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Ornithine decarboxylase (ODC1), a critical regulatory enzyme in polyamine biosynthesis, is a direct transcriptional target of MYCN, amplification of which is a powerful marker of aggressive neuroblastoma. A single nucleotide polymorphism (SNP), G316A, within the first intron of ODC1 , results in genotypes wildtype GG, and variants AG/AA. CRISPR-cas9 technology was used to investigate the effects of AG clones from wildtype MYCN -amplified SK-N-BE(2)-C cells and the effect of the SNP on MYCN binding, and promoter activity was investigated using EMSA and luciferase assays. AG clones exhibited decreased ODC1 expression, growth rates, and histone acetylation and increased sensitivity to ODC1 inhibition. MYCN was a stronger transcriptional regulator of the ODC1 promoter containing the G allele, and preferentially bound the G allele over the A. Two neuroblastoma cohorts were used to investigate the clinical impact of the SNP. In the study cohort, the minor AA genotype was associated with improved survival, while poor prognosis was associated with the GG genotype and AG/GG genotypes in MYCN -amplified and non-amplified patients, respectively. These effects were lost in the GWAS cohort. We have demonstrated that the ODC1 G316A polymorphism has functional significance in neuroblastoma and is subject to allele-specific regulation by the MYCN oncoprotein.

Neuroblastoma (NB) is a neural crest-derived tumor, which develops before birth or in early childhood, with metastatic dissemination typically preceding diagnosis. Tumors are characterized by a highly heterogeneous combination of cellular phenotypes demonstrating varying degrees of differentiation along different lineage pathways, and possessing distinct super-enhancers and core regulatory circuits, thereby leading to highly varied malignant potential and divergent clinical outcomes. Cytoskeletal reorganization is fundamental to cellular transformations, including the processes of cellular differentiation and epithelial to mesenchymal transition (EMT), previously reported by our lab and others to coincide with chemotherapy resistance and enhanced metastatic ability of tumor cells. This study set out to investigate the ability of the neuronal miR-124-3p to reverse the cellular transformation associated with drug resistance development and assess the anti-oncogenic role of this miRNA in in vitro models of drug-resistant adrenergic (ADRN) and mesenchymal (MES) neuroblastoma cell lines. Low expression of miR-124-3p in a cohort of neuroblastomas was significantly associated with poor overall and progression-free patient survival. Over-expression of miR-124-3p in vitro inhibited cell viability through the promotion of cell cycle arrest and induction of apoptosis in addition to sensitizing drug-resistant cells to chemotherapeutics in a panel of morphologically distinct neuroblastoma cell lines. Finally, we describe miR-124-3p direct targeting and repression of key up-regulated cytoskeletal genes including MYH9 , ACTN4 and PLEC and the reversal of the resistance-associated EMT and enhanced invasive capacity previously reported in our in vitro model (SK-N-ASCis24)., (Copyright © 2020 Nolan, Salvucci, Carberry, Barat, Segura, Fenn, Prehn, Stallings and Piskareva.)

Current therapies for most non-infectious diseases are directed at or affect functionality of the human translated genome, barely 2% of all genetic information. By contrast, the therapeutic potential of targeting the transcriptome, ~ 70% of the genome, remains largely unexplored. RNA therapeutics is an emerging field that widens the range of druggable targets and includes elements such as microRNA. Here, we sought to screen for microRNA with tumor-suppressive functions in neuroblastoma, an aggressive pediatric tumor of the sympathetic nervous system that requires the development of new therapies. We found miR-323a-5p and miR-342-5p to be capable of reducing cell proliferation in multiple neuroblastoma cell lines in vitro and in vivo, thereby providing a proof of concept for miRNA-based therapies for neuroblastoma. Furthermore, the combined inhibition of the direct identified targets such as CCND1, CHAF1A, INCENP and BCL-XL could reveal new vulnerabilities of high-risk neuroblastoma.

3D scaffold-based in vitro cell culturing is a recent technological advancement in cancer research bridging the gap between conventional 2D culture and in vivo tumours. The main challenge in treating neuroblastoma, a paediatric cancer of the sympathetic nervous system, is to combat tumour metastasis and resistance to multiple chemotherapeutic drugs. The aim of this study was to establish a physiologically relevant 3D neuroblastoma tissue-engineered system and explore its therapeutic relevance. Two neuroblastoma cell lines, chemotherapeutic sensitive Kelly and chemotherapeutic resistant KellyCis83 were cultured in a 3D in vitro model on two collagen-based scaffolds containing either glycosaminoglycan (Coll-GAG) or nanohydroxyapatite (Coll-nHA) and compared to 2D cell culture and an orthotopic murine model. Both neuroblastoma cell lines actively infiltrated the scaffolds and proliferated displaying >100-fold increased resistance to cisplatin treatment when compared to 2D cultures, exhibiting chemosensitivity similar to orthotopic xenograft in vivo models. This model demonstrated its applicability to validate miRNA-based gene delivery. The efficacy of liposomes bearing miRNA mimics uptake and gene knockdown was similar in both 2D and 3D in vitro culturing models highlighting the proof-of-principle for the applicability of 3D collagen-based scaffolds cell system for validation of miRNA function. Collectively, this data shows the successful development and characterisation of a physiologically relevant, scaffold-based 3D tissue-engineered neuroblastoma cell model, strongly supporting its value in the evaluation of chemotherapeutics, targeted therapies and investigation of neuroblastoma pathogenesis. While neuroblastoma is the specific disease being focused upon, the platform may have multi-functionality beyond this tumour type., Statement of Significance: Traditional 2D cell cultures do not completely capture the 3D architecture of cells and extracellular matrix contributing to a gap in our understanding of mammalian biology at the tissue level and may explain some of the discrepancies between in vitro and in vivo results. Here, we demonstrated the successful development and characterisation of a physiologically relevant, scaffold-based 3D tissue-engineered neuroblastoma cell model, strongly supporting its value in the evaluation of chemotherapeutics, targeted therapies and investigation of neuroblastoma pathogenesis. The ability to test drugs in this reproducible and controllable tissue-engineered model system will help reduce the attrition rate of the drug development process and lead to more effective and tailored therapies. Importantly, such 3D cell models help to reduce and replace animals for pre-clinical research addressing the principles of the 3Rs., (Copyright © 2018 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.)

