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RNA interference (RNAi) is a potent cellular system against viruses in various organisms. Although common traits are observed in plants, insects, and nematodes, the situation observed in mammals appears more complex. In mammalian somatic cells, RNAi is implicated in endonucleolytic cleavage mediated by artificially delivered small interfering RNAs (siRNAs) as well as in translation repression mediated by microRNAs (miRNAs). Because siRNAs and miRNAs recognize viral mRNAs, RNAi inherently limits virus production and participates in antiviral defense. However, several observations made in the cases of hepatitis C virus and retroviruses (including the human immunodeficiency virus and the primate foamy virus) bring evidence that this relationship is much more complex and that certain components of the RNAi effector complex [called the RNA-induced silencing complex (RISC)], such as AGO2, are also required for viral replication. Here, we summarize recent discoveries that have revealed this dual implication in virus biology. We further discuss their potential implications for the functions of RNAi-related proteins, with special emphasis on retrotransposition and genome stability.

Abstract Background To understand biological processes and diseases, it is crucial to unravel the concerted interplay of transcription factors (TFs), microRNAs (miRNAs) and their targets within regulatory networks and fundamental sub-networks. An integrative computational resource generating a comprehensive view of these regulatory molecular interactions at a genome-wide scale would be of great interest to biologists, but is not available to date. Results To identify and analyze molecular interaction networks, we developed MIR@NT@N, an integrative approach based on a meta-regulation network model and a large-scale database. MIR@NT@N uses a graph-based approach to predict novel molecular actors across multiple regulatory processes (i.e. TFs acting on protein-coding or miRNA genes, or miRNAs acting on messenger RNAs). Exploiting these predictions, the user can generate networks and further analyze them to identify sub-networks, including motifs such as feedback and feedforward loops (FBL and FFL). In addition, networks can be built from lists of molecular actors with an a priori role in a given biological process to predict novel and unanticipated interactions. Analyses can be contextualized and filtered by integrating additional information such as microarray expression data. All results, including generated graphs, can be visualized, saved and exported into various formats. MIR@NT@N performances have been evaluated using published data and then applied to the regulatory program underlying epithelium to mesenchyme transition (EMT), an evolutionary-conserved process which is implicated in embryonic development and disease. Conclusions MIR@NT@N is an effective computational approach to identify novel molecular regulations and to predict gene regulatory networks and sub-networks including conserved motifs within a given biological context. Taking advantage of the M@IA environment, MIR@NT@N is a user-friendly web resource freely available at http://mironton.uni.lu which will be updated on a regular basis.

Abstract The anti-viral function of RNA silencing was first discovered in plants as a natural manifestation of the artificial 'co-suppression', which refers to the extinction of endogenous gene induced by homologous transgene. Because silencing components are conserved among most, if not all, eukaryotes, the question rapidly arose as to determine whether this process fulfils anti-viral functions in animals, such as insects and mammals. It appears that, whereas the anti-viral process seems to be similarly conserved from plants to insects, even in worms, RNA silencing does influence the replication of mammalian viruses but in a particular mode: micro(mi)RNAs, endogenous small RNAs naturally implicated in translational control, rather than virus-derived small interfering (si)RNAs like in other organisms, are involved. In fact, these recent studies even suggest that RNA silencing may be beneficial for viral replication. Accordingly, several large DNA mammalian viruses have been shown to encode their own miRNAs. Here, we summarize the seminal studies that have implicated RNA silencing in viral infection and compare the different eukaryotic responses.

Characterizing the binding preferences of transcription factors (TFs) in different cell types and conditions is key to understand how they orchestrate gene expression. Here, we develop TFscope, a machine learning approach that identifies sequence features explaining the binding differences observed between two ChIP-seq experiments targeting either the same TF in two conditions or two TFs with similar motifs (paralogous TFs). TFscope systematically investigates differences in the core motif, nucleotide environment and co-factor motifs, and provides the contribution of each key feature in the two experiments. TFscope was applied to > 305 ChIP-seq pairs, and several examples are discussed., (© 2024. The Author(s).)

