Targeting oncogenic microRNAs from the miR-371~373 and miR-302/367 clusters in malignant germ cell tumours causes growth inhibition through cell cycle disruption.

BACKGROUND: MiR-371~373 and miR-302/367 cluster over-expression occurs in all malignant germ cell tumours (GCTs), regardless of age (paediatric/adult), site (gonadal/extragonadal), or subtype [seminoma, yolk sac tumour (YST), embryonal carcinoma (EC)]. Six of eight microRNAs from these clusters contain the seed sequence 'AAGUGC', determining mRNA targeting. Here we sought to identify the significance of these observations by targeting these microRNAs functionally. METHODS: We targeted miR-371~373 and/or miR-302/367 clusters in malignant GCT cell lines, using CRISPR-Cas9, gapmer primary miR-302/367 transcript inhibition, and peptide nucleic acid (PNA) or locked nucleic acid (LNA)-DNA inhibition targeting miR-302a-d-3p, and undertook relevant functional assays. RESULTS: MiR-302/367 cluster microRNAs made the largest contribution to AAGUGC seed abundance in malignant GCT cells, regardless of subtype (seminoma/YST/EC). Following the unsuccessful use of CRISPR-Cas9, gapmer, and PNA systems, LNA-DNA-based targeting resulted in growth inhibition in seminoma and YST cells. This was associated with the de-repression of multiple mRNAs targeted by AAGUGC seed-containing microRNAs, with pathway analysis confirming predominant disruption of Rho-GTPase signalling, vesicle organisation/transport, and cell cycle regulation, findings corroborated in clinical samples. Further LNA-DNA inhibitor studies confirmed direct cell cycle effects, with an increase of cells in G0/G1-phase and a decrease in S-phase. CONCLUSION: Targeting of specific miR-371~373 and miR-302/367 microRNAs in malignant GCTs demonstrated their functional significance, with growth inhibition mediated through cell cycle disruption.

Small non-coding RNA sequencing reveals global dysregulation of piwi-interacting RNA (piRNA) expression in gonadal malignant germ cell tumours.

BACKGROUND: Analyses of small non-coding RNA (ncRNA) expression in malignant germ cell tumours (GCTs) have focused on microRNAs (miRNAs). As GCTs all arise from primordial germ cells, and piwi-interacting RNAs (piRNAs) have important roles in maintaining germline integrity via transposon silencing, we hypothesised that malignant GCTs are characterised by fundamental piRNA dysregulation. AIMS: We undertook global small ncRNA sequencing in malignant GCTs, in order to describe small ncRNA expression changes for both miRNAs and piRNAs. MATERIALS AND METHODS: We performed small ncRNA next generation sequencing on a representative panel of 47 samples, comprising malignant GCT (n = 31) and control (n = 16) tissues/cell lines. Following quality control and normalisation, filtered count reads were used for differential miRNA and piRNA expression analyses via DESeq2. Predicted mRNA targets for piRNAs were identified and utilised for pathway enrichment analyses. RESULTS: Overall, miRNAs and piRNAs comprised 21.9% and 43.0% of small ncRNA species, respectively. There were 749 differentially expressed miRNAs in malignant GCTs, of which 536 (72%) were over-expressed and 213 (28%) under-expressed. The top-ranking over-expressed miRNAs were exclusively from the miR-371∼373 and miR-302/367 clusters. The most significantly under-expressed miRNAs were miR-100-5p, miR-214-3p, miR-125b-5p and let-7 family members, including miR-202-3p. There were 1,121 differentially expressed piRNAs in malignant GCTs, of which 167 (15%) were over-expressed and 954 (85%) under-expressed. Of note, of the top-20 differentially expressed piRNAs, 16 were over-expressed, of which piR-hsa-2506793 was both top-ranking and most abundant. Mobile element (ME; i.e., transposon)-associated piRNAs comprised 166 (15%) of the 1,121 differentially expressed piRNAs, of which 165 (>99%) were down-regulated. The remaining 955 (85%) non-ME-associated piRNAs may have wider cellular roles. To explore this, predicted mRNA targets of differentially expressed piRNAs identified putative involvement in cancer-associated pathways. CONCLUSION: This study confirms previous miRNA observations, giving credence to our novel demonstration of global piRNA dysregulation in gonadal malignant GCTs, through both ME and non-ME-associated pathways, which likely contributes to GCT pathogenesis.

