Extracellular vesicles from neural stem cells transfer IFN-γ via Ifngr1 to activate Stat1 signaling in target cells.

The idea that stem cell therapies work only via cell replacement is challenged by the observation of consistent intercellular molecule exchange between the graft and the host. Here we defined a mechanism of cellular signaling by which neural stem/precursor cells (NPCs) communicate with the microenvironment via extracellular vesicles (EVs), and we elucidated its molecular signature and function. We observed cytokine-regulated pathways that sort proteins and mRNAs into EVs. We described induction of interferon gamma (IFN-γ) pathway in NPCs exposed to proinflammatory cytokines that is mirrored in EVs. We showed that IFN-γ bound to EVs through Ifngr1 activates Stat1 in target cells. Finally, we demonstrated that endogenous Stat1 and Ifngr1 in target cells are indispensable to sustain the activation of Stat1 signaling by EV-associated IFN-γ/Ifngr1 complexes. Our study identifies a mechanism of cellular signaling regulated by EV-associated IFN-γ/Ifngr1 complexes, which grafted stem cells may use to communicate with the host immune system.

Visualization and analysis of RNA-Seq assembly graphs.

RNA-Seq is a powerful transcriptome profiling technology enabling transcript discovery and quantification. Whilst most commonly used for gene-level quantification, the data can be used for the analysis of transcript isoforms. However, when the underlying transcript assemblies are complex, current visualization approaches can be limiting, with splicing events a challenge to interpret. Here, we report on the development of a graph-based visualization method as a complementary approach to understanding transcript diversity from short-read RNA-Seq data. Following the mapping of reads to a reference genome, a read-to-read comparison is performed on all reads mapping to a given gene, producing a weighted similarity matrix between reads. This is used to produce an RNA assembly graph, where nodes represent reads and edges similarity scores between them. The resulting graphs are visualized in 3D space to better appreciate their sometimes large and complex topology, with other information being overlaid on to nodes, e.g. transcript models. Here we demonstrate the utility of this approach, including the unusual structure of these graphs and how they can be used to identify issues in assembly, repetitive sequences within transcripts and splice variants. We believe this approach has the potential to significantly improve our understanding of transcript complexity.

Extracellular vesicles are independent metabolic units with asparaginase activity.

Extracellular vesicles (EVs) are membrane particles involved in the exchange of a broad range of bioactive molecules between cells and the microenvironment. Although it has been shown that cells can traffic metabolic enzymes via EVs, much remains to be elucidated with regard to their intrinsic metabolic activity. Accordingly, herein we assessed the ability of neural stem/progenitor cell (NSC)-derived EVs to consume and produce metabolites. Our metabolomics and functional analyses both revealed that EVs harbor L-asparaginase activity, catalyzed by the enzyme asparaginase-like protein 1 (Asrgl1). Critically, we show that Asrgl1 activity is selective for asparagine and is devoid of glutaminase activity. We found that mouse and human NSC EVs traffic Asrgl1. Our results demonstrate, for the first time, that NSC EVs function as independent metabolic units that are able to modify the concentrations of critical nutrients, with the potential to affect the physiology of their microenvironment.

Transposon-driven transcription is a conserved feature of vertebrate spermatogenesis and transcript evolution.

Spermatogenesis is associated with major and unique changes to chromosomes and chromatin. Here, we sought to understand the impact of these changes on spermatogenic transcriptomes. We show that long terminal repeats (LTRs) of specific mouse endogenous retroviruses (ERVs) drive the expression of many long non-coding transcripts (lncRNA). This process occurs post-mitotically predominantly in spermatocytes and round spermatids. We demonstrate that this transposon-driven lncRNA expression is a conserved feature of vertebrate spermatogenesis. We propose that transposon promoters are a mechanism by which the genome can explore novel transcriptional substrates, increasing evolutionary plasticity and allowing for the genesis of novel coding and non-coding genes. Accordingly, we show that a small fraction of these novel ERV-driven transcripts encode short open reading frames that produce detectable peptides. Finally, we find that distinct ERV elements from the same subfamilies act as differentially activated promoters in a tissue-specific context. In summary, we demonstrate that LTRs can act as tissue-specific promoters and contribute to post-mitotic spermatogenic transcriptome diversity.

