-5p is highly associated with poor patient survival, indicating a potential tumor suppressive function. Here, we demonstrate that ectopic over-expression of this miRNA decreases the invasive potential of neuroblastoma cell lines in vitro, along with primary tumor growth and metastases in an orthotopic mouse xenograft model, providing the first functional evidence for the involvement of miR-542-5p as a tumor suppressor in any type of cancer.

MicroRNAs function as negative regulators of post-transcriptional gene expression, playing major roles in cellular differentiation. Several neuroblastoma cell lines can be induced to undergo differentiation by all-trans-retinoic acid (ATRA) and are used for modelling signalling pathways involved in this process. To identify miRNAs contributing to differentiation, we profiled 364 loci following ATRA treatment of neuroblastoma cell lines and found miR-10a and miR-10b to be highly over-expressed in SK-N-BE, LAN5, and SHSY-5Y. Ectopic over-expression of these miRNAs led to a major reprogramming of the transcriptome and a differentiated phenotype that was similar to that induced by ATRA in each of these cell lines. One of the predicted down-regulated miR-10a/b targets was nuclear receptor co-repressor 2 (NCOR2), a co-repressor of gene transcription which is known to suppress neurite outgrowth. NCOR2 was experimentally validated as a direct target of miR-10a/b, and siRNA mediated inhibition of this mRNA alone resulted in neural cell differentiation. Moreover, induction of differentiation could be blocked by ectopic up-regulation of NCOR2 using an expression construct lacking the miR-10a/b 3’ UTR target site. We conclude that miR-10a/b play major roles in the process of neural cell differentiation through direct targeting of NCOR2, which in turn induces a cascade of primary and secondary transcriptional alterations, including the down-regulation of MYCN.

Neuroblastoma is an often fatal pediatric cancer arising from precursor cells of the sympathetic nervous system. 13-Cis retinoic acid is included in the treatment regime for patients with high-risk disease, and a similar derivative, all-trans retinoic acid (ATRA) causes neuroblastoma cell lines to undergo differentiation. The molecular signaling pathways involved with ATRA induced differentiation are complex, and the role that DNA methylation changes might play are unknown. The purpose of this study was to evaluate the genome-wide effects of ATRA on DNA methylation using methylated DNA immunoprecipitation applied to microarrays representing all known promoter and CpG islands. 402 gene promoters became demethylated, while 88 were hypermethylated post-ATRA. mRNA expression microarrays revealed that 82 of the demethylated genes were over-expressed by >2 fold, while 13 of the hyper methylated genes were under-expressed. Gene ontology analysis indicated that de-methylated and re-expressed genes were enriched for signal transduction pathways, including NOS1, which is required for neural cell differentiation. As a potential mechanism for the DNA methylation changes, we demonstrate the down-regulation of methyltransferases, DNMT1 and DNMT3B, along with the up-regulation of endogenous microRNAs targeting them. Ectopic over-expression of miR-152, targeting DNMT1, also negatively impacted cell invasiveness and anchorage independent growth, contributing in part to the differentiated phenotype. We conclude that functionally important, miRNA-mediated DNA de-methylation changes contribute to the process of ATRA induced differentiation resulting in the activation of NOS1, a critical determinant of neural cell differentiation. Our findings illustrate the plasticity and dynamic nature of the epigenome during cancer cell differentiation.

Background

MYCN is a transcription factor that is expressed during the development of the neural crest and its dysregulation plays a major role in the pathogenesis of pediatric cancers such as neuroblastoma, medulloblastoma and rhabdomyosarcoma. MeCP2 is a CpG methyl binding protein which has been associated with a number of cancers and developmental disorders, particularly Rett syndrome.