Short- and long-range cis interactions between integrated HPV genomes and cellular chromatin dysregulate host gene expression in early cervical carcinogenesis.

Development of cervical cancer is directly associated with integration of human papillomavirus (HPV) genomes into host chromosomes and subsequent modulation of HPV oncogene expression, which correlates with multi-layered epigenetic changes at the integrated HPV genomes. However, the process of integration itself and dysregulation of host gene expression at sites of integration in our model of HPV16 integrant clone natural selection has remained enigmatic. We now show, using a state-of-the-art 'HPV integrated site capture' (HISC) technique, that integration likely occurs through microhomology-mediated repair (MHMR) mechanisms via either a direct process, resulting in host sequence deletion (in our case, partially homozygously) or via a 'looping' mechanism by which flanking host regions become amplified. Furthermore, using our 'HPV16-specific Region Capture Hi-C' technique, we have determined that chromatin interactions between the integrated virus genome and host chromosomes, both at short- (<500 kbp) and long-range (>500 kbp), appear to drive local host gene dysregulation through the disruption of host:host interactions within (but not exceeding) host structures known as topologically associating domains (TADs). This mechanism of HPV-induced host gene expression modulation indicates that integration of virus genomes near to or within a 'cancer-causing gene' is not essential to influence their expression and that these modifications to genome interactions could have a major role in selection of HPV integrants at the early stage of cervical neoplastic progression.

Identification of Recurrent Mutations in the microRNA-Binding Sites of B-Cell Lymphoma-Associated Genes in Follicular Lymphoma.

Follicular lymphoma (FL) is a common indolent B-cell lymphoma that can transform into the more aggressive transformed FL (tFL). However, the molecular process driving this transformation is uncertain. In this work, we aimed to identify microRNA (miRNA)-binding sites recurrently mutated in follicular lymphoma patients, as well as in transformed FL patients. Using whole-genome sequencing data from FL tumors, we discovered 544 mutations located in bioinformatically predicted microRNA-binding sites. We then studied these specific regions using targeted sequencing in a cohort of 55 FL patients, found 16 recurrent mutations, and identified a further 69 variants. After filtering for QC, we identified 21 genes with mutated miRNA-binding sites that were also enriched for B-cell-associated genes by Gene Ontology. Over 40% of mutations identified in these genes were present exclusively in tFL patients. We validated the predicted miRNA-binding sites of five of the genes by luciferase assay and demonstrated that the identified mutations in BCL2 and EZH2 genes impaired the binding efficiency of miR-5008 and miR-144 and regulated the endogenous levels of messenger RNA (mRNA).

Genomic positional conservation identifies topological anchor point RNAs linked to developmental loci.