Detection of secondary binding sites in proteins using fragment screening.

Proteins need to be tightly regulated as they control biological processes in most normal cellular functions. The precise mechanisms of regulation are rarely completely understood but can involve binding of endogenous ligands and/or partner proteins at specific locations on a protein that can modulate function. Often, these additional secondary binding sites appear separate to the primary binding site, which, for example for an enzyme, may bind a substrate. In previous work, we have uncovered several examples in which secondary binding sites were discovered on proteins using fragment screening approaches. In each case, we were able to establish that the newly identified secondary binding site was biologically relevant as it was able to modulate function by the binding of a small molecule. In this study, we investigate how often secondary binding sites are located on proteins by analyzing 24 protein targets for which we have performed a fragment screen using X-ray crystallography. Our analysis shows that, surprisingly, the majority of proteins contain secondary binding sites based on their ability to bind fragments. Furthermore, sequence analysis of these previously unknown sites indicate high conservation, which suggests that they may have a biological function, perhaps via an allosteric mechanism. Comparing the physicochemical properties of the secondary sites with known primary ligand binding sites also shows broad similarities indicating that many of the secondary sites may be druggable in nature with small molecules that could provide new opportunities to modulate potential therapeutic targets.

Emergence of resistance to tyrosine kinase inhibitors in non-small-cell lung cancer can be delayed by an upfront combination with the HSP90 inhibitor onalespib.

BACKGROUND: Tyrosine kinase inhibitors, such as crizotinib and erlotinib, are widely used to treat non-small-cell lung cancer, but after initial response, relapse is common because of the emergence of resistance through multiple mechanisms. Here, we investigated whether a frontline combination with an HSP90 inhibitor could delay the emergence of resistance to these inhibitors in preclinical lung cancer models. METHODS: The HSP90 inhibitor, onalespib, was combined with either crizotinib or erlotinib in ALK- or EGFR-activated xenograft models respectively (H2228, HCC827). RESULTS: In both models, after initial response to the monotherapy kinase inhibitors, tumour relapse was observed. In contrast, tumour growth remained inhibited when treated with an onalespib/kinase inhibitor combination. Analysis of H2228 tumours, which had relapsed on crizotinib monotherapy, identified a number of clinically relevant crizotinib resistance mechanisms, suggesting that HSP90 inhibitor treatment was capable of suppressing multiple mechanisms of resistance. Resistant cell lines, derived from these tumours, retained sensitivity to onalespib (proliferation and signalling pathways were inhibited), indicating that, despite their resistance to crizotinib, they were still sensitive to HSP90 inhibition. CONCLUSIONS: Together, these preclinical data suggest that frontline combination with an HSP90 inhibitor may be a method for delaying the emergence of resistance to targeted therapies.

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.

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.

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.

Dual-Mechanism ERK1/2 Inhibitors Exploit a Distinct Binding Mode to Block Phosphorylation and Nuclear Accumulation of ERK1/2.