Methods and Findings

Using an integrative global genomics approach involving chromatin immunoprecipitation applied to microarrays, we have determined that MYCN and MeCP2 co-localize to gene promoter regions, as well as inter/intragenic sites, within the neuroblastoma genome (MYCN amplified Kelly cells) at high frequency (70.2% of MYCN sites were also positive for MeCP2). Intriguingly, the frequency of co-localization was significantly less at promoter regions exhibiting substantial hypermethylation (8.7%), as determined by methylated DNA immunoprecipitation (MeDIP) applied to the same microarrays. Co-immunoprecipitation of MYCN using an anti-MeCP2 antibody indicated that a MYCN/MeCP2 interaction occurs at protein level. mRNA expression profiling revealed that the median expression of genes with promoters bound by MYCN was significantly higher than for genes bound by MeCP2, and that genes bound by both proteins had intermediate expression. Pathway analysis was carried out for genes bound by MYCN, MeCP2 or MYCN/MeCP2, revealing higher order functions.

Conclusions

Our results indicate that MYCN and MeCP2 protein interact and co-localize to similar genomic sites at very high frequency, and that the patterns of binding of these proteins can be associated with significant differences in transcriptional activity. Although it is not yet known if this interaction contributes to neuroblastoma disease pathogenesis, it is intriguing that the interaction occurs at the promoter regions of several genes important for the development of neuroblastoma, including ALK, AURKA and BDNF.

Purpose

The purpose of this study was to further define the biology of the 11q− neuroblastoma tumor subgroup by the integration of aCGH with miRNA expression profiling data to determine if improved patient stratification is possible.

Experimental Design

A set of primary neuroblastoma (n=160) which was broadly representative of all genetic subtypes was analyzed by aCGH and for the expression of 430 miRNAs. A 15 miRNA expression signature previously demonstrated to be predictive of clinical outcome was used to analyze an independent cohort of 11q− tumors (n=37).

Results

Loss of 4p and gain of 7q occurred at a significantly higher frequency in the 11q−tumors, further defining the genetic characteristics of this subtype. The 11q− tumors could be split into two subgroups using a miRNA expression survival signature which differed significantly in both clinical outcome and the overall frequency of large scale genomic imbalances, with the poor survival subgroup having significantly more imbalances. MiRNAs from the expression signature which were up-regulated in unfavorable tumors were predicted to target down-regulated genes from a published mRNA expression classifier of clinical outcome at a higher than expected frequency, indicating the miRNAs might contribute to the regulation of genes within the signature.

Conclusion

We demonstrate that two distinct biological subtypes of neuroblastoma with loss of 11q occur which differ in their miRNA expression profiles, frequency of segmental imbalances and clinical outcome. A miRNA expression signature, combined with an analysis of segmental imbalances, provides greater prediction of EFS and OS outcomes than 11q status by itself, improving patient stratification.

Background

Neuroblastoma is a paediatric cancer which originates from precursor cells of the sympathetic nervous system and accounts for 15% of childhood cancer mortalities. With regards to the role of miRNAs in neuroblastoma, miR-34a, mapping to a chromosome 1p36 region that is commonly deleted, has been found to act as a tumor suppressor through targeting of numerous genes associated with cell proliferation and apoptosis.

Methods

A synthetic miR-34a (or negative control) precursor molecule was transfected into NB1691luc and SK-N-ASluc neuroblastoma cells. Quantitative PCR was used to verify increased miR-34a levels in NB1691luc and SK-N-ASluc cell lines prior to in vitro and in vivo analysis. In vitro analysis of the effects of miR-34a over expression on cell growth, cell cycle and phosphoprotein activation in signal transduction pathways was performed. Neuroblastoma cells over expressing miR-34a were injected retroperitoneally into immunocompromised CB17-SCID mice and tumor burden was assessed over a 21 day period by measuring bioluminescence (photons/sec/cm2).

Results

Over expression of miR-34a in both NB1691luc and SK-N-ASluc neuroblastoma cell lines led to a significant decrease in cell number relative to premiR-negative control treated cells over a 72 hour period. Flow cytometry results indicated that miR-34a induced cell cycle arrest and subsequent apoptosis activation. Phosphoprotein analysis highlighted key elements involved in signal transduction, whose activation was dysregulated as a result of miR-34a introduction into cells. As a potential mechanism of miR-34a action on phosphoprotein levels, we demonstrate that miR-34a over-expression results in a significant reduction of MAP3K9 mRNA and protein levels. Although MAP3K9 is a predicted target of miR-34a, direct targeting could not be validated with luciferase reporter assays. Despite this fact, any functional effects of reduced MAP3K9 expression as a result of miR-34a would be expected to be similar regardless of the mechanism involved. Most notably, in vivo studies showed that tumor growth was significantly repressed after exogenous miR-34a administration in retroperitoneal neuroblastoma tumors.