BACKGROUND: The mammalian genome is transcribed into large numbers of long noncoding RNAs (lncRNAs), but the definition of functional lncRNA groups has proven difficult, partly due to their low sequence conservation and lack of identified shared properties. Here we consider promoter conservation and positional conservation as indicators of functional commonality. RESULTS: We identify 665 conserved lncRNA promoters in mouse and human that are preserved in genomic position relative to orthologous coding genes. These positionally conserved lncRNA genes are primarily associated with developmental transcription factor loci with which they are coexpressed in a tissue-specific manner. Over half of positionally conserved RNAs in this set are linked to chromatin organization structures, overlapping binding sites for the CTCF chromatin organiser and located at chromatin loop anchor points and borders of topologically associating domains (TADs). We define these RNAs as topological anchor point RNAs (tapRNAs). Characterization of these noncoding RNAs and their associated coding genes shows that they are functionally connected: they regulate each other's expression and influence the metastatic phenotype of cancer cells in vitro in a similar fashion. Furthermore, we find that tapRNAs contain conserved sequence domains that are enriched in motifs for zinc finger domain-containing RNA-binding proteins and transcription factors, whose binding sites are found mutated in cancers. CONCLUSIONS: This work leverages positional conservation to identify lncRNAs with potential importance in genome organization, development and disease. The evidence that many developmental transcription factors are physically and functionally connected to lncRNAs represents an exciting stepping-stone to further our understanding of genome regulation.

Activation of the TGFß pathway impairs endothelial to haematopoietic transition.

The endothelial to haematopoietic transition (EHT) is a key developmental process where a drastic change of endothelial cell morphology leads to the formation of blood stem and progenitor cells during embryogenesis. As TGFß signalling triggers a similar event during embryonic development called epithelial to mesenchymal transition (EMT), we hypothesised that TGFß activity could play a similar role in EHT as well. We used the mouse embryonic stem cell differentiation system for in vitro recapitulation of EHT and performed gain and loss of function analyses of the TGFß pathway. Quantitative proteomics analysis showed that TGFß treatment during EHT increased the secretion of several proteins linked to the vascular lineage. Live cell imaging showed that TGFß blocked the formation of round blood cells. Using gene expression profiling we demonstrated that the TGFß signalling activation decreased haematopoietic genes expression and increased the transcription of endothelial and extracellular matrix genes as well as EMT markers. Finally we found that the expression of the transcription factor Sox17 was up-regulated upon TGFß signalling activation and showed that its overexpression was enough to block blood cell formation. In conclusion we showed that triggering the TGFß pathway does not enhance EHT as we hypothesised but instead impairs it.

Solid tumors of childhood display specific serum microRNA profiles.

BACKGROUND: Serum biomarkers for diagnosis and risk stratification of childhood solid tumors would improve the accuracy/timeliness of diagnosis and reduce the need for invasive biopsies. We hypothesized that differential expression and/or release of microRNAs (miRNAs) by such tumors may be detected as altered serum miRNA profiles. METHODS: We undertook global quantitative reverse transcription PCR (qRT-PCR) miRNA profiling (n = 741) on RNA from 53 serum samples, representing 33 diagnostic cases of common childhood cancers plus 20 controls. Technical confirmation was performed in a subset of 21 cases, plus four independent samples. RESULTS: We incorporated robust quality control steps for RNA extraction, qRT-PCR efficiency and hemolysis quantification. We evaluated multiple methods to normalize global profiling data and identified the 'global mean' approach as optimal. We generated a panel of six miRNAs that were most stable in pediatric serum samples and therefore most suitable for normalization of targeted miRNA qRT-PCR data. Tumor-specific serum miRNA profiles were identified for each tumor type and selected miRNAs underwent confirmatory testing. We identified a panel of miRNAs (miR-124-3p/miR-9-3p/miR-218-5p/miR-490-5p/miR-1538) of potential importance in the clinical management of neuroblastoma, as they were consistently highly overexpressed in MYCN-amplified high-risk cases (MYCN-NB). We also derived candidate miRNA panels for noninvasive differential diagnosis of a liver mass (hepatoblastoma vs. combined MYCN-NB/NB), an abdominal mass (Wilms tumor vs. combined MYCN-NB/NB), and sarcoma subtypes. CONCLUSIONS: This study describes a pipeline for robust diagnostic serum miRNA profiling in childhood solid tumors, and has identified candidate miRNA profiles for prospective testing. IMPACT: We propose a new noninvasive method with the potential to diagnose childhood solid tumors.

Integrated analysis of microRNA and mRNA expression and association with HIF binding reveals the complexity of microRNA expression regulation under hypoxia.