Motivations: Gene regulatory networks (GRNs) are traditionally inferred from gene expression profiles monitoring a specific condition or treatment. In the last decade, integrative strategies have successfully emerged to guide GRN inference from gene expression with complementary prior data. However, datasets used as prior information and validation gold standards are often related and limited to a subset of genes. This lack of complete and independent evaluation calls for new criteria to robustly estimate the optimal intensity of prior data integration in the inference process., Results: We address this issue for two regression-based GRN inference models, a weighted random forest (weigthedRF) and a generalized linear model estimated under a weighted LASSO penalty with stability selection (weightedLASSO). These approaches are applied to data from the root response to nitrate induction in Arabidopsis thaliana. For each gene, we measure how the integration of transcription factor binding motifs influences model prediction. We propose a new approach, DIOgene, that uses model prediction error and a simulated null hypothesis in order to optimize data integration strength in a hypothesis-driven, gene-specific manner. This integration scheme reveals a strong diversity of optimal integration intensities between genes, and offers good performance in minimizing prediction error as well as retrieving experimental interactions. Experimental results show that DIOgene compares favorably against state-of-the-art approaches and allows to recover master regulators of nitrate induction., Availability and Implementation: The R code and notebooks demonstrating the use of the proposed approaches are available in the repository https://github.com/OceaneCsn/integrative_GRN_N_induction., (© The Author(s) 2024. Published by Oxford University Press.)

The action of cis-regulatory elements with either activation or repression functions underpins the precise regulation of gene expression during normal development and cell differentiation. Gene activation by the combined activities of promoters and distal enhancers has been extensively studied in normal and pathological contexts. In sharp contrast, gene repression by cis-acting silencers, defined as genetic elements that negatively regulate gene transcription in a position-independent fashion, is less well understood. Here, we repurpose the STARR-seq approach as a novel high-throughput reporter strategy to quantitatively assess silencer activity in mammals. We assessed silencer activity from DNase hypersensitive I sites in a mouse T cell line. Identified silencers were associated with either repressive or active chromatin marks and enriched for binding motifs of known transcriptional repressors. CRISPR-mediated genomic deletions validated the repressive function of distinct silencers involved in the repression of non-T cell genes and genes regulated during T cell differentiation. Finally, we unravel an association of silencer activity with short tandem repeats, highlighting the role of repetitive elements in silencer activity. Our results provide a general strategy for genome-wide identification and characterization of silencer elements., (© The Author(s) 2023. Published by Oxford University Press on behalf of Nucleic Acids Research.)

The ability to visualize RNA in its native subcellular environment by using single-molecule fluorescence in situ hybridization (smFISH) has reshaped our understanding of gene expression and cellular functions. A major hindrance of smFISH is the difficulty to perform systematic experiments in medium- or high-throughput formats, principally because of the high cost of generating the individual fluorescent probe sets. Here, we present high-throughput smFISH (HT-smFISH), a simple and cost-efficient method for imaging hundreds to thousands of single endogenous RNA molecules in 96-well plates. HT-smFISH uses RNA probes transcribed in vitro from a large pool of unlabeled oligonucleotides. This allows the generation of individual probes for many RNA species, replacing commercial DNA probe sets. HT-smFISH thus reduces costs per targeted RNA compared with many smFISH methods and is easily scalable and flexible in design. We provide a protocol that combines oligo pool design, probe set generation, optimized hybridization conditions and guidelines for image acquisition and analysis. The pipeline requires knowledge of standard molecular biology tools, cell culture and fluorescence microscopy. It is achievable in ~20 d. In brief, HT-smFISH is tailored for medium- to high-throughput screens that image RNAs at single-molecule sensitivity., (© 2022. Springer Nature Limited.)

Using the Cap Analysis of Gene Expression (CAGE) technology, the FANTOM5 consortium provided one of the most comprehensive maps of transcription start sites (TSSs) in several species. Strikingly, ~72% of them could not be assigned to a specific gene and initiate at unconventional regions, outside promoters or enhancers. Here, we probe these unassigned TSSs and show that, in all species studied, a significant fraction of CAGE peaks initiate at microsatellites, also called short tandem repeats (STRs). To confirm this transcription, we develop Cap Trap RNA-seq, a technology which combines cap trapping and long read MinION sequencing. We train sequence-based deep learning models able to predict CAGE signal at STRs with high accuracy. These models unveil the importance of STR surrounding sequences not only to distinguish STR classes, but also to predict the level of transcription initiation. Importantly, genetic variants linked to human diseases are preferentially found at STRs with high transcription initiation level, supporting the biological and clinical relevance of transcription initiation at STRs. Together, our results extend the repertoire of non-coding transcription associated with DNA tandem repeats and complexify STR polymorphism.