Conclusion

We demonstrate for the first time that miR-34a significantly reduces tumor growth in an in vivo orthotopic murine model of neuroblastoma and identified novel effects that miR-34a has on phospho-activation of key proteins involved with apoptosis.

Neuroblastoma arises from precursor cells of the sympathetic nervous system and presently accounts for 15% of all childhood cancer deaths. These tumors display remarkable heterogeneity in clinical behavior, ranging from spontaneous regression to rapid progression and resistance to therapy. The clinical behavior of these tumors is associated with many factors, including patient age, histopathology and genetic abnormalities such as MYCN amplification. More recently, the dysregulation of some miRNAs, including the miR-17-5p-92 cluster and miR-34a, has been implicated in the pathobiology of neuroblastoma. MiR-17-5p-92 family members act in an oncogenic manner while miR-34a has tumor suppressor functions. The evidence for the contribution of miRNAs in the aggressive neuroblastoma phenotype is reviewed in this article, along with exciting possibilities for miRNA mediated therapeutics.

Primary effusion lymphomas (PEL) form a subset of AIDS-related lymphomas and usually have a poor prognosis. Although Kaposi’s sarcoma–associated herpes virus (KSHV) is often associated with PEL, very little is known about the exact mechanisms or causative effects of these associations. We investigated the chromosomal imbalances in six KSHV-positive PEL cell lines using comparative genomic hybridization analysis. We defined the shortest regions of overlaps for genomic gains on six chromosomes: 1q31, 4q31~q33, 7q10~q21, 8q21.1, 12q0~q23, and Xp11~q21. The recurrent nature of the gains found in these chromosomal regions suggests that these imbalances play roles in the pathogenesis of PEL.

A correction to Human fetal neuroblast and neuroblastoma transcriptome analysis confirms neuroblast origin and highlights neuroblastoma candidate genes by K De Preter, J Vandesompele, P Heimann, N Yigit, S Beckman, A Schramm, A Eggert, RL Stallings, Y Benoit, M Renard, A De Paepe, G Laureys, S Påhlman and F Speleman. Genome Biology 2006 7:R84

Background

Accurate outcome prediction in neuroblastoma, which is necessary to enable the optimal choice of risk-related therapy, remains a challenge. To improve neuroblastoma patient stratification, this study aimed to identify prognostic tumor DNA methylation biomarkers.

Results

To identify genes silenced by promoter methylation, we first applied two independent genome-wide methylation screening methodologies to eight neuroblastoma cell lines. Specifically, we used re-expression profiling upon 5-aza-2'-deoxycytidine (DAC) treatment and massively parallel sequencing after capturing with a methyl-CpG-binding domain (MBD-seq). Putative methylation markers were selected from DAC-upregulated genes through a literature search and an upfront methylation-specific PCR on 20 primary neuroblastoma tumors, as well as through MBD- seq in combination with publicly available neuroblastoma tumor gene expression data. This yielded 43 candidate biomarkers that were subsequently tested by high-throughput methylation-specific PCR on an independent cohort of 89 primary neuroblastoma tumors that had been selected for risk classification and survival. Based on this analysis, methylation of KRT19, FAS, PRPH, CNR1, QPCT, HIST1H3C, ACSS3 and GRB10 was found to be associated with at least one of the classical risk factors, namely age, stage or MYCN status. Importantly, HIST1H3C and GNAS methylation was associated with overall and/or event-free survival.

Conclusions

This study combines two genome-wide methylation discovery methodologies and is the most extensive validation study in neuroblastoma performed thus far. We identified several novel prognostic DNA methylation markers and provide a basis for the development of a DNA methylation-based prognostic classifier in neuroblastoma.

