Ontogeny of small RNA in the regulation of mammalian brain development.

BACKGROUND: MicroRNAs (miRNAs) play a pivotal role in coordinating messenger RNA (mRNA) transcription and stability in almost all known biological processes, including the development of the central nervous system. Despite our broad understanding of their involvement, we still have a very sparse understanding of specifically how miRNA contribute to the strict regional and temporal regulation of brain development. Accordingly, in the current study we have examined the contribution of miRNA in the developing rat telencephalon and mesencephalon from just after neural tube closure till birth using a genome-wide microarray strategy. RESULTS: We identified temporally distinct expression patterns in both the telencephalon and mesencephalon for both miRNAs and their target genes. We demonstrate direct miRNA targeting of several genes involved with the migration, differentiation and maturation of neurons. CONCLUSIONS: Our findings suggest that miRNA have significant implications for the development of neural structure and support important mechanisms that if disrupted, may contribute to or drive neurodevelopmental disorders.

Design and interpretation of microRNA-reporter gene activity.

MicroRNAs (miRNAs) are small noncoding RNA molecules that act as sequence specificity guides to direct post-transcriptional gene silencing. In doing so, miRNAs regulate many critical developmental processes, including cellular proliferation, differentiation, migration, and apoptosis, as well as more specialized biological functions such as dendritic spine development and synaptogenesis. Interactions between miRNAs and their miRNA recognition elements occur via partial complementarity, rendering tremendous redundancy in targeting such that miRNAs are predicted to regulate 60% of the genome, with each miRNA estimated to regulate more than 200 genes. Because these predictions are prone to false positives and false negatives, there is an ever present need to provide material support to these assertions to firmly establish the biological function of specific miRNAs in both normal and pathophysiological contexts. Using schizophrenia-associated miR-181b as an example, we present detailed guidelines and novel insights for the rapid establishment of a streamlined miRNA-reporter gene assay and explore various design concepts for miRNA-reporter gene applications, including bidirectional miRNA modulation. In exemplifying this approach, we report seven novel miR-181b target sites for five schizophrenia candidate genes (DISC1, BDNF, ENKUR, GRIA1, and GRIK1) and dissect a number of vital concepts regarding future developments for miRNA-reporter gene assays and the interpretation of their results.

Alternative mRNA fates identified in microRNA-associated transcriptome analysis.

BACKGROUND: MicroRNA (miRNA) are small non-coding RNA molecules which function as nucleic acid-based specificity factors in the universal RNA binding complex known as the RNA induced silencing complex (RISC). In the canonical gene-silencing pathway, these activated RISC particles are associated with RNA decay and gene suppression, however, there is evidence to suggest that in some circumstances they may also stabilise their target RNA and even enhance translation. To further explore the role of miRNA in this context, we performed a genome-wide expression analysis to investigate the molecular consequences of bidirectional modulation of the disease-associated miRNAs miR-181b and miR-107 in multiple human cell lines. RESULTS: This data was subjected to pathways analysis and correlated against miRNA targets predicted through seed region homology. This revealed a large number of both conserved and non-conserved miRNA target genes, a selection of which were functionally validated through reporter gene assays. Contrary to expectation we also identified a significant proportion of predicted target genes with both conserved and non-conserved recognition elements that were positively correlated with the modulated miRNA. Finally, a large proportion of miR-181b associated genes devoid of the corresponding miRNA recognition element, were enriched with binding motifs for the E2F1 transcription factor, which is encoded by a miR-181b target gene. CONCLUSIONS: These findings suggest that miRNA regulate target genes directly through interactions with both conserved and non-conserved target recognition elements, and can lead to both a decrease and increase in transcript abundance. They also multiply their influence through interaction with transcription factor genes exemplified by the observed miR-181b/E2F1 relationship.

Dysregulation of miRNA 181b in the temporal cortex in schizophrenia.

Analysis of global microRNA (miRNA) expression in postmortem cortical grey matter from the superior temporal gyrus, revealed significant up-regulation of miR-181b expression in schizophrenia. This finding was supported by quantitative real-time RT-PCR analysis of miRNA expression in a cohort of 21 matched pairs of schizophrenia and non-psychiatric controls. The implications of this finding are substantial, as this miRNA is predicted to regulate many target genes with potential significance to the development of schizophrenia. They include the calcium sensor gene visinin-like 1 (VSNL1) and the ionotropic AMPA glutamate receptor subunit (GRIA2), which were found to be down-regulated in the same cortical tissue from the schizophrenia group. Both of these genes were also suppressed in miR-181b transfected cells and shown to contain functional miR-181b miRNA recognition elements by reporter gene assay. This study suggests altered miRNA levels could be a significant factor in the dysregulation of cortical gene expression in schizophrenia.

Paraneoplastic bullous pemphigoid - A sign of clear cell renal carcinoma.