BACKGROUND: In mammalians, HIF is a master regulator of hypoxia gene expression through direct binding to DNA, while its role in microRNA expression regulation, critical in the hypoxia response, is not elucidated genome wide. Our aim is to investigate in depth the regulation of microRNA expression by hypoxia in the breast cancer cell line MCF-7, establish the relationship between microRNA expression and HIF binding sites, pri-miRNA transcription and microRNA processing gene expression. METHODS: MCF-7 cells were incubated at 1% Oxygen for 16, 32 and 48 h. SiRNA against HIF-1α and HIF-2α were performed as previously published. MicroRNA and mRNA expression were assessed using microRNA microarrays, small RNA sequencing, gene expression microarrays and Real time PCR. The Kraken pipeline was applied for microRNA-seq analysis along with Bioconductor packages. Microarray data was analysed using Limma (Bioconductor), ChIP-seq data were analysed using Gene Set Enrichment Analysis and multiple testing correction applied in all analyses. RESULTS: Hypoxia time course microRNA sequencing data analysis identified 41 microRNAs significantly up- and 28 down-regulated, including hsa-miR-4521, hsa-miR-145-3p and hsa-miR-222-5p reported in conjunction with hypoxia for the first time. Integration of HIF-1α and HIF-2α ChIP-seq data with expression data showed overall association between binding sites and microRNA up-regulation, with hsa-miR-210-3p and microRNAs of miR-27a/23a/24-2 and miR-30b/30d clusters as predominant examples. Moreover the expression of hsa-miR-27a-3p and hsa-miR-24-3p was found positively associated to a hypoxia gene signature in breast cancer. Gene expression analysis showed no full coordination between pri-miRNA and microRNA expression, pointing towards additional levels of regulation. Several transcripts involved in microRNA processing were found regulated by hypoxia, of which DICER (down-regulated) and AGO4 (up-regulated) were HIF dependent. DICER expression was found inversely correlated to hypoxia in breast cancer. CONCLUSIONS: Integrated analysis of microRNA, mRNA and ChIP-seq data in a model cell line supports the hypothesis that microRNA expression under hypoxia is regulated at transcriptional and post-transcriptional level, with the presence of HIF binding sites at microRNA genomic loci associated with up-regulation. The identification of hypoxia and HIF regulated microRNAs relevant for breast cancer is important for our understanding of disease development and design of therapeutic interventions.

Depletion of HPV16 early genes induces autophagy and senescence in a cervical carcinogenesis model, regardless of viral physical state.

In cervical carcinomas, high-risk human papillomavirus (HR-HPV) may be integrated into host chromosomes or remain extra-chromosomal (episomal). We used the W12 cervical keratinocyte model to investigate the effects of HPV16 early gene depletion on in vitro cervical carcinogenesis pathways, particularly effects shared by cells with episomal versus integrated HPV16 DNA. Importantly, we were able to study the specific cellular consequences of viral gene depletion by using short interfering RNAs known not to cause phenotypic or transcriptional off-target effects in keratinocytes. We found that while cervical neoplastic progression in vitro was characterized by dynamic changes in HPV16 transcript levels, viral early gene expression was required for cell survival at all stages of carcinogenesis, regardless of viral physical state, levels of early gene expression or histology in organotypic tissue culture. Moreover, HPV16 early gene depletion induced changes in host gene expression that were common to both episome-containing and integrant-containing cells. In particular, we observed up-regulation of autophagy genes, associated with enrichment of senescence and innate immune-response pathways, including the senescence-associated secretory phenotype (SASP). In keeping with these observations, HPV16 early gene depletion induced autophagy in both episome-containing and integrant-containing W12 cells, as evidenced by the appearance of autophagosomes, punctate expression of the autophagy marker LC3, conversion of LC3B-I to LC3B-II, and reduced levels of the autophagy substrate p62. Consistent with the reported association between autophagy and senescence pathways, HPV16 early gene depletion induced expression of the senescence marker beta-galactosidase and increased secretion of the SASP-related protein IGFBP3. Together, these data indicate that depleting HR-HPV early genes would be of potential therapeutic benefit in all cervical carcinogenesis pathways, regardless of viral physical state. In addition, the senescence/SASP response associated with autophagy induction may promote beneficial immune effects in bystander cells.