Background

Neuroblastoma is one of the most challenging malignancies of childhood, being associated with the highest death rate in paediatric oncology, underlining the need for novel therapeutic approaches. Typically, patients with high risk disease undergo an initial remission in response to treatment, followed by disease recurrence that has become refractory to further treatment. Here, we demonstrate the first silica nanoparticle-based targeted delivery of a tumor suppressive, pro-apoptotic microRNA, miR-34a, to neuroblastoma tumors in a murine orthotopic xenograft model. These tumors express high levels of the cell surface antigen disialoganglioside GD2 (GD2), providing a target for tumor-specific delivery.

Principal Findings

Nanoparticles encapsulating miR-34a and conjugated to a GD2 antibody facilitated tumor-specific delivery following systemic administration into tumor bearing mice, resulted in significantly decreased tumor growth, increased apoptosis and a reduction in vascularisation. We further demonstrate a novel, multi-step molecular mechanism by which miR-34a leads to increased levels of the tissue inhibitor metallopeptidase 2 precursor (TIMP2) protein, accounting for the highly reduced vascularisation noted in miR-34a-treated tumors.

Significance

These novel findings highlight the potential of anti-GD2-nanoparticle-mediated targeted delivery of miR-34a for both the treatment of GD2-expressing tumors, and as a basic discovery tool for elucidating biological effects of novel miRNAs on tumor growth.

Hippocampal sclerosis (HS) is a common pathological finding in patients with temporal lobe epilepsy (TLE) and is associated with altered expression of genes controlling neuronal excitability, glial function, neuroinflammation and cell death. MicroRNAs (miRNAs), a class of small non-coding RNAs, function as post-transcriptional regulators of gene expression and are critical for normal brain development and function. Production of mature miRNAs requires Dicer, an RNAase III, loss of which has been shown to cause neuronal and glial dysfunction, seizures, and neurodegeneration. Here we investigated miRNA biogenesis in hippocampal and neocortical resection specimens from pharmacoresistant TLE patients and autopsy controls. Western blot analysis revealed protein levels of Dicer were significantly lower in certain TLE patients with HS. Dicer levels were also reduced in the hippocampus of mice subject to experimentally-induced epilepsy. To determine if Dicer loss was associated with altered miRNA processing, we profiled levels of 380 mature miRNAs in control and TLE-HS samples. Expression of nearly 200 miRNAs was detected in control human hippocampus. In TLE-HS samples there was a large-scale reduction of miRNA expression, with 51% expressed at lower levels and a further 24% not detectable. Primary transcript (pri-miRNAs) expression levels for several tested miRNAs were not different between control and TLE-HS samples. These findings suggest loss of Dicer and failure of mature miRNA expression may be a feature of the pathophysiology of HS in patients with TLE.

Background

Protein tyrosine phosphatase receptor delta (PTPRD) is a member of a large family of protein tyrosine phosphatases which negatively regulate tyrosine phosphorylation. Neuroblastoma is a major childhood cancer arising from precursor cells of the sympathetic nervous system which is known to acquire deletions and alterations in the expression patterns of PTPRD, indicating a potential tumor suppressor function for this gene. The molecular mechanism, however, by which PTPRD renders a tumor suppressor effect in neuroblastoma is unknown.

Results

As a molecular mechanism, we demonstrate that PTPRD interacts with aurora kinase A (AURKA), an oncogenic protein that is over-expressed in multiple forms of cancer, including neuroblastoma. Ectopic up-regulation of PTPRD in neuroblastoma dephosphorylates tyrosine residues in AURKA resulting in a destabilization of this protein culminating in interfering with one of AURKA's primary functions in neuroblastoma, the stabilization of MYCN protein, the gene of which is amplified in approximately 25 to 30% of high risk neuroblastoma.

Conclusions

PTPRD has a tumor suppressor function in neuroblastoma through AURKA dephosphorylation and destabilization and a downstream destabilization of MYCN protein, representing a novel mechanism for the function of PTPRD in neuroblastoma.

Background

MicroRNAs are small molecules which regulate gene expression post-transcriptionally and aberrant expression of several miRNAs is associated with neuroblastoma, a childhood cancer arising from precursor cells of the sympathetic nervous system. Amplification of the MYCN transcription factor characterizes the most clinically aggressive subtype of this disease, and although alteration of p53 signaling is not commonly found in primary tumors, deregulation of proteins involved in this pathway frequently arise in recurrent disease after pharmacological treatment. TH-MYCN is a well-characterized transgenic model of MYCN-driven neuroblastoma which recapitulates many clinicopathologic features of the human disease. Here, we evaluate the dysregulation of miRNAs in tumors from TH-MYCN mice that are either wild-type (TH-MYCN) or deficient (TH-MYCN/p53ERTAM) for the p53 tumor suppressor gene.