Paraneoplastic bullous pemphigoid is a rare paraneoplastic syndrome. Rash is pruritic, with erythematous eruption of large subepidermal bullae over skin and often mucosal surfaces. We present an 84y woman with a three week history of erythematous rash, and 48hrs of bullae. A left clear cell renal cell carcinoma was identified on CT imaging during the presentation. Subsequent removal of the tumour resulted in resolution of bullous pemphigoid symptoms. This first-of-kind case and successful result strengthens the association between renal cell carcinoma and paraneoplastic bullous pemphigoid, arguing for a high degree of clinical suspicion in unexplained presentations of bullous pemphigoid.

Schizophrenia-associated MicroRNA-Gene Interactions in the Dorsolateral Prefrontal Cortex.

Schizophrenia-associated anomalies in gene expression in postmortem brain can be attributed to a combination of genetic and environmental influences. Given the small effect size of common variants, it is likely that we may only see the combined impact of some of these at the pathway level in small postmortem studies. At the gene level, however, there may be more impact from common environmental exposures mediated by influential epigenomic modifiers, such as microRNA (miRNA). We hypothesise that dysregulation of miRNAs and their alteration of gene expression have significant implications in the pathophysiology of schizophrenia. In this study, we integrate changes in cortical gene and miRNA expression to identify regulatory interactions and networks associated with the disorder. Gene expression analysis in post-mortem prefrontal dorsolateral cortex (BA 46) (n = 74 matched pairs of schizophrenia, schizoaffective, and control samples) was integrated with miRNA expression in the same cohort to identify gene-miRNA regulatory networks. A significant gene-miRNA interaction network was identified, including miR-92a, miR-495, and miR-134, which converged with differentially expressed genes in pathways involved in neurodevelopment and oligodendrocyte function. The capacity for miRNA to directly regulate gene expression through respective binding sites in BCL11A, PLP1, and SYT11 was also confirmed to support the biological relevance of this integrated network model. The observations in this study support the hypothesis that miRNA dysregulation is an important factor in the complex pathophysiology of schizophrenia.

miR-518f-5p decreases tetraspanin CD9 protein levels and differentially affects non-tumourigenic prostate and prostate cancer cell migration and adhesion.

Tetraspanin CD9 is generally considered to be a metastasis suppressor, with decreased levels associated with progression and metastasis in many advanced stage cancers. Little is known about the cause of CD9 dysregulation in prostate cancer, however there are several miRNA-binding sites in the 3´UTR of the transcript suggesting it could be post-transcriptionally regulated. Using microarrays and luciferase assays in tumourigenic and non-tumourigenic prostate cell lines we identified miR-518f-5p as a regulator of the CD9 3'UTR gene expression, and decreased expression of endogenous CD9 in non-tumorigenic prostate RWPE1 and prostate cancer DU145 cells. This resulted in differential functional effects, in which RWPE1 cells showed increased migration and decreased adhesion to extracellular matrix substrates, whereas DU145 cells showed decreased migration and increased adhesion. Moreover, overexpression of miR-518f-5p significantly increased proliferation between 48h and 72h in normal RWPE1 cells, with no effect on tumourigenic DU145 cell proliferation. These results show that tetraspanin CD9 is regulated by miRNAs in prostate cell lines and that due to differential functional effects in non-tumourigenic versus tumourigenic prostate cells, miR-518f-5p may be an effective biomarker and/or therapeutic target for prostate cancer progression.

Understanding principles of miRNA target recognition and function through integrated biological and bioinformatics approaches.

In recent times, microRNA (miRNA) have emerged as primary regulators of fundamental biological processes including cellular differentiation, proliferation, apoptosis, as well as synaptic plasticity. However, miRNAs bind their targets with only partial complementarity, making it very challenging to determine exactly how a miRNA is functioning in specific biological environments. This review discusses key principles of miRNA target recognition and function which have emerged through the progressive advancement of biological and bioinformatics approaches. Ultimately, the integration of gene expression and biochemical methods with sequence- and systems-based bioinformatics approaches will reveal profound insights regarding the importance of target contextual features in determining miRNA target recognition and regulatory outcome, as well as the importance of RNA interaction networks in enabling miRNA to regulate different target genes and functions in specific biological contexts. There is therefore a demand for the elegant design of future experiments such that principles of context-specific miRNA target recognition and regulatory outcome can be accurately modeled in normal developmental and disease states.

Context-specific microRNA function in developmental complexity.