LIN28 Expression in malignant germ cell tumors downregulates let-7 and increases oncogene levels.

Despite their clinicopathologic heterogeneity, malignant germ cell tumors (GCT) share molecular abnormalities that are likely to be functionally important. In this study, we investigated the potential significance of downregulation of the let-7 family of tumor suppressor microRNAs in malignant GCTs. Microarray results from pediatric and adult samples (n = 45) showed that LIN28, the negative regulator of let-7 biogenesis, was abundant in malignant GCTs, regardless of patient age, tumor site, or histologic subtype. Indeed, a strong negative correlation existed between LIN28 and let-7 levels in specimens with matched datasets. Low let-7 levels were biologically significant, as the sequence complementary to the 2 to 7 nt common let-7 seed "GAGGUA" was enriched in the 3' untranslated regions of mRNAs upregulated in pediatric and adult malignant GCTs, compared with normal gonads (a mixture of germ cells and somatic cells). We identified 27 mRNA targets of let-7 that were upregulated in malignant GCT cells, confirming significant negative correlations with let-7 levels. Among 16 mRNAs examined in a largely independent set of specimens by quantitative reverse transcription PCR, we defined negative-associations with let-7e levels for six oncogenes, including MYCN, AURKB, CCNF, RRM2, MKI67, and C12orf5 (when including normal control tissues). Importantly, LIN28 depletion in malignant GCT cells restored let-7 levels and repressed all of these oncogenic let-7 mRNA targets, with LIN28 levels correlating with cell proliferation and MYCN levels. Conversely, ectopic expression of let-7e was sufficient to reduce proliferation and downregulate MYCN, AURKB, and LIN28, the latter via a double-negative feedback loop. We conclude that the LIN28/let-7 pathway has a critical pathobiologic role in malignant GCTs and therefore offers a promising target for therapeutic intervention.

miR-221 affects multiple cancer pathways by modulating the level of hundreds messenger RNAs.

microRNA miR-221 is frequently over-expressed in a variety of human neoplasms. Aim of this study was to identify new miR-221 gene targets to improve our understanding on the molecular tumor-promoting mechanisms affected by miR-221. Gene expression profiling of miR-221-transfected-SNU-398 cells was analyzed by the Sylamer algorithm to verify the enrichment of miR-221 targets among down-modulated genes. This analysis revealed that enforced expression of miR-221 in SNU-398 cells caused the down-regulation of 602 mRNAs carrying sequences homologous to miR-221 seed sequence within their 3'UTRs. Pathways analysis performed on these genes revealed their prominent involvement in cell proliferation and apoptosis. Activation of E2F, MYC, NFkB, and ß-catenin pathways was experimentally proven. Some of the new miR-221 target genes, including RB1, WEE1 (cell cycle inhibitors), APAF1 (pro-apoptotic), ANXA1, CTCF (transcriptional repressor), were individually validated as miR-221 targets in SNU-398, HepG2, and HEK293 cell lines. By identifying a large set of miR-221 gene targets, this study improves our knowledge about miR-221 molecular mechanisms involved in tumorigenesis. The modulation of mRNA level of 602 genes confirms the ability of miR-221 to promote cancer by affecting multiple oncogenic pathways.

MicroRNA-9 inhibition of cell proliferation and identification of novel miR-9 targets by transcriptome profiling in breast cancer cells.

