miR-126 Is Downregulated in Cystic Fibrosis Airway Epithelial Cells and Regulates TOM1 Expression.

Cystic fibrosis (CF) is one of the most common lethal genetic diseases in which the role of microRNAs has yet to be explored. Predicted to be regulated by miR-126, TOM1 (target of Myb1) has been shown to interact with Toll-interacting protein, forming a complex to regulate endosomal trafficking of ubiquitinated proteins. TOM1 has also been proposed as a negative regulator of IL-1beta and TNF-alpha-induced signaling pathways. MiR-126 is highly expressed in the lung, and we now show for the first time differential expression of miR-126 in CF versus non-CF airway epithelial cells both in vitro and in vivo. MiR-126 downregulation in CF bronchial epithelial cells correlated with a significant upregulation of TOM1 mRNA, both in vitro and in vivo when compared with their non-CF counterparts. Introduction of synthetic pre-miR-126 inhibited luciferase activity in a reporter system containing the full length 3\u27-untranslated region of TOM1 and resulted in decreased TOM1 protein production in CF bronchial epithelial cells. Following stimulation with LPS or IL-1beta, overexpression of TOM1 was found to downregulate NF-kappaB luciferase activity. Conversely, TOM1 knockdown resulted in a significant increase in NF-kappaB regulated IL-8 secretion. These data show that miR-126 is differentially regulated in CF versus non-CF airway epithelial cells and that TOM1 is a miR-126 target that may have an important role in regulating innate immune responses in the CF lung. To our knowledge, this study is the first to report of a role for TOM1 in the TLR2/4 signaling pathways and the first to describe microRNA involvement in CF

MicroRNA-184 inhibits neuroblastoma cell survival through targeting the serine/threonine kinase AKT2

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\u27UTR 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

Global MYCN Transcription Factor Binding Analysis in Neuroblastoma Reveals Association with Distinct E-Box Motifs and Regions of DNA Hypermethylation

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\u3c0.0016), 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

Inhibition of Neuroblastoma Tumor Growth by Targeted Delivery of MicroRNA-34a Using Anti-Disialoganglioside GD2 Coated Nanoparticles

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.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.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

Reduced Mature MicroRNA Levels in Association with Dicer Loss in Human Temporal Lobe Epilepsy with Hippocampal Sclerosis

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

Metastasis suppressor microRNA-335 targets the formin family of actin nucleators.

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

Discovery and visualization of miRNA-mRNA functional modules within integrated data using bicluster analysis

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 diseas