Principal Findings

We analyzed the expression of 591 miRNAs in control (adrenal) and neuroblastoma tumor tissues derived from either TH-MYCN or TH-MYCN/p53ERTAM mice, respectively wild-type or deficient in p53. Comparing miRNA expression in tumor and control samples, we identified 159 differentially expressed miRNAs. Using data previously obtained from human neuroblastoma samples, we performed a comparison of miRNA expression between murine and human tumors to assess the concordance between murine and human expression data. Notably, the miR-17-5p-92 oncogenic polycistronic cluster, which is over-expressed in human MYCN amplified tumors, was over-expressed in mouse tumors. Moreover, analyzing miRNAs expression in a mouse model (TH-MYCN/p53ERTAM) possessing a transgenic p53 allele that drives the expression of an inactive protein, we identified miR-125b-3p and miR-676 as directly or indirectly regulated by the level of functional p53.

Significance

Our study represents the first miRNA profiling of an important mouse model of neuroblastoma. Similarities and differences in miRNAs expression between human and murine neuroblastoma were identified, providing important insight into the efficacy of this mouse model for assessing miRNA involvement in neuroblastoma and their potential effectiveness as therapeutic targets.

Background

Neuroblastoma is a paediatric cancer of the sympathetic nervous system. The single most important genetic indicator of poor clinical outcome is amplification of the MYCN transcription factor. One of many down-stream MYCN targets is miR-184, which is either directly or indirectly repressed by this transcription factor, possibly due to its pro-apoptotic effects when ectopically over-expressed in neuroblastoma cells. The purpose of this study was to elucidate the molecular mechanism by which miR-184 conveys pro-apoptotic effects.

Results

We demonstrate that the knock-down of endogenous miR-184 has the opposite effect of ectopic up-regulation, leading to enhanced neuroblastoma cell numbers. As a mechanism of how miR-184 causes apoptosis when over-expressed, and increased cell numbers when inhibited, we demonstrate direct targeting and degradation of AKT2, a major downstream effector of the phosphatidylinositol 3-kinase (PI3K) pathway, one of the most potent pro-survival pathways in cancer. The pro-apoptotic effects of miR-184 ectopic over-expression in neuroblastoma cell lines is reproduced by siRNA inhibition of AKT2, while a positive effect on cell numbers similar to that obtained by the knock-down of endogenous miR-184 can be achieved by ectopic up-regulation of AKT2. Moreover, co-transfection of miR-184 with an AKT2 expression vector lacking the miR-184 target site in the 3'UTR rescues cells from the pro-apoptotic effects of miR-184.

Conclusions

MYCN contributes to tumorigenesis, in part, by repressing miR-184, leading to increased levels of AKT2, a direct target of miR-184. Thus, two important genes with positive effects on cell growth and survival, MYCN and AKT2, can be linked into a common genetic pathway through the actions of miR-184. As an inhibitor of AKT2, miR-184 could be of potential benefit in miRNA mediated therapeutics of MYCN amplified neuroblastoma and other forms of cancer.

Background

Neuroblastoma, a cancer derived from precursor cells of the sympathetic nervous system, is a major cause of childhood cancer related deaths. The single most important prognostic indicator of poor clinical outcome in this disease is genomic amplification of MYCN, a member of a family of oncogenic transcription factors.

Methodology

We applied MYCN chromatin immunoprecipitation to microarrays (ChIP-chip) using MYCN amplified/non-amplified cell lines as well as a conditional knockdown cell line to determine the distribution of MYCN binding sites within all annotated promoter regions.