Although underexpression of miR-9 in cancer cells is reported in many cancer types, it is currently difficult to classify miR-9 as a tumor suppressor or an oncomir. We demonstrate that miR-9 expression is down-regulated in MCF-7 and MDA-MB-231 breast cancer cells compared with MCF-10-2A normal breast cell line. Increasing miR-9 expression levels in breast cancer cells induced anti-proliferative, anti-invasive, and pro-apoptotic activity. In addition, microarray profiling of the transcriptome of MCF-7 cells overexpressing miR-9 identified six novel direct miR-9 targets (AP3B1, CCNG1, LARP1, MTHFD1L, MTHFD2, and SRPK1). Among these, MTHFD2 was identified as a miR-9 target gene that affects cell proliferation. Knockdown of MTHFD2 mimicked the effect observed when miR-9 was overexpressed by decreasing cell viability and increasing apoptotic activity. Despite variable effects on different cell lines, proliferative and anti-apoptotic activity of MTHFD2 was demonstrated whereby it could escape from miR-9-directed suppression (by overexpression of MTHFD2 with mutated miR-9 binding sites). Furthermore, endogenous expression levels of miR-9 and MTHFD2 displayed inverse expression profiles in primary breast tumor samples compared with normal breast samples; miR-9 was down-regulated, and MTHFD2 was up-regulated. These results indicate anti-proliferative and pro-apoptotic activity of miR-9 and that direct targeting of MTHFD2 can contribute to tumor suppressor-like activity of miR-9 in breast cancer cells.

MiR-221 influences effector functions and actin cytoskeleton in mast cells.

Mast cells have essential effector and immunoregulatory functions in IgE-associated allergic disorders and certain innate and adaptive immune responses, but the role of miRNAs in regulating mast cell functions is almost completely unexplored. To examine the role of the activation-induced miRNA miR-221 in mouse mast cells, we developed robust lentiviral systems for miRNA overexpression and depletion. While miR-221 favored mast cell adhesion and migration towards SCF or antigen in trans-well migration assays, as well as cytokine production and degranulation in response to IgE-antigen complexes, neither miR-221 overexpression, nor its ablation, interfered with mast cell differentiation. Transcriptional profiling of miR-221-overexpressing mast cells revealed modulation of many transcripts, including several associated with the cytoskeleton; indeed, miR-221 overexpression was associated with reproducible increases in cortical actin in mast cells, and with altered cellular shape and cell cycle in murine fibroblasts. Our bioinformatics analysis showed that this effect was likely mediated by the composite effect of miR-221 on many primary and secondary targets in resting cells. Indeed, miR-221-induced cellular alterations could not be recapitulated by knockdown of one of the major targets of miR-221. We propose a model in which miR-221 has two different roles in mast cells: in resting cells, basal levels of miR-221 contribute to the regulation of the cell cycle and cytoskeleton, a general mechanism probably common to other miR-221-expressing cell types, such as fibroblasts. Vice versa, upon induction in response to mast cell stimulation, miR-221 effects are mast cell-specific and activation-dependent, contributing to the regulation of degranulation, cytokine production and cell adherence. Our studies provide new insights into the roles of miR-221 in mast cell biology, and identify novel mechanisms that may contribute to mast cell-related pathological conditions, such as asthma, allergy and mastocytosis.

The two most common histological subtypes of malignant germ cell tumour are distinguished by global microRNA profiles, associated with differential transcription factor expression.