Long regulatory elements (LREs), such as CpG islands, polydA:dT tracts or AU-rich elements, are thought to play key roles in gene regulation but, as opposed to conventional binding sites of transcription factors, few methods have been proposed to formally and automatically characterize them. We present here a computational approach named DExTER (Domain Exploration To Explain gene Regulation) dedicated to the identification of candidate LREs (cLREs) and apply it to the analysis of the genomes of P. falciparum and other eukaryotes. Our analyses show that all tested genomes contain several cLREs that are somewhat conserved along evolution, and that gene expression can be predicted with surprising accuracy on the basis of these long regions only. Regulation by cLREs exhibits very different behaviours depending on species and conditions. In P. falciparum and other Apicomplexan organisms as well as in Dictyostelium discoideum, the process appears highly dynamic, with different cLREs involved at different phases of the life cycle. For multicellular organisms, the same cLREs are involved in all tissues, but a dynamic behavior is observed along embryonic development stages. In P. falciparum, whose genome is known to be strongly depleted of transcription factors, cLREs are predictive of expression with an accuracy above 70%, and our analyses show that they are associated with both transcriptional and post-transcriptional regulation signals. Moreover, we assessed the biological relevance of one LRE discovered by DExTER in P. falciparum using an in vivo reporter assay. The source code (python) of DExTER is available at https://gite.lirmm.fr/menichelli/DExTER., Competing Interests: The authors have declared that no competing interests exist.

The ubiquitous family of dimeric transcription factors AP-1 is made up of Fos and Jun family proteins. It has long been thought to operate principally at gene promoters and how it controls transcription is still ill-understood. The Fos family protein Fra-1 is overexpressed in triple negative breast cancers (TNBCs) where it contributes to tumor aggressiveness. To address its transcriptional actions in TNBCs, we combined transcriptomics, ChIP-seqs, machine learning and NG Capture-C. Additionally, we studied its Fos family kin Fra-2 also expressed in TNBCs, albeit much less. Consistently with their pleiotropic effects, Fra-1 and Fra-2 up- and downregulate individually, together or redundantly many genes associated with a wide range of biological processes. Target gene regulation is principally due to binding of Fra-1 and Fra-2 at regulatory elements located distantly from cognate promoters where Fra-1 modulates the recruitment of the transcriptional co-regulator p300/CBP and where differences in AP-1 variant motif recognition can underlie preferential Fra-1- or Fra-2 bindings. Our work also shows no major role for Fra-1 in chromatin architecture control at target gene loci, but suggests collaboration between Fra-1-bound and -unbound enhancers within chromatin hubs sometimes including promoters for other Fra-1-regulated genes. Our work impacts our view of AP-1., (© The Author(s) 2021. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Local translation allows for a spatial control of gene expression. Here, we use high-throughput smFISH to screen centrosomal protein-coding genes, and we describe 8 human mRNAs accumulating at centrosomes. These mRNAs localize at different stages during cell cycle with a remarkable choreography, indicating a finely regulated translational program at centrosomes. Interestingly, drug treatments and reporter analyses reveal a common translation-dependent localization mechanism requiring the nascent protein. Using ASPM and NUMA1 as models, single mRNA and polysome imaging reveals active movements of endogenous polysomes towards the centrosome at the onset of mitosis, when these mRNAs start localizing. ASPM polysomes associate with microtubules and localize by either motor-driven transport or microtubule pulling. Remarkably, the Drosophila orthologs of the human centrosomal mRNAs also localize to centrosomes and also require translation. These data identify a conserved family of centrosomal mRNAs that localize by active polysome transport mediated by nascent proteins.