Systemic lupus erythematosus (SLE) is a complex disease targeting multiple organs as a result of overactivation of the type I interferon (IFN) system, a feature currently being targeted by multiple biologic therapies against IFN-α. We have identified an estrogen-regulated microRNA, miR-302d, whose expression is decreased in SLE patient monocytes and identify its target as interferon regulatory factor (IRF)-9, a critical component of the transcriptional complex that regulates expression of interferon-stimulated genes (ISGs). In keeping with the reduced expression of miR-302d in SLE patient monocytes, IRF9 levels were increased, as was expression of a number of ISGs including MX1 and OAS1. In vivo evaluation revealed that miR-302d protects against pristane-induced inflammation in mice by targeting IRF9 and hence ISG expression. Importantly, patients with enhanced disease activity have markedly reduced expression of miR-302d and enhanced IRF9 and ISG expression, with miR-302d negatively correlating with IFN score. Together these findings identify miR-302d as a key regulator of type I IFN driven gene expression via its ability to target IRF9 and regulate ISG expression, underscoring the importance of non-coding RNA in regulating the IFN pathway in SLE., (Copyright © 2017 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Defects within a self-assembled monolayer (SAM) of dodecanethiol on gold have been used as nucleation sites for the electrodeposition of mushroom shaped platinum nanoparticles (PtNPs). The top surfaces of these PtNPs were then decorated with a layer of silver creating a hemispherical - platinum : silver core : shell nanoparticle (Pt-AgNP). Thiolated probe strand miRNA was then immobilised onto the upper silver surface. These regioselectively modified particles were desorbed by applying a current jump to yield nanoparticles capable of hybridising to a complementary miRNA target with electrocatalysis occurring on the non-functionalized lower surface. A second electrode was functionalized with single stranded capture miRNA that has a sequence that is complementary to an miRNA, miR-132, associated with the childhood cancer, Neuroblastoma but leaves a section of the target available to bind the nucleic acid sequence on the core : shell Pt-AgNPs. Following hybridization of the target and capture strands the surface was exposed to the miRNA labelled electrocatalytic Pt-AgNPs. The concentration of the target was then determined by monitoring the current associated with the reduction of hydrogen peroxide in a solution of H 2 SO 4 . Calibration plots of the log[miRNA] vs. faradaic current were linear from 1 aM to 1 μM and aM concentrations could be detected without the need for chemical amplification of the target, e.g., using PCR or NASBA. The regioselectively modified particles were also immobilised within the interior of gold microcavity arrays via miRNA hybridisation and their Raman properties investigated.

Assessment of cell viability and proliferation under different miRNA expression levels is an important step in the evaluation of basic miRNA functional effects within the cell. Here, we describe the overexpression of miRNA in question in cells achieved by transfection with subsequent examination of cell viability and proliferation over a period of time using the acid phosphatase assay.

Disturbance of homeostasis within the endoplasmic reticulum (ER) lumen leads to the accumulation of unfolded and misfolded proteins. This results in the activation of an evolutionary conserved stress response termed ER stress that, if unresolved, induces apoptosis. Previously the Bcl-2 homology domain 3-Only Protein Puma was identified as a mediator of ER stress-induced apoptosis in neurons. In the search of alternative contributors to ER stress-induced apoptosis, a downregulation of the anti-apoptotic Bcl-2 family protein Mcl-1 was noted during ER stress in both mouse cortical neurons and human SH-SY5Y neuroblastoma cells. Downregulation of Mcl-1 was associated with an upregulation of microRNA-29a (miR-29a) expression, and subsequent experiments showed that miR-29a targeted the 3'-untranslated region of the anti-apoptotic Bcl-2 family protein, Mcl-1. Inhibition of miR-29a expression using sequence-specific antagomirs or the overexpression of Mcl-1 decreased cell death following tunicamycin treatment, while gene silencing of Mcl-1 increased cell death. miR-29a did not alter the signalling branches of the ER stress response, rather its expression was controlled by the ER stress-induced transcription factor activating-transcription-factor-4 (ATF4). The current data demonstrate that the ATF4-mediated upregulation of miR-29a enhances the sensitivity of neurons to ER stress-induced apoptosis., (© 2016 Federation of European Neuroscience Societies and John Wiley & Sons Ltd.)

Despite multimodal therapies, a high percentage of high-risk neuroblastoma (NB) become refractory to current treatments, most of which interfere with cell cycle and DNA synthesis or function, activating the DNA damage response (DDR). In cancer, this process is frequently altered by deregulated expression or function of several genes which contribute to multidrug resistance (MDR). MicroRNAs are outstanding candidates for therapy since a single microRNA can modulate the expression of multiple genes of the same or different pathways, thus hindering the development of resistance mechanisms by the tumor. We found several genes implicated in the MDR to be overexpressed in high-risk NB which could be targeted by microRNAs simultaneously. Our functional screening identified several of those microRNAs that reduced proliferation of chemoresistant NB cell lines, the best of which was miR-497. Low expression of miR-497 correlated with poor patient outcome. The overexpression of miR-497 reduced the proliferation of multiple chemoresistant NB cell lines and induced apoptosis in MYCN-amplified cell lines. Moreover, the conditional expression of miR-497 in NB xenografts reduced tumor growth and inhibited vascular permeabilization. MiR-497 targets multiple genes related to the DDR, cell cycle, survival and angiogenesis, which renders this molecule a promising candidate for NB therapy.

Accurate assessment of neuroblastoma outcome prediction remains challenging. Therefore, this study aims at establishing novel prognostic tumor DNA methylation biomarkers. In total, 396 low- and high-risk primary tumors were analyzed, of which 87 were profiled using methyl-CpG-binding domain (MBD) sequencing for differential methylation analysis between prognostic patient groups. Subsequently, methylation-specific PCR (MSP) assays were developed for 78 top-ranking differentially methylated regions and tested on two independent cohorts of 132 and 177 samples, respectively. Further, a new statistical framework was used to identify a robust set of MSP assays of which the methylation score (i.e. the percentage of methylated assays) allows accurate outcome prediction. Survival analyses were performed on the individual target level, as well as on the combined multimarker signature. As a result of the differential DNA methylation assessment by MBD sequencing, 58 of the 78 MSP assays were designed in regions previously unexplored in neuroblastoma, and 36 are located in non-promoter or non-coding regions. In total, 5 individual MSP assays (located in CCDC177, NXPH1, lnc-MRPL3-2, lnc-TREX1-1 and one on a region from chromosome 8 with no further annotation) predict event-free survival and 4 additional assays (located in SPRED3, TNFAIP2, NPM2 and CYYR1) also predict overall survival. Furthermore, a robust 58-marker methylation signature predicting overall and event-free survival was established. In conclusion, this study encompasses the largest DNA methylation biomarker study in neuroblastoma so far. We identified and independently validated several novel prognostic biomarkers, as well as a prognostic 58-marker methylation signature.