BACKGROUND: We hypothesised that differences in microRNA expression profiles contribute to the contrasting natural history and clinical outcome of the two most common types of malignant germ cell tumour (GCT), yolk sac tumours (YSTs) and germinomas. RESULTS: By direct comparison, using microarray data for paediatric GCT samples and published qRT-PCR data for adult samples, we identified microRNAs significantly up-regulated in YSTs (n = 29 paediatric, 26 adult, 11 overlapping) or germinomas (n = 37 paediatric). By Taqman qRT-PCR we confirmed differential expression of 15 of 16 selected microRNAs and further validated six of these (miR-302b, miR-375, miR-200b, miR-200c, miR-122, miR-205) in an independent sample set. Interestingly, the miR-302 cluster, which is over-expressed in all malignant GCTs, showed further over-expression in YSTs versus germinomas, representing six of the top eight microRNAs over-expressed in paediatric YSTs and seven of the top 11 in adult YSTs. To explain this observation, we used mRNA expression profiles of paediatric and adult malignant GCTs to identify 10 transcription factors (TFs) consistently over-expressed in YSTs versus germinomas, followed by linear regression to confirm associations between TF and miR-302 cluster expression levels. Using the sequence motif analysis environment iMotifs, we identified predicted binding sites for four of the 10 TFs (GATA6, GATA3, TCF7L2 and MAF) in the miR-302 cluster promoter region. Finally, we showed that miR-302 family over-expression in YST is likely to be functionally significant, as mRNAs down-regulated in YSTs were enriched for 3' untranslated region sequences complementary to the common seed of miR-302a~miR-302d. Such mRNAs included mediators of key cancer-associated processes, including tumour suppressor genes, apoptosis regulators and TFs. CONCLUSIONS: Differential microRNA expression is likely to contribute to the relatively aggressive behaviour of YSTs and may enable future improvements in clinical diagnosis and/or treatment.

Malignant germ cell tumors display common microRNA profiles resulting in global changes in expression of messenger RNA targets.

Despite their extensive clinical and pathologic heterogeneity, all malignant germ cell tumors (GCT) are thought to originate from primordial germ cells. However, no common biological abnormalities have been identified to date. We profiled 615 microRNAs (miRNA) in pediatric malignant GCTs, controls, and GCT cell lines (48 samples in total) and re-analyzed available miRNA expression data in adult gonadal malignant GCTs. We applied the bioinformatic algorithm Sylamer to identify miRNAs that are of biological importance by inducing global shifts in mRNA levels. The most significant differentially expressed miRNAs in malignant GCTs were all from the miR-371-373 and miR-302 clusters (adjusted P < 0.00005), which were overexpressed regardless of histologic subtype [yolk sac tumor (YST)/seminoma/embryonal carcinoma (EC)], site (gonadal/extragonadal), or patient age (pediatric/adult). Sylamer revealed that the hexamer GCACTT, complementary to the 2- to 7-nucleotide miRNA seed AAGUGC shared by six members of the miR-371-373 and miR-302 clusters, was the only sequence significantly enriched in the 3'-untranslated region of mRNAs downregulated in pediatric malignant GCTs (as a group), YSTs and ECs, and in adult YSTs (all versus nonmalignant tissue controls; P < 0.05). For the pediatric samples, downregulated genes containing the 3'-untranslated region GCACTT showed significant overrepresentation of Gene Ontology terms related to cancer-associated processes, whereas for downregulated genes lacking GCACTT, Gene Ontology terms generally represented metabolic processes only, with few genes per term (adjusted P < 0.05). We conclude that the miR-371-373 and miR-302 clusters are universally overexpressed in malignant GCTs and coordinately downregulate mRNAs involved in biologically significant pathways.

Systematic analysis of off-target effects in an RNAi screen reveals microRNAs affecting sensitivity to TRAIL-induced apoptosis.