Background: In eukaryotic cells, transcription factors (TFs) are thought to act in a combinatorial way, by competing and collaborating to regulate common target genes. However, several questions remain regarding the conservation of these combinations among different gene classes, regulatory regions and cell types., Results: We propose a new approach named TFcoop to infer the TF combinations involved in the binding of a target TF in a particular cell type. TFcoop aims to predict the binding sites of the target TF upon the nucleotide content of the sequences and of the binding affinity of all identified cooperating TFs. The set of cooperating TFs and model parameters are learned from ChIP-seq data of the target TF. We used TFcoop to investigate the TF combinations involved in the binding of 106 TFs on 41 cell types and in four regulatory regions: promoters of mRNAs, lncRNAs and pri-miRNAs, and enhancers. We first assess that TFcoop is accurate and outperforms simple PWM methods for predicting TF binding sites. Next, analysis of the learned models sheds light on important properties of TF combinations in different promoter classes and in enhancers. First, we show that combinations governing TF binding on enhancers are more cell-type specific than that governing binding in promoters. Second, for a given TF and cell type, we observe that TF combinations are different between promoters and enhancers, but similar for promoters of mRNAs, lncRNAs and pri-miRNAs. Analysis of the TFs cooperating with the different targets show over-representation of pioneer TFs and a clear preference for TFs with binding motif composition similar to that of the target. Lastly, our models accurately distinguish promoters associated with specific biological processes., Conclusions: TFcoop appears as an accurate approach for studying TF combinations. Its use on ENCODE and FANTOM data allowed us to discover important properties of human TF combinations in different promoter classes and in enhancers. The R code for learning a TFcoop model and for reproducing the main experiments described in the paper is available in an R Markdown file at address https://gite.lirmm.fr/brehelin/TFcoop .

miRNAs are master regulators of gene expression that play key roles in cancer metastasis. During bone metastasis, metastatic tumor cells must rewire their biology and express genes that are normally expressed by bone cells (a process called osteomimicry), which endow tumor cells with full competence for outgrowth in the bone marrow. Here, we establish miR-30 family members miR-30a, miR-30b, miR-30c, miR-30d, and miR-30e as suppressors of breast cancer bone metastasis that regulate multiple pathways, including osteomimicry. Low expression of miR-30 in primary tumors from patients with breast cancer were associated with poor relapse-free survival. In addition, estrogen receptor (ER)-negative/progesterone receptor (PR)-negative breast cancer cells expressed lower miR-30 levels than their ER/PR-positive counterparts. Overexpression of miR-30 in ER/PR-negative breast cancer cells resulted in the reduction of bone metastasis burden in vivo In vitro , miR-30 did not affect tumor cell proliferation, but did inhibit tumor cell invasion. Furthermore, overexpression of miR-30 restored bone homeostasis by reversing the effects of tumor cell-conditioned medium on osteoclastogenesis and osteoblastogenesis. A number of genes associated with osteoclastogenesis stimulation ( IL8, IL11 ), osteoblastogenesis inhibition ( DKK-1 ), tumor cell osteomimicry ( RUNX2, CDH11 ), and invasiveness ( CTGF, ITGA5, ITGB3 ) were identified as targets for repression by miR-30. Among these genes, silencing CDH11 or ITGA5 in ER-/PR-negative breast cancer cells recapitulated inhibitory effects of miR-30 on skeletal tumor burden in vivo Overall, our findings provide evidence that miR-30 family members employ multiple mechanisms to impede breast cancer bone metastasis and may represent attractive targets for therapeutic intervention. Significance: These findings suggest miR-30 family members may serve as an effective means to therapeutically attenuate metastasis in triple-negative breast cancer. Cancer Res; 78(18); 5259-73. ©2018 AACR ., (©2018 American Association for Cancer Research.)

The FANTOM5 consortium recently characterized 65,423 human enhancers from 1829 cell and tissue samples using the Cap Analysis of Gene Expression technology. We showed that the guanine and cytosine content at enhancer regions distinguishes two classes of enhancers harboring distinct DNA structural properties at flanking regions. A functional analysis of their predicted gene targets highlighted one class of enhancers as significantly enriched for associations with immune response genes. Moreover, these enhancers were specifically enriched for regulatory motifs recognized by transcription factors involved in immune response. We observed that enhancers enriched for links to immune response genes were more cell-type specific, preferentially activated upon bacterial infection, and with specific response activity. Looking at chromatin capture data, we found that the two classes of enhancers were lying in distinct topologically associating domains and chromatin loops. Our results suggest that specific nucleotide compositions encode for classes of enhancers that are functionally distinct and specifically organized in the human genome., (Copyright © 2018 by the Genetics Society of America.)