Neuroblastoma is the most common extra-cranial solid tumour in children. Natural killer (NK) cells are innate lymphocytes that are known to mediate the direct cytotoxicity of neuroblastoma tumour cells. Natural variation in the highly polymorphic killer immunoglobulin-like receptors (KIR) and their cognate human leukocyte antigen (HLA) class I ligands results in considerable diversity in NK cell function. As the early onset of neuroblastoma suggests the contribution of genetic factors, we investigated if individual KIR genes, combined KIR gene haplotypes or compound KIR-HLA ligand genotypes could influence susceptibility to neuroblastoma. Genotype analysis of the KIR genes as well as their three major HLA class I ligand groups, HLA-C1, HLA-C2 and HLA-Bw4, was carried out in a cohort of 201 neuroblastoma patients compared with 240 healthy control subjects using polymerase chain reaction with sequence-specific primers. We found a significant increase in the frequency of KIR2DL2 (P = 0.019) as well as KIR2DS2 (P = 0.008) in patients with neuroblastoma compared with the healthy control group. While the incidence of the least inhibitory compound KIR-HLA-C genotype, KIR2DL3 in the presence of HLA-C1 was slightly reduced in neuroblastoma patients, this did not reach statistical significance (P = 0.069). In summary, while KIR-HLA compound genotypes have previously been implicated in predicting treatment outcomes in neuroblastoma, here we show that the presence of the individual KIR genes, KIR2DL2 and KIR2DS2, irrespective of HLA-C genotype is associated with the onset of this embryonal malignancy., (© 2015 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

Neuroblastoma is a challenging childhood malignancy, with a very high percentage of patients relapsing following acquisition of drug resistance, thereby necessitating the identification of mechanisms of drug resistance as well as new biological targets contributing to the aggressive pathogenicity of the disease. In order to investigate the molecular pathways that are involved with drug resistance in neuroblastoma, we have developed and characterised cisplatin resistant sublines SK-N-ASCis24, KellyCis83 and CHP-212Cis100, integrating data of cell behaviour, cytotoxicity, genomic alterations and modulation of protein expression. All three cisplatin resistant cell lines demonstrated cross resistance to temozolomide, etoposide and irinotecan, all of which are drugs in re-initiation therapy. Array CGH analysis indicated that resistant lines have acquired additional genomic imbalances. Differentially expressed proteins were identified by mass spectrometry and classified by bioinformatics tools according to their molecular and cellular functions and their involvement into biological pathways. Significant changes in the expression of proteins involved with pathways such as actin cytoskeletal signalling (p = 9.28E-10), integrin linked kinase (ILK) signalling (p = 4.01E-8), epithelial adherens junctions signalling (p = 5.49E-8) and remodelling of epithelial adherens junctions (p = 5.87E-8) pointed towards a mesenchymal phenotype developed by cisplatin resistant SK-N-ASCis24. Western blotting and confocal microscopy of MYH9, ACTN4 and ROCK1 coupled with invasion assays provide evidence that elevated levels of MYH9 and ACTN4 and reduced levels of ROCK1 contribute to the increased ROCK1-independent migratory potential of SK-N-ASCis24. Therefore, our results suggest that epithelial-to-mesenchymal transition is a feature during the development of drug resistance in neuroblastoma., (Copyright © 2015 The Authors. Published by Elsevier Ireland Ltd.. All rights reserved.)

The acquisition of multidrug resistance is a major impediment to the successful treatment of neuroblastoma, a clinically heterogeneous cancer accounting for ∼15% of all pediatric cancer deaths. The MYCN transcription factor, whose gene is amplified in ∼30% of high-risk neuroblastoma cases, influences drug resistance by regulating a cadre of genes, including those involved with drug efflux, however, other high-risk subtypes of neuroblastoma lacking MYCN amplification, such as those with chromosome 11q deletions, also acquire multidrug resistance. To elucidate additional mechanisms involved with drug resistance in non-MYCN amplified tumour cells, an SK-N-AS subline (SK-N-AsCis24) that is significantly resistant to cisplatin and cross resistant to etoposide was developed through a pulse-selection process. High resolution aCGH analysis of SK-N-AsCis24 revealed a focal gain on chromosome 5 containing the coding sequence for the neural apoptosis inhibitory protein (NAIP). Significant overexpression of NAIP mRNA and protein was documented, while experimental modulation of NAIP levels in both SK-N-AsCis24 and in parental SK-N-AS cells confirmed that NAIP was responsible for the drug resistant phenotype by apoptosis inhibition. Furthermore, a decrease in the NAIP targeting microRNA, miR-520f, was also demonstrated to be partially responsible for increased NAIP levels in SK-N-AsCis24. Interestingly, miR-520f levels were determined to be significantly lower in postchemotherapy treatment tumours relative to matched prechemotherapy samples, consistent with a role for this miRNA in the acquisition of drug resistance in vivo, potentially through decreased NAIP targeting. Our findings provide biological novel insight into neuroblastoma drug-resistance and have implications for future therapeutic research., (© 2014 UICC.)

Temporal lobe epilepsy is associated with large-scale, wide-ranging changes in gene expression in the hippocampus. Epigenetic changes to DNA are attractive mechanisms to explain the sustained hyperexcitability of chronic epilepsy. Here, through methylation analysis of all annotated C-phosphate-G islands and promoter regions in the human genome, we report a pilot study of the methylation profiles of temporal lobe epilepsy with or without hippocampal sclerosis. Furthermore, by comparative analysis of expression and promoter methylation, we identify methylation sensitive non-coding RNA in human temporal lobe epilepsy. A total of 146 protein-coding genes exhibited altered DNA methylation in temporal lobe epilepsy hippocampus (n = 9) when compared to control (n = 5), with 81.5% of the promoters of these genes displaying hypermethylation. Unique methylation profiles were evident in temporal lobe epilepsy with or without hippocampal sclerosis, in addition to a common methylation profile regardless of pathology grade. Gene ontology terms associated with development, neuron remodelling and neuron maturation were over-represented in the methylation profile of Watson Grade 1 samples (mild hippocampal sclerosis). In addition to genes associated with neuronal, neurotransmitter/synaptic transmission and cell death functions, differential hypermethylation of genes associated with transcriptional regulation was evident in temporal lobe epilepsy, but overall few genes previously associated with epilepsy were among the differentially methylated. Finally, a panel of 13, methylation-sensitive microRNA were identified in temporal lobe epilepsy including MIR27A, miR-193a-5p (MIR193A) and miR-876-3p (MIR876), and the differential methylation of long non-coding RNA documented for the first time. The present study therefore reports select, genome-wide DNA methylation changes in human temporal lobe epilepsy that may contribute to the molecular architecture of the epileptic brain., (© The Author (2014). Published by Oxford University Press on behalf of the Guarantors of Brain. All rights reserved. For Permissions, please email: journals.permissions@oup.com.)