BACKGROUND: RNA inhibition by siRNAs is a frequently used approach to identify genes required for specific biological processes. However RNAi screening using siRNAs is hampered by non-specific or off target effects of the siRNAs, making it difficult to separate genuine hits from false positives. It is thought that many of the off-target effects seen in RNAi experiments are due to siRNAs acting as microRNAs (miRNAs), causing a reduction in gene expression of unintended targets via matches to the 6 or 7 nt 'seed' sequence. We have conducted a careful examination of off-target effects during an siRNA screen for novel regulators of the TRAIL apoptosis induction pathway(s). RESULTS: We identified 3 hexamers and 3 heptamer seed sequences that appeared multiple times in the top twenty siRNAs in the TRAIL apoptosis screen. Using a novel statistical enrichment approach, we systematically identified a further 17 hexamer and 13 heptamer seed sequences enriched in high scoring siRNAs. The presence of one of these seeds sequences (which could explain 6 of 8 confirmed off-target effects) is sufficient to elicit a phenotype. Three of these seed sequences appear in the human miRNAs miR-26a, miR-145 and miR-384. Transfection of mimics of these miRNAs protects several cell types from TRAIL-induced cell death. CONCLUSIONS: We have demonstrated a role for miR-26a, miR-145 and miR-26a in TRAIL-induced apoptosis. Further these results show that RNAi screening enriches for siRNAs with relevant off-target effects. Some of these effects can be identified by the over-representation of certain seed sequences in high-scoring siRNAs and we demonstrate the usefulness of such systematic analysis of enriched seed sequences.

A logic-based diagram of signalling pathways central to macrophage activation.

BACKGROUND: The complex yet flexible cellular response to pathogens is orchestrated by the interaction of multiple signalling and metabolic pathways. The molecular regulation of this response has been studied in great detail but comprehensive and unambiguous diagrams describing these events are generally unavailable. Four key signalling cascades triggered early-on in the innate immune response are the toll-like receptor, interferon, NF-kappaB and apoptotic pathways, which co-operate to defend cells against a given pathogen. However, these pathways are commonly viewed as separate entities rather than an integrated network of molecular interactions. RESULTS: Here we describe the construction of a logically represented pathway diagram which attempts to integrate these four pathways central to innate immunity using a modified version of the Edinburgh Pathway Notation. The pathway map is available in a number of electronic formats and editing is supported by yEd graph editor software. CONCLUSION: The map presents a powerful visual aid for interpreting the available pathway interaction knowledge and underscores the valuable contribution well constructed pathway diagrams make to communicating large amounts of molecular interaction data. Furthermore, we discuss issues with the limitations and scalability of pathways presented in this fashion, explore options for automated layout of large pathway networks and demonstrate how such maps can aid the interpretation of functional studies.

microRNA-155 regulates the generation of immunoglobulin class-switched plasma cells.

microRNA-155 (miR-155) is expressed by cells of the immune system after activation and has been shown to be required for antibody production after vaccination with attenuated Salmonella. Here we show the intrinsic requirement for miR-155 in B cell responses to thymus-dependent and -independent antigens. B cells lacking miR-155 generated reduced extrafollicular and germinal center responses and failed to produce high-affinity IgG1 antibodies. Gene-expression profiling of activated B cells indicated that miR-155 regulates an array of genes with diverse function, many of which are predicted targets of miR-155. The transcription factor Pu.1 is validated as a direct target of miR155-mediated inhibition. When Pu.1 is overexpressed in wild-type B cells, fewer IgG1 cells are produced, indicating that loss of Pu.1 regulation is a contributing factor to the miR-155-deficient phenotype. Our results implicate post-transcriptional regulation of gene expression for establishing the terminal differentiation program of B cells.

Prediction of microRNA targets.

Recently, microRNAs (miRNAs) have been shown to be important regulators of genes in many organisms and have already been implicated in a growing number of diseases. MiRNAs are short (21-23 nucleotides) RNAs that bind to the 3' untranslated regions of target genes. This binding event causes translational repression of the target gene and, evidence now suggests, also stimulates rapid degradation of the target transcript. miRNAs represent a new species of regulator, controlling the levels of potentially large numbers of proteins, many of which might be important drug targets. The expression of miRNAs shows that they are highly differentially expressed, with specific miRNAs active in certain tissues at certain times. In many cancers, miRNA expression is significantly altered, and this has been shown to be a useful diagnostic tool. Several computational approaches have been developed for the prediction of miRNA targets.