The RAS-regulated RAF-MEK1/2-ERK1/2 signaling pathway is frequently deregulated in cancer due to activating mutations of growth factor receptors, RAS or BRAF. Both RAF and MEK1/2 inhibitors are clinically approved and various ERK1/2 inhibitors (ERKi) are currently undergoing clinical trials. To date, ERKi display two distinct mechanisms of action (MoA): catalytic ERKi solely inhibit ERK1/2 catalytic activity, whereas dual mechanism ERKi additionally prevents the activating phosphorylation of ERK1/2 at its T-E-Y motif by MEK1/2. These differences may impart significant differences in biological activity because T-E-Y phosphorylation is the signal for nuclear entry of ERK1/2, allowing them to access many key transcription factor targets. Here, we characterized the MoA of five ERKi and examined their functional consequences in terms of ERK1/2 signaling, gene expression, and antiproliferative efficacy. We demonstrate that catalytic ERKi promote a striking nuclear accumulation of p-ERK1/2 in KRAS-mutant cell lines. In contrast, dual-mechanism ERKi exploits a distinct binding mode to block ERK1/2 phosphorylation by MEK1/2, exhibit superior potency, and prevent the nuclear accumulation of ERK1/2. Consequently, dual-mechanism ERKi exhibit more durable pathway inhibition and enhanced suppression of ERK1/2-dependent gene expression compared with catalytic ERKi, resulting in increased efficacy across BRAF- and RAS-mutant cell lines.

Annotation of mammalian primary microRNAs.

BACKGROUND: MicroRNAs (miRNAs) are important regulators of gene expression and have been implicated in development, differentiation and pathogenesis. Hundreds of miRNAs have been discovered in mammalian genomes. Approximately 50% of mammalian miRNAs are expressed from introns of protein-coding genes; the primary transcript (pri-miRNA) is therefore assumed to be the host transcript. However, very little is known about the structure of pri-miRNAs expressed from intergenic regions. Here we annotate transcript boundaries of miRNAs in human, mouse and rat genomes using various transcription features. The 5' end of the pri-miRNA is predicted from transcription start sites, CpG islands and 5' CAGE tags mapped in the upstream flanking region surrounding the precursor miRNA (pre-miRNA). The 3' end of the pri-miRNA is predicted based on the mapping of polyA signals, and supported by cDNA/EST and ditags data. The predicted pri-miRNAs are also analyzed for promoter and insulator-associated regulatory regions. RESULTS: We define sets of conserved and non-conserved human, mouse and rat pre-miRNAs using bidirectional BLAST and synteny analysis. Transcription features in their flanking regions are used to demarcate the 5' and 3' boundaries of the pri-miRNAs. The lengths and boundaries of primary transcripts are highly conserved between orthologous miRNAs. A significant fraction of pri-miRNAs have lengths between 1 and 10 kb, with very few introns. We annotate a total of 59 pri-miRNA structures, which include 82 pre-miRNAs. 36 pri-miRNAs are conserved in all 3 species. In total, 18 of the confidently annotated transcripts express more than one pre-miRNA. The upstream regions of 54% of the predicted pri-miRNAs are found to be associated with promoter and insulator regulatory sequences. CONCLUSION: Little is known about the primary transcripts of intergenic miRNAs. Using comparative data, we are able to identify the boundaries of a significant proportion of human, mouse and rat pri-miRNAs. We confidently predict the transcripts including a total of 77, 58 and 47 human, mouse and rat pre-miRNAs respectively. Our computational annotations provide a basis for subsequent experimental validation of predicted pri-miRNAs.

Computational prediction of protein-protein interactions.

Recently a number of computational approaches have been developed for the prediction of protein-protein interactions. Complete genome sequencing projects have provided the vast amount of information needed for these analyses. These methods utilize the structural, genomic, and biological context of proteins and genes in complete genomes to predict protein interaction networks and functional linkages between proteins. Given that experimental techniques remain expensive, time-consuming, and labor-intensive, these methods represent an important advance in proteomics. Some of these approaches utilize sequence data alone to predict interactions, while others combine multiple computational and experimental datasets to accurately build protein interaction maps for complete genomes. These methods represent a complementary approach to current high-throughput projects whose aim is to delineate protein interaction maps in complete genomes. We will describe a number of computational protocols for protein interaction prediction based on the structural, genomic, and biological context of proteins in complete genomes, and detail methods for protein interaction network visualization and analysis.

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.

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.

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.