Background: Neuroblastoma is a pediatric cancer that exhibits a wide clinical spectrum ranging from spontaneous regression in low-risk patients to fatal disease in high-risk patients. The identification of single nucleotide polymorphisms (SNPs) may help explain the heterogeneity of neuroblastoma and assist in identifying patients at higher risk for poor survival. SNPs in the TP53 pathway are of special importance, as several studies have reported associations between TP53 pathway SNPs and cancer. Of note, less than 2% of neuroblastoma tumors have a TP53 mutation at diagnosis., Patients and Methods: We selected 21 of the most frequently studied SNPs in the TP53 pathway and evaluated their association with outcome in 500 neuroblastoma patients using TaqMan allelic discrimination assays., Results and Conclusion: We investigated the impact of 21 SNPs on overall survival, event-free survival, age at diagnosis, MYCN status, and stage of the disease in 500 neuroblastoma patients. A missense SNP in exon 10 of the CASP8 gene SNP D302H was associated with worse overall and event-free survival in patients with MYCN-amplified neuroblastoma tumors.

Identifying relevant signatures for clinical patient outcome is a fundamental task in high-throughput studies. Signatures, composed of features such as mRNAs, miRNAs, SNPs or other molecular variables, are often non-overlapping, even though they have been identified from similar experiments considering samples with the same type of disease. The lack of a consensus is mostly due to the fact that sample sizes are far smaller than the numbers of candidate features to be considered, and therefore signature selection suffers from large variation. We propose a robust signature selection method that enhances the selection stability of penalized regression algorithms for predicting survival risk. Our method is based on an aggregation of multiple, possibly unstable, signatures obtained with the preconditioned lasso algorithm applied to random (internal) subsamples of a given cohort data, where the aggregated signature is shrunken by a simple thresholding strategy. The resulting method, RS-PL, is conceptually simple and easy to apply, relying on parameters automatically tuned by cross validation. Robust signature selection using RS-PL operates within an (external) subsampling framework to estimate the selection probabilities of features in multiple trials of RS-PL. These probabilities are used for identifying reliable features to be included in a signature. Our method was evaluated on microarray data sets from neuroblastoma, lung adenocarcinoma, and breast cancer patients, extracting robust and relevant signatures for predicting survival risk. Signatures obtained by our method achieved high prediction performance and robustness, consistently over the three data sets. Genes with high selection probability in our robust signatures have been reported as cancer-relevant. The ordering of predictor coefficients associated with signatures was well-preserved across multiple trials of RS-PL, demonstrating the capability of our method for identifying a transferable consensus signature. The software is available as an R package rsig at CRAN (http://cran.r-project.org).

While a polymorphism located within the promoter region of the MDM2 proto-oncogene, SNP309 (T > G), has previously been associated with increased risk and aggressiveness of neuroblastoma and other tumor entities, a protective effect has also been reported in certain other cancers. In this study, we evaluated the association of MDM2 SNP309 with outcome in 496 patients with neuroblastoma and its effect on MDM2 expression. No significant difference in overall or event-free survival was observed among patients with neuroblastoma with or without MDM2 SNP309. The presence of SNP309 does not affect MDM2 expression in neuroblastoma., (© 2014 Wiley Periodicals, Inc.)

Neuroblastoma (NBL) is the most common solid tumor in infants and accounts for 15% of all pediatric cancer deaths. Several risk factors predict NBL outcome: age at the time of diagnosis, stage, chromosome alterations and MYCN (V-Myc Avian Myelocytomatosis Viral Oncogene Neuroblastoma-Derived Homolog) amplification, which characterizes the subset of the most aggressive NBLs with an overall survival below 30%. MYCN-amplified tumors develop exceptional chemoresistance and metastatic capacity. These properties have been linked to defects in the apoptotic machinery, either by silencing components of the extrinsic apoptotic pathway (e.g. caspase-8) or by overexpression of antiapoptotic regulators (e.g. Bcl-2, Mcl-1 or FLIP). Very little is known on the implication of death receptors and their antagonists in NBL. In this work, the expression levels of several death receptor antagonists were analyzed in multiple human NBL data sets. We report that Lifeguard (LFG/FAIM2 (Fas apoptosis inhibitory molecule 2)/NMP35) is downregulated in the most aggressive and undifferentiated tumors. Intringuingly, although LFG has been initially characterized as an antiapoptotic protein, we have found a new association with NBL differentiation. Moreover, LFG repression resulted in reduced cell adhesion, increased sphere growth and enhanced migration, thus conferring a higher metastatic capacity to NBL cells. Furthermore, LFG expression was found to be directly repressed by MYCN at the transcriptional level. Our data, which support a new functional role for a hitherto undiscovered MYCN target, provide a new link between MYCN overexpression and increased NBL metastatic properties.

MicroRNAs (miRNAs) are small non-coding RNA molecules that regulate gene expression at a post-transcriptional level. An miRNA may target many messenger RNA (mRNA) transcripts, and each transcript may be targeted by multiple miRNAs. Our understanding of miRNA regulation is evolving to consider modules of miRNAs that regulate groups of functionally related mRNAs. Here we expand the model of miRNA functional modules and use it to guide the integration of miRNA and mRNA expression and target prediction data. We present evidence of cooperativity between miRNA classes within this integrated miRNA-mRNA association matrix. We then apply bicluster analysis to uncover miRNA functional modules within this integrated data set and develop a novel application to visualize and query these results. We show that this wholly unsupervised approach can discover a network of miRNA-mRNA modules that are enriched for both biological processes and miRNA classes. We apply this method to investigate the interplay of miRNAs and mRNAs in integrated data sets derived from neuroblastoma and human immune cells. This study is the first to apply the technique of biclustering to model functional modules within an integrated miRNA-mRNA association matrix. Results provide evidence of an extensive modular miRNA functional network and enable characterization of miRNA function and dysregulation in disease.

MiRNAs can have pleiotropic effects by targeting multiple genes belonging to diverse signalling networks. Alternatively, miRNAs can enhance the potency of their cellular effects by targeting multiple genes within the same genetic pathway. Previously, we and others have demonstrated that miR-335 is a potent suppressor of tumour cell migration, invasion and metastasis, in part by targeting several genes involved in these cellular processes, including ROCK1, MAPK1, LRG1, SP1 and SOX4. Here, we demonstrate that direct targeting of multiple members of the formin family of actin nucleators contributes to the inhibitory effects of miR-335 in neuroblastoma cells. We demonstrate that miR-335 regulates the expression of at least five formin family members and validate three family members, FMNL3, FMN2 and DAAM2, as direct targets of miR-335. The contribution of the formin family genes to cancer progression and metastasis has recently begun to emerge and here we demonstrate for the first time the ability of FMN2 and DAAM2 to regulate tumour cell migration and invasion, using siRNA-mediated inhibition of each of these formin genes. Finally, we demonstrate that the formin genes, in particular FMNL3, are responsible for the protrusion of actin-rich filopodia structures that contribute to the enhanced migratory and invasive potential associated with reduced expression of miR-335. Thus, direct targeting of the formin family contributes to the metastasis suppressing abilities of miR-335 by providing a direct regulatory link to the actin assembly machinery of the cell. We conclude that miR-335 is a master regulator of tumour cell migration and invasion by directly targeting a plethora of genes that effectively control cell migratory processes.