Oxidative phosphorylation (OXPHOS) generates ROS as a byproduct of mitochondrial complex I activity. ROS-detoxifying enzymes are made available through the activation of their antioxidant response elements (ARE) in their gene promoters. NRF2 binds to AREs and induces this anti-oxidant response. We show that cells from multiple origins performing OXPHOS induced NRF2 expression and its transcriptional activity. The NRF2 promoter contains MEF2 binding sites and the MAPK ERK5 induced MEF2-dependent NRF2 expression. Blocking OXPHOS in a mouse model decreased Erk5 and Nrf2 expression. Furthermore, fibroblasts derived from patients with mitochondrial disorders also showed low expression of ERK5 and NRF2 mRNAs. Notably, in cells lacking functional mitochondrial complex I activity OXPHOS did not induce ERK5 expression and failed to generate this anti-oxidant response. Complex I activity induces ERK5 expression through fumarate accumulation. Eukaryotic cells have evolved a genetic program to prevent oxidative stress directly linked to OXPHOS and not requiring ROS.

Progress in assisted reproductive technologies strongly relies on understanding the regulation of the dialogue between oocyte and cumulus cells (CCs). Little is known about the role of long non-coding RNAs (lncRNAs) in the human cumulus-oocyte complex (COC). To this aim, publicly available RNA-sequencing data were analyzed to identify lncRNAs that were abundant in metaphase II (MII) oocytes (BCAR4, C3orf56, TUNAR, OOEP-AS1, CASC18, and LINC01118) and CCs (NEAT1, MALAT1, ANXA2P2, MEG3, IL6STP1, and VIM-AS1). These data were validated by RT-qPCR analysis using independent oocytes and CC samples. The functions of the identified lncRNAs were then predicted by constructing lncRNA-mRNA co-expression networks. This analysis suggested that MII oocyte lncRNAs could be involved in chromatin remodeling, cell pluripotency and in driving early embryonic development. CC lncRNAs were co-expressed with genes involved in apoptosis and extracellular matrix-related functions. A bioinformatic analysis of RNA-sequencing data to identify CC lncRNAs that are affected by maternal age showed that lncRNAs with age-related altered expression in CCs are essential for oocyte growth. This comprehensive analysis of lncRNAs expressed in human MII oocytes and CCs could provide biomarkers of oocyte quality for the development of non-invasive tests to identify embryos with high developmental potential.

Gene expression is orchestrated by distinct regulatory regions to ensure a wide variety of cell types and functions. A challenge is to identify which regulatory regions are active, what are their associated features and how they work together in each cell type. Several approaches have tackled this problem by modeling gene expression based on epigenetic marks, with the ultimate goal of identifying driving regions and associated genomic variations that are clinically relevant in particular in precision medicine. However, these models rely on experimental data, which are limited to specific samples (even often to cell lines) and cannot be generated for all regulators and all patients. In addition, we show here that, although these approaches are accurate in predicting gene expression, inference of TF combinations from this type of models is not straightforward. Furthermore these methods are not designed to capture regulation instructions present at the sequence level, before the binding of regulators or the opening of the chromatin. Here, we probe sequence-level instructions for gene expression and develop a method to explain mRNA levels based solely on nucleotide features. Our method positions nucleotide composition as a critical component of gene expression. Moreover, our approach, able to rank regulatory regions according to their contribution, unveils a strong influence of the gene body sequence, in particular introns. We further provide evidence that the contribution of nucleotide content can be linked to co-regulations associated with genome 3D architecture and to associations of genes within topologically associated domains.