Conclusion

Assessment of E-box usage within consistently positive MYCN binding sites revealed a predominance for the CATGTG motif (p<0.0016), with significant enrichment of additional motifs CATTTG, CATCTG, CAACTG in the MYCN amplified state. For cell lines over-expressing MYCN, gene ontology analysis revealed enrichment for the binding of MYCN at promoter regions of numerous molecular functional groups including DNA helicases and mRNA transcriptional regulation. In order to evaluate MYCN binding with respect to other genomic features, we determined the methylation status of all annotated CpG islands and promoter sequences using methylated DNA immunoprecipitation (MeDIP). The integration of MYCN ChIP-chip and MeDIP data revealed a highly significant positive correlation between MYCN binding and DNA hypermethylation. This association was also detected in regions of hemizygous loss, indicating that the observed association occurs on the same homologue. In summary, these findings suggest that MYCN binding occurs more commonly at CATGTG as opposed to the classic CACGTG E-box motif, and that disease associated over expression of MYCN leads to aberrant binding to additional weaker affinity E-box motifs in neuroblastoma. The co-localization of MYCN binding and DNA hypermethylation further supports the dual role of MYCN, namely that of a classical transcription factor affecting the activity of individual genes, and that of a mediator of global chromatin structure.

MiRNAs regulate gene expression at a post-transcriptional level and their dysregulation can play major roles in the pathogenesis of many different forms of cancer, including neuroblastoma, an often fatal paediatric cancer originating from precursor cells of the sympathetic nervous system. We have analyzed a set of neuroblastoma (n = 145) that is broadly representative of the genetic subtypes of this disease for miRNA expression (430 loci by stem-loop RT qPCR) and for DNA copy number alterations (array CGH) to assess miRNA involvement in disease pathogenesis. The tumors were stratified and then randomly split into a training set (n = 96) and a validation set (n = 49) for data analysis. Thirty-seven miRNAs were significantly over- or under-expressed in MYCN amplified tumors relative to MYCN single copy tumors, indicating a potential role for the MYCN transcription factor in either the direct or indirect dysregulation of these loci. In addition, we also determined that there was a highly significant correlation between miRNA expression levels and DNA copy number, indicating a role for large-scale genomic imbalances in the dysregulation of miRNA expression. In order to directly assess whether miRNA expression was predictive of clinical outcome, we used the Random Forest classifier to identify miRNAs that were most significantly associated with poor overall patient survival and developed a 15 miRNA signature that was predictive of overall survival with 72.7% sensitivity and 86.5% specificity in the validation set of tumors. We conclude that there is widespread dysregulation of miRNA expression in neuroblastoma tumors caused by both over-expression of the MYCN transcription factor and by large-scale chromosomal imbalances. MiRNA expression patterns are also predicative of clinical outcome, highlighting the potential for miRNA mediated diagnostics and therapeutics.

Transcriptome profiling of neuroblasts and neuroblastoma tumor cells provides strong support for a neuroblast origin of neuroblastoma and highlights new candidate neuroblastoma genes

Background

Neuroblastoma tumor cells are assumed to originate from primitive neuroblasts giving rise to the sympathetic nervous system. Because these precursor cells are not detectable in postnatal life, their transcription profile has remained inaccessible for comparative data mining strategies in neuroblastoma. This study provides the first genome-wide mRNA expression profile of these human fetal sympathetic neuroblasts. To this purpose, small islets of normal neuroblasts were isolated by laser microdissection from human fetal adrenal glands.

Results

Expression of catecholamine metabolism genes, and neuronal and neuroendocrine markers in the neuroblasts indicated that the proper cells were microdissected. The similarities in expression profile between normal neuroblasts and malignant neuroblastomas provided strong evidence for the neuroblast origin hypothesis of neuroblastoma. Next, supervised feature selection was used to identify the genes that are differentially expressed in normal neuroblasts versus neuroblastoma tumors. This approach efficiently sifted out genes previously reported in neuroblastoma expression profiling studies; most importantly, it also highlighted a series of genes and pathways previously not mentioned in neuroblastoma biology but that were assumed to be involved in neuroblastoma pathogenesis.

Conclusion

This unique dataset adds power to ongoing and future gene expression studies in neuroblastoma and will facilitate the identification of molecular targets for novel therapies. In addition, this neuroblast transcriptome resource could prove useful for the further study of human sympathoadrenal biogenesis.