Skeletogenesis is initiated during fetal development and persists through adult life as either a remodeling process in response to homeostatic regulation or as a regenerative process in response to physical injury. Mesenchymal stem cells (MSCs) play a crucial role providing progenitor cells from which osteoblasts, bone matrix forming cells are differentiated. The mechanical environment plays an important role in regulating stem cell differentiation into osteoblasts, however, the mechanisms by which MSCs respond to mechanical stimuli are yet to be fully elucidated. To increase understanding of MSC mechanotransuction and osteogenic differentiation, this study aimed to identify novel, mechanically augmented genes and pathways with pro-osteogenic functionality. Using collagen glycoaminoglycan scaffolds as mimics of native extracellular matrix, to create a 3D environment more representative of that found in bone, MSC-seeded constructs were mechanically stimulated in a flow-perfusion bioreactor. Global gene expression profiling techniques were used to identify potential candidates warranting further investigation. Of these, placental growth factor (PGF) was selected and expression levels were shown to strongly correlate to both the magnitude and duration of mechanical stimulation. We demonstrated that PGF gene expression was modulated through an actin polymerization-mediated mechanism. The functional role of PGF in modulating MSC osteogenic differentiation was interrogated, and we showed a concentration-dependent response whereby low concentrations exhibited the strongest pro-osteogenic effect. Furthermore, pre-osteoclast migration and differentiation, as well as endothelial cell tubule formation also maintained concentration-dependent responses to PGF, suggesting a potential role for PGF in bone resorption and angiogenesis, processes key to bone remodeling and fracture repair., (Copyright © 2013 AlphaMed Press.)

Neuroblastoma is the most common extracranial solid tumor of childhood, and accounts for ∼15% of all childhood cancer deaths. The histone demethylase, lysine-specific demethylase 1 (KDM1A, previously known as LSD1), is strongly expressed in neuroblastomas, and overexpression correlates with poor patient prognosis. Inducing differentiation in neuroblastoma cells has previously been shown to down regulate KDM1A, and siRNA-mediated KDM1A knockdown inhibited neuroblastoma cell viability. The microRNA, miR-137, has been reported to be downregulated in several human cancers, and KDM1A mRNA was reported as a putative target of miR-137 in colon cancer. We hypothesized that miR-137 might have a tumor-suppressive role in neuroblastoma mediated via downregulation of KDM1A. Indeed, low levels of miR-137 expression in primary neuroblastomas correlated with poor patient prognosis. Re-expressing miR-137 in neuroblastoma cell lines increased apoptosis and decreased cell viability and proliferation. KDM1A mRNA was repressed by miR-137 in neuroblastoma cells, and was validated as a direct target of miR-137 using reporter assays in SHEP and HEK293 cells. Furthermore, siRNA-mediated KDM1A knockdown phenocopied the miR-137 re-expression phenotype in neuroblastoma cells. We conclude that miR-137 directly targets KDM1A mRNA in neuroblastoma cells, and activates cell properties consistent with tumor suppression. Therapeutic strategies to re-express miR-137 in neuroblastomas could be useful to reduce tumor aggressiveness., (Copyright © 2013 UICC.)

Aims/hypothesis: HNF1A-MODY is a monogenic form of diabetes caused by mutations in the HNF1A gene. Here we identify, for the first time, HNF1A-MODY-associated microRNAs (miRNAs) that can be detected in the serum of HNF1A-MODY carriers., Methods: An miRNA array was carried out in rat INS-1 insulinoma cells inducibly expressing the common human Pro291fsinsC-HNF1A frame shift mutation. Differentially expressed miRNAs were validated by quantitative real-time PCR. Expression of miRNAs in the serum of HNF1A-MODY carriers (n = 31), MODY-negative family members (n = 10) and individuals with type 2 diabetes mellitus (n = 17) was quantified by absolute real-time PCR analysis., Results: Inducible expression of Pro291fsinsC-HNF1A in INS-1 cells caused a significant upregulation of three miRNAs (miR-103, miR-224, miR-292-3p). The differential expression of two miRNAs (miR-103 and miR-224) was validated in vitro. Strongly elevated levels of miR-103 and miR-224 could be detected in the serum of HNF1A-MODY carriers compared with MODY-negative family controls. Serum levels of miR-103 distinguished HNF1A-MODY carriers from HbA1c-matched individuals with type 2 diabetes mellitus., Conclusions/interpretation: Our study demonstrates that the pathophysiology of HNF1A-MODY is associated with the overexpression of miR-103 and miR-224. Furthermore, our study demonstrates that these miRNAs can be readily detected in the serum of HNF1A-MODY carriers.

Background: Neuroblastoma (NB) tumours are commonly divided into three cytogenetic subgroups. However, by unsupervised principal components analysis of gene expression profiles we recently identified four distinct subgroups, r1-r4. In the current study we characterized these different subgroups in more detail, with a specific focus on the fourth divergent tumour subgroup (r4)., Methods: Expression microarray data from four international studies corresponding to 148 neuroblastic tumour cases were subject to division into four expression subgroups using a previously described 6-gene signature. Differentially expressed genes between groups were identified using Significance Analysis of Microarray (SAM). Next, gene expression network modelling was performed to map signalling pathways and cellular processes representing each subgroup. Findings were validated at the protein level by immunohistochemistry and immunoblot analyses., Results: We identified several significantly up-regulated genes in the r4 subgroup of which the tyrosine kinase receptor ERBB3 was most prominent (fold change: 132-240). By gene set enrichment analysis (GSEA) the constructed gene network of ERBB3 (n = 38 network partners) was significantly enriched in the r4 subgroup in all four independent data sets. ERBB3 was also positively correlated to the ErbB family members EGFR and ERBB2 in all data sets, and a concurrent overexpression was seen in the r4 subgroup. Further studies of histopathology categories using a fifth data set of 110 neuroblastic tumours, showed a striking similarity between the expression profile of r4 to ganglioneuroblastoma (GNB) and ganglioneuroma (GN) tumours. In contrast, the NB histopathological subtype was dominated by mitotic regulating genes, characterizing unfavourable NB subgroups in particular. The high ErbB3 expression in GN tumour types was verified at the protein level, and showed mainly expression in the mature ganglion cells., Conclusions: Conclusively, this study demonstrates the importance of performing unsupervised clustering and subtype discovery of data sets prior to analyses to avoid a mixture of tumour subtypes, which may otherwise give distorted results and lead to incorrect conclusions. The current study identifies ERBB3 as a clear-cut marker of a GNB/GN-like expression profile, and we suggest a 7-gene expression signature (including ERBB3) as a complement to histopathology analysis of neuroblastic tumours. Further studies of ErbB3 and other ErbB family members and their role in neuroblastic differentiation and pathogenesis are warranted.