Since their discovery, microRNAs (miRNA) have been implicated in a vast array of biological processes in animals, from fundamental developmental functions including cellular proliferation and differentiation, to more complex and specialized roles such as long-term potentiation and synapse-specific modifications in neurons. This review recounts the history behind this paradigm shift, which has seen small non-coding RNA molecules coming to the forefront of molecular biology, and introduces their role in establishing developmental complexity in animals. The fundamental mechanisms of miRNA biogenesis and function are then considered, leading into a discussion of recent discoveries transforming our understanding of how these molecules regulate gene network behaviour throughout developmental and pathophysiological processes. The emerging complexity of this mechanism is also examined with respect to the influence of cellular context on miRNA function. This discussion highlights the absolute imperative for experimental designs to appreciate the significance of context-specific factors when determining what genes are regulated by a particular miRNA. Moreover, by establishing the timing, location, and mechanism of these regulatory events, we may ultimately understand the true biological function of a specific miRNA in a given cellular environment.

MicroRNA-16 is down-regulated in mutated FLT3 expressing murine myeloid FDC-P1 cells and interacts with Pim-1.

Activating mutations in the receptor tyrosine kinase FLT3 are one of the most frequent somatic mutations in acute myeloid leukemia (AML). Internal tandem duplications of the juxtamembrane region of FLT3 (FLT3/ITD) constitutively activate survival and proliferation pathways, and are associated with a poor prognosis in AML. We suspected that alteration of small non-coding microRNA (miRNA) expression in these leukemia cells is involved in the transformation process and used miRNA microarrays to determine the miRNA signature from total RNA harvested from FLT3/ITD expressing FDC-P1 cells (FD-FLT3/ITD). This revealed that a limited set of miRNAs appeared to be affected by expression of FLT3/ITD compared to the control group consisting of FDC-P1 parental cells transfected with an empty vector (FD-EV). Among differentially expressed miRNAs, we selected miR-16, miR-21 and miR-223 to validate the microarray data by quantitative real-time RT-PCR showing a high degree of correlation. We further analyzed miR-16 expression with FLT3 inhibitors in FLT3/ITD expressing cells. MiR-16 was found to be one of most significantly down-regulated miRNAs in FLT3/ITD expressing cells and was up-regulated upon FLT3 inhibition. The data suggests that miR-16 is acting as a tumour suppressor gene in FLT3/ITD-mediated leukemic transformation. Whilst miR-16 has been reported to target multiple mRNAs, computer models from public bioinformatic resources predicted a potential regulatory mechanism between miR-16 and Pim-1 mRNA. In support of this interaction, miR-16 was shown to suppress Pim-1 reporter gene expression. Further, our data demonstrated that over-expression of miR-16 mimics suppressed Pim-1 expression in FD-FLT3/ITD cells suggesting that increased miR-16 expression contributes to depletion of Pim-1 after FLT3 inhibition and that miR-16 repression may be associated with up-regulated Pim-1 in FLT3/ITD expressing cells.

MicroRNAs miR-17 and miR-20a inhibit T cell activation genes and are under-expressed in MS whole blood.

It is well established that Multiple Sclerosis (MS) is an immune mediated disease. Little is known about what drives the differential control of the immune system in MS patients compared to unaffected individuals. MicroRNAs (miRNAs) are small non-coding nucleic acids that are involved in the control of gene expression. Their potential role in T cell activation and neurodegenerative disease has recently been recognised and they are therefore excellent candidates for further studies in MS. We investigated the transcriptome of currently known miRNAs using miRNA microarray analysis in peripheral blood samples of 59 treatment naïve MS patients and 37 controls. Of these 59, 18 had a primary progressive, 17 a secondary progressive and 24 a relapsing remitting disease course. In all MS subtypes miR-17 and miR-20a were significantly under-expressed in MS, confirmed by RT-PCR. We demonstrate that these miRNAs modulate T cell activation genes in a knock-in and knock-down T cell model. The same T cell activation genes are also up-regulated in MS whole blood mRNA, suggesting these miRNAs or their analogues may provide useful targets for new therapeutic approaches.

Down-regulation of miR-17 family expression in response to retinoic acid induced neuronal differentiation.

Whole-genome microRNA and gene expression analyses were used to monitor changes during retinoic acid induced differentiation of neuroblasts in vitro. Interestingly, the entire miR-17 family was over-represented among the down-regulated miRNA. The implications of these changes are considerable, as target gene prediction suggests that the miR-17 family is involved in the regulation of the mitogen-activated protein kinase (MAPK) signaling pathway, synaptic plasticity and other markers of neuronal differentiation. Significantly, many of the target responses predicted by changes in miRNA expression were supported by the observed changes in gene expression. As expected, markers of neuronal differentiation such as anti-apoptotic protein B-cell lymphoma 2 (BCL2), myocyte enhancer factor-2D (MEF2D) and zipper protein kinase (MAP3K12; aka ZPK/MUK/DLK) were each up-regulated in response to differentiation. The expression of these genes was also reduced in response to miR-17 and miR-20a transfection, and more specifically they were also shown to contain functional miRNA recognition elements for members of the miR-17 family by reporter gene assay. This suggests that the miR-17 family have an integral role in fine-tuning the pathways involved in the regulation of neuronal differentiation.