MicroRNAs (miRNAs) contribute to the pathogenesis of many forms of cancer, including the pediatric cancer neuroblastoma, but the underlying mechanisms leading to altered miRNA expression are often unknown. Here, a novel integrated approach for analyzing DNA methylation coupled with miRNA and mRNA expression data sets identified 67 epigenetically regulated miRNA in neuroblastoma. A large proportion (42%) of these miRNAs was associated with poor patient survival when underexpressed in tumors. Moreover, we demonstrate that this panel of epigenetically silenced miRNAs targets a large set of genes that are overexpressed in tumors from patients with poor survival in a highly redundant manner. The genes targeted by the epigenetically regulated miRNAs are enriched for a number of biological processes, including regulation of cell differentiation. Functional studies involving ectopic overexpression of several of the epigenetically silenced miRNAs had a negative impact on neuroblastoma cell viability, providing further support to the concept that inactivation of these miRNAs is important for neuroblastoma disease pathogenesis. One locus, miR-340, induced either differentiation or apoptosis in a cell context dependent manner, indicating a tumor suppressive function for this miRNA. Intriguingly, it was determined that miR-340 is upregulated by demethylation of an upstream genomic region that occurs during the process of neuroblastoma cell differentiation induced by all-trans retinoic acid (ATRA). Further biological studies of miR-340 revealed that it directly represses the SOX2 transcription factor by targeting of its 3'-untranslated region, explaining the mechanism by which SOX2 is downregulated by ATRA. Although SOX2 contributes to the maintenance of stem cells in an undifferentiated state, we demonstrate that miR-340-mediated downregulation of SOX2 is not required for ATRA induced differentiation to occur. In summary, our results exemplify the dynamic nature of the miRNA epigenome and identify a remarkable network of miRNA/mRNA interactions that significantly contribute to neuroblastoma disease pathogenesis.

Background: Ultra-conserved regions (UCRs) are segments of the genome (≥ 200 bp) that exhibit 100% DNA sequence conservation between human, mouse and rat. Transcribed UCRs (T-UCRs) have been shown to be differentially expressed in cancers versus normal tissue, indicating a possible role in carcinogenesis. All-trans-retinoic acid (ATRA) causes some neuroblastoma (NB) cell lines to undergo differentiation and leads to a significant decrease in the oncogenic transcription factor MYCN. Here, we examine the impact of ATRA treatment on T-UCR expression and investigate the biological significance of these changes., Methods: We designed a custom tiling microarray to profile the expression of 481 T-UCRs in sense and anti-sense orientation (962 potential transcripts) in untreated and ATRA-treated neuroblastoma cell lines (SH-SY5Y, SK-N-BE, LAN-5). Following identification of significantly differentially expressed T-UCRs, we carried out siRNA knockdown and gene expression microarray analysis to investigate putative functional roles for selected T-UCRs., Results: Following ATRA-induced differentiation, 32 T-UCRs were differentially expressed (16 up-regulated, 16 down-regulated) across all three cell lines. Further insight into the possible role of T-UC.300A, an independent transcript whose expression is down-regulated following ATRA was achieved by siRNA knockdown, resulting in the decreased viability and invasiveness of ATRA-responsive cell lines. Gene expression microarray analysis following knockdown of T-UC.300A revealed a number of genes whose expression was altered by changing T-UC.300A levels and that might play a role in the increased proliferation and invasion of NB cells prior to ATRA-treatment., Conclusions: Our results indicate that significant numbers of T-UCRs have altered expression levels in response to ATRA. While the precise roles that T-UCRs might play in cancer or in normal development are largely unknown and an important area for future study, our findings strongly indicate that the function of non-coding RNA T-UC.300A is connected with proliferation, invasion and the inhibition of differentiation of neuroblastoma cell lines prior to ATRA treatment.

Neuroblastoma is a genetically and clinically heterogeneous tumor of childhood, arising from precursor cells of the sympathetic nervous system. It is still a challenging cancer for pediatric oncology, as some tumors will spontaneously regress, while others will become refractory to all forms of therapy. The clinical course of this disease is greatly influenced by both patient age and the genetic abnormalities that occur within the tumors. MYCN (v-myc myelocytomatosis viral related oncogene, neuroblastoma derived (avian)) amplification and loss of chromosome 11q heterozygosity have been known to be indicative of poor prognosis. In this article, we review how mutations and structural alterations in specific genes contribute to inheritable predisposition to neuroblastoma and/or to aggressive disease pathogenesis, as well as implications for diagnosis and therapy. These genes include PHOX2B (paired-like homeobox 2b), ALK (anaplastic lymphoma receptor tyrosine kinase), and ATRX (alpha thalassemia/mental retardation syndrome X-linked).

Background: Neuroblastoma is responsible for 15% of all childhood cancer deaths. Despite advances in treatment and disease management, the overall 5-year survival rates remain poor in high-risk disease (25-40%). MiR-497 was previously identified by our laboratory as a member of a miRNA expression signature, predictive of neuroblastoma patient survival and has been reported as a tumor suppressor in a variety of other cancers. WEE1, a tyrosine kinase regulator of the cell cycle and predicted target of miR-497, has emerged as an oncogene in several cancer types and therefore represents an attractive potential target for novel therapy approaches in high-risk neuroblastoma. Our aim was to investigate the potential tumor suppressive role of miR-497 in high-risk neuroblastoma., Methods: Expression levels of miR-497 and WEE1 in tissues and cells were determined using RT-PCR. The effect of miR-497 and siWEE1 on cell viability was evaluated using MTS assays, apoptosis levels were determined using FACS analysis of Annexin V/PI stained cells, and target protein expression was determined using western blot. Luciferase reporter plasmids were constructed to confirm direct targeting. Results were reported as mean±S.E.M and differences were tested for significance using 2-tailed Students t-test., Results: We determined that miR-497 expression was significantly lower in high-risk MYCN amplified (MNA) tumors and that low miR-497 expression was associated with worse EFS and OS in our cohort. Over-expression of miR-497 reduced cell viability and increased apoptosis in MNA cells. We identified WEE1 as a novel target for miR-497 in neuroblastoma. Furthermore, our analysis showed that high WEE1 levels are significantly associated with poor EFS and OS in neuroblastoma and that siRNA knockdown of WEE1 in MNA cell lines results in significant levels of apoptosis, supporting an oncogenic role of WEE1 in neuroblastoma. Cisplatin (CDDP) treatment of both miR-497 over-expressing cells and WEE1 inhibited cells, resulted in a significant increase in apoptosis in MNA cells, describing a synergistic effect and therefore a potential therapeutic for high-risk neuroblastoma., Conclusion: Our study's results are consistent with miR-497 being a candidate tumor suppressor in neuroblastoma, through the direct targeting of WEE1. These findings re-enforce the proposal of WEE1 as a therapeutic target in neuroblastoma.

Neuroblastoma is a highly heterogeneous tumor accounting for 15 % of all pediatric cancer deaths. Clinical behavior ranges from the spontaneous regression of localized, asymptomatic tumors, as well as metastasized tumors in infants, to rapid progression and resistance to therapy. Genomic amplification of the MYCN oncogene has been used to predict outcome in neuroblastoma for over 30 years, however, recent methodological advances including miRNA and mRNA profiling, comparative genomic hybridization (array-CGH), and whole-genome sequencing have enabled the detailed analysis of the neuroblastoma genome, leading to the identification of new prognostic markers and better patient stratification. In this review, we will describe the main genetic factors responsible for these diverse clinical phenotypes in neuroblastoma, the chronology of their discovery, and the impact on patient prognosis.

Epigenetic modification of DNA by methylation of the cytosine present in CG dinucleotides constitutes a key regulatory mechanism in the control of gene expression in neurological diseases. In this chapter, we describe an in-depth methodology of methylated DNA immunoprecipitation used in combination with tiling microarrays (MeDIP-chip) in order to analyze genome-wide gene promoter methylation in the hippocampus of mice following status epilepticus (prolonged seizure). While a specific mouse model and array format are described, the method can be applied to DNA from many tissues to analyze the methylation status of promoter regions across whole genomes, using a wide range of available array formats (both custom designed and commercially catalogued). We conclude the chapter with the description of bisulfite sequencing validation of MeDIP-chip results.

LIN28B regulates developmental processes by modulating microRNAs (miRNAs) of the let-7 family. A role for LIN28B in cancer has been proposed but has not been established in vivo. Here, we report that LIN28B showed genomic aberrations and extensive overexpression in high-risk neuroblastoma compared to several other tumor entities and normal tissues. High LIN28B expression was an independent risk factor for adverse outcome in neuroblastoma. LIN28B signaled through repression of the let-7 miRNAs and consequently resulted in elevated MYCN protein expression in neuroblastoma cells. LIN28B-let-7-MYCN signaling blocked differentiation of normal neuroblasts and neuroblastoma cells. These findings were fully recapitulated in a mouse model in which LIN28B expression in the sympathetic adrenergic lineage induced development of neuroblastomas marked by low let-7 miRNA levels and high MYCN protein expression. Interference with this pathway might offer therapeutic perspectives.

Developing targeted therapies for high grade gliomas (HGG), the most common primary brain tumor in adults, relies largely on glioma cultures. However, it is unclear if HGG tumorigenic signaling pathways are retained under in-vitro conditions. Using array comparative genomic hybridization and immunohistochemical profiling, we contrasted the epidermal and platelet-derived growth factor receptor (EGFR/PDGFR) in-vitro pathway status of twenty-six primary HGG cultures with the pathway status of their original HGG biopsies. Genomic gains or amplifications were lost during culturing while genomic losses were more likely to be retained. Loss of EGFR amplification was further verified immunohistochemically when EGFR over expression was decreased in the majority of cultures. Conversely, PDGFRα and PDGFRβ were more abundantly expressed in primary cultures than in the original tumor (p<0.05). Despite these genomic and proteomic differences, primary HGG cultures retained key aspects of dysregulated tumorigenic signaling. Both in-vivo and in-vitro the presence of EGFR resulted in downstream activation of P70s6K while reduced downstream activation was associated with the presence of PDGFR and the tumor suppressor, PTEN. The preserved pathway dysregulation make this glioma model suitable for further studies of glioma tumorigenesis, however individual culture related differences must be taken into consideration when testing responsiveness to chemotherapeutic agents., (Copyright © 2012 Elsevier Inc. All rights reserved.)

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.

Brief seizures (epileptic/seizure preconditioning) are capable of activating endogenous protective pathways in the brain which can temporarily generate a damage-refractory state against subsequent and otherwise harmful episodes of prolonged seizures (tolerance). Altered expression of microRNAs, a class of non-coding RNAs that function post-transcriptionally to regulate mRNA translation has recently been implicated in the molecular mechanism of epileptic tolerance. Here we characterized the effect of seizure preconditioning induced by low-dose systemic kainic acid on microRNA expression in the hippocampus of mice. Seizure preconditioning resulted in up-regulation of 25 mature microRNAs in the CA3 subfield of the mouse hippocampus, with the highest levels detected for miR-184. This finding was supported by real time PCR and in situ hybridization showing increased neuronal miR-184 levels and a reduction in protein levels of a miR-184 target. Inhibiting miR-184 expression in vivo resulted in the emergence of neuronal death after preconditioning seizures and increased seizure-induced neuronal death following status epilepticus in previously preconditioned animals, without altered electrographic seizure durations. The present study suggests miRNA up-regulation after preconditioning may contribute to development of epileptic tolerance and identifies miR-184 as a novel contributor to neuronal survival following both mild and severe seizures., (Copyright © 2012 Elsevier Inc. All rights reserved.)

Background: Neuroblastoma remains a major cause of cancer-linked mortality in children. miR-204 has been used in microRNA expression signatures predictive of neuroblastoma patient survival. The aim of this study was to explore the independent association of miR-204 with survival in a neuroblastoma cohort, and to investigate the phenotypic effects mediated by miR-204 expression in neuroblastoma., Methods: Neuroblastoma cell lines were transiently transfected with miR-204 mimics and assessed for cell viability using MTS assays. Apoptosis levels in cell lines were evaluated by FACS analysis of Annexin V-/propidium iodide-stained cells transfected with miR-204 mimics and treated with chemotherapy drug or vehicle control. Potential targets of miR-204 were validated using luciferase reporter assays., Results: miR-204 expression in primary neuroblastoma tumours was predictive of patient event-free and overall survival, independent of established known risk factors. Ectopic miR-204 expression significantly increased sensitivity to cisplatin and etoposide in vitro. miR-204 direct targeting of the 3' UTR of BCL2 and NTRK2 (TrkB) was confirmed., Conclusion: miR-204 is a novel predictor of outcome in neuroblastoma, functioning, at least in part, through increasing sensitivity to cisplatin by direct targeting and downregulation of anti-apoptotic BCL2. miR-204 also targets full-length NTRK2, a potent oncogene involved with chemotherapy drug resistance in neuroblastoma., (© 2012 Cancer Research UK)

Temporal lobe epilepsy is a common, chronic neurological disorder characterized by recurrent spontaneous seizures. MicroRNAs (miRNAs) are small, noncoding RNAs that regulate post-transcriptional expression of protein-coding mRNAs, which may have key roles in the pathogenesis of neurological disorders. In experimental models of prolonged, injurious seizures (status epilepticus) and in human epilepsy, we found upregulation of miR-134, a brain-specific, activity-regulated miRNA that has been 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 in mice reduced the later occurrence of spontaneous seizures by over 90% and mitigated the attendant pathological features of temporal lobe epilepsy. Thus, silencing miR-134 exerts prolonged seizure-suppressant and neuroprotective actions; determining whether these are anticonvulsant effects or are truly antiepileptogenic effects requires additional experimentation.

Transforming growth factor-β (TGF-β) signaling regulates many diverse cellular activities through both canonical (SMAD-dependent) and non-canonical branches, which includes the mitogen-activated protein kinase (MAPK), Rho-like guanosine triphosphatase and phosphatidylinositol-3-kinase/AKT pathways. Here, we demonstrate that miR-335 directly targets and downregulates genes in the TGF-β non-canonical pathways, including the Rho-associated coiled-coil containing protein (ROCK1) and MAPK1, resulting in reduced phosphorylation of downstream pathway members. Specifically, inhibition of ROCK1 and MAPK1 reduces phosphorylation levels of the motor protein myosin light chain (MLC) leading to a significant inhibition of the invasive and migratory potential of neuroblastoma cells. Additionally, miR-335 targets the leucine-rich alpha-2-glycoprotein 1 (LRG1) messenger RNA, which similarly results in a significant reduction in the phosphorylation status of MLC and a decrease in neuroblastoma cell migration and invasion. Thus, we link LRG1 to the migratory machinery of the cell, altering its activity presumably by exerting its effect within the non-canonical TGF-β pathway. Moreover, we demonstrate that the MYCN transcription factor, whose coding sequence is highly amplified in a particularly clinically aggressive neuroblastoma tumor subtype, directly binds to a region immediately upstream of the miR-335 transcriptional start site, resulting in transcriptional repression. We conclude that MYCN contributes to neuroblastoma cell migration and invasion, by directly downregulating miR-335, resulting in the upregulation of the TGF-β signaling pathway members ROCK1, MAPK1 and putative member LRG1, which positively promote this process. Our results provide novel insight into the direct regulation of TGF-β non-canonical signaling by miR-335, which in turn is downregulated by MYCN.

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.

Prolonged seizures (status epilepticus) produce pathophysiological changes in the hippocampus that are associated with large-scale, wide-ranging changes in gene expression. Epileptic tolerance is an endogenous program of cell protection that can be activated in the brain by previous exposure to a non-harmful seizure episode before status epilepticus. A major transcriptional feature of tolerance is gene downregulation. Here, through methylation analysis of 34,143 discrete loci representing all annotated CpG islands and promoter regions in the mouse genome, we report the genome-wide DNA methylation changes in the hippocampus after status epilepticus and epileptic tolerance in adult mice. A total of 321 genes showed altered DNA methylation after status epilepticus alone or status epilepticus that followed seizure preconditioning, with >90% of the promoters of these genes undergoing hypomethylation. These profiles included genes not previously associated with epilepsy, such as the polycomb gene Phc2. Differential methylation events generally occurred throughout the genome without bias for a particular chromosomal region, with the exception of a small region of chromosome 4, which was significantly overrepresented with genes hypomethylated after status epilepticus. Surprisingly, only few genes displayed differential hypermethylation in epileptic tolerance. Nevertheless, gene ontology analysis emphasized the majority of differential methylation events between the groups occurred in genes associated with nuclear functions, such as DNA binding and transcriptional regulation. The present study reports select, genome-wide DNA methylation changes after status epilepticus and in epileptic tolerance, which may contribute to regulating the gene expression environment of the seizure-damaged hippocampus.

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 (GD(2)), providing a target for tumor-specific delivery., Principal Findings: Nanoparticles encapsulating miR-34a and conjugated to a GD(2) 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-GD(2)-nanoparticle-mediated targeted delivery of miR-34a for both the treatment of GD(2)-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.

Purpose: More accurate assessment of prognosis is important to further improve the choice of risk-related therapy in neuroblastoma (NB) patients. In this study, we aimed to establish and validate a prognostic miRNA signature for children with NB and tested it in both fresh frozen and archived formalin-fixed paraffin-embedded (FFPE) samples., Experimental Design: Four hundred-thirty human mature miRNAs were profiled in two patient subgroups with maximally divergent clinical courses. Univariate logistic regression analysis was used to select miRNAs correlating with NB patient survival. A 25-miRNA gene signature was built using 51 training samples, tested on 179 test samples, and validated on an independent set of 304 fresh frozen tumor samples and 75 archived FFPE samples., Results: The 25-miRNA signature significantly discriminates the test patients with respect to progression-free and overall survival (P < 0.0001), both in the overall population and in the cohort of high-risk patients. Multivariate analysis indicates that the miRNA signature is an independent predictor of patient survival after controlling for current risk factors. The results were confirmed in an external validation set. In contrast to a previously published mRNA classifier, the 25-miRNA signature was found to be predictive for patient survival in a set of 75 FFPE neuroblastoma samples., Conclusions: In this study, we present the largest NB miRNA expression study so far, including more than 500 NB patients. We established and validated a robust miRNA classifier, able to identify a cohort of high-risk NB patients at greater risk for adverse outcome using both fresh frozen and archived material., (©2011 AACR.)

When an otherwise harmful insult to the brain is preceded by a brief, noninjurious stimulus, the brain becomes tolerant, and the resulting damage is reduced. Epileptic tolerance develops when brief seizures precede an episode of prolonged seizures (status epilepticus). MicroRNAs (miRNAs) are small, noncoding RNAs that function as post-transcriptional regulators of gene expression. We investigated how prior seizure preconditioning affects the miRNA response to status epilepticus evoked by intra-amygdalar kainic acid in mice. The miRNA was extracted from the ipsilateral CA3 subfield 24 hours after focal-onset status epilepticus in animals that had previously received either seizure preconditioning (tolerance) or no preconditioning (injury), and mature miRNA levels were measured using TaqMan low-density arrays. Expression of 21 miRNAs was increased, relative to control, after status epilepticus alone, and expression of 12 miRNAs was decreased. Increased miR-132 levels were matched with increased binding to Argonaute-2, a constituent of the RNA-induced silencing complex. In tolerant animals, expression responses of >40% of the injury-group-detected miRNAs differed, being either unchanged relative to control or down-regulated, and this included miR-132. In vivo microinjection of locked nucleic acid-modified oligonucleotides (antagomirs) against miR-132 depleted hippocampal miR-132 levels and reduced seizure-induced neuronal death. Thus, our data strongly suggest that miRNAs are important regulators of seizure-induced neuronal death., (Copyright © 2011 American Society for Investigative Pathology. Published by Elsevier Inc. All rights reserved.)