Controlling cholesterol levels is a major challenge in human health, since hypercholesterolemia can lead to serious cardiovascular disease. Drugs that target carbohydrate metabolism can also modify lipid metabolism and hence cholesterol plasma levels. In this sense, dichloroacetate (DCA), a pyruvate dehydrogenase kinase (PDK) inhibitor, augments usage of the glycolysis-produced pyruvate in the mitochondria increasing oxidative phosphorylation (OXPHOS). In several animal models, DCA decreases plasma cholesterol and triglycerides. Thus, DCA was used in the 70 s to treat diabetes mellitus, hyperlipoproteinemia and hypercholesterolemia with satisfactory results. However, the mechanism of action remained unknown and we describe it here. DCA increases LDLR mRNA and protein levels as well as LDL intake in several cell lines, primary human hepatocytes and two different mouse models. This effect is mediated by transcriptional activation as evidenced by H3 acetylation on lysine 27 on the LDLR promoter. DCA induces expression of the MAPK ERK5 that turns on the transcription factor MEF2. Inhibition of this ERK5/MEF2 pathway by genetic or pharmacological means decreases LDLR expression and LDL intake. In summary, our results indicate that DCA, by inducing OXPHOS, promotes ERK5/MEF2 activation leading to LDLR expression. The ERK5/MEF2 pathway offers an interesting pharmacological target for drug development.

MicroRNAs (miRNAs) are short non-coding RNAs with key roles in cellular regulation. As part of the fifth edition of the Functional Annotation of Mammalian Genome (FANTOM5) project, we created an integrated expression atlas of miRNAs and their promoters by deep-sequencing 492 short RNA (sRNA) libraries, with matching Cap Analysis Gene Expression (CAGE) data, from 396 human and 47 mouse RNA samples. Promoters were identified for 1,357 human and 804 mouse miRNAs and showed strong sequence conservation between species. We also found that primary and mature miRNA expression levels were correlated, allowing us to use the primary miRNA measurements as a proxy for mature miRNA levels in a total of 1,829 human and 1,029 mouse CAGE libraries. We thus provide a broad atlas of miRNA expression and promoters in primary mammalian cells, establishing a foundation for detailed analysis of miRNA expression patterns and transcriptional control regions.

Metabolic changes drive monocyte differentiation and fate. Although abnormal mitochondria metabolism and innate immune responses participate in the pathogenesis of many inflammatory disorders, molecular events regulating mitochondrial activity to control life and death in monocytes remain poorly understood. We show here that, in human monocytes, microRNA-125b (miR-125b) attenuates the mitochondrial respiration through the silencing of the BH3-only proapoptotic protein BIK and promotes the elongation of the mitochondrial network through the targeting of the mitochondrial fission process 1 protein MTP18, leading to apoptosis. Proinflammatory activation of monocyte-derived macrophages is associated with a concomitant increase in miR-125b expression and decrease in BIK and MTP18 expression, which lead to reduced oxidative phosphorylation and enhanced mitochondrial fusion. In a chronic inflammatory systemic disorder, CD14 + blood monocytes display reduced miR-125b expression as compared with healthy controls, inversely correlated with BIK and MTP18 messenger RNA expression. Our findings not only identify BIK and MTP18 as novel targets for miR-125b that control mitochondrial metabolism and dynamics, respectively, but also reveal a novel function for miR-125b in regulating metabolic adaptation of monocytes to inflammation. Together, these data unravel new molecular mechanisms for a proapoptotic role of miR-125b in monocytes and identify potential targets for interfering with excessive inflammatory activation of monocytes in inflammatory disorders., (© 2016 by The American Society of Hematology.)

At the request of the University of Luxembourg and following an external investigation, the Editor and Publisher have agreed to retract this paper owing to unreliable data.

Tumor cell metabolism is altered during leukemogenesis. Cells performing oxidative phosphorylation (OXPHOS) generate reactive oxygen species (ROS) through mitochondrial activity. To limit the deleterious effects of excess ROS, certain gene promoters contain antioxidant response elements (ARE), e.g. the genes NQO-1 and HO-1. ROS induces conformational changes in KEAP1 and releases NRF2, which activates AREs. We show in vitro and in vivo that OXPHOS induces, both in primary leukemic cells and cell lines, de novo expression of NQO-1 and HO-1 and also the MAPK ERK5 and decreases KEAP1 mRNA. ERK5 activates the transcription factor MEF2, which binds to the promoter of the miR-23a-27a-24-2 cluster. Newly generated miR-23a destabilizes KEAP1 mRNA by binding to its 3'UTR. Lower KEAP1 levels increase the basal expression of the NRF2-dependent genes NQO-1 and HO-1. Hence, leukemic cells performing OXPHOS, independently of de novo ROS production, generate an antioxidant response to protect themselves from ROS.

microRNA deregulations are often, if not invariably, associated with human malignancies, including cancers. Though most of these deregulations may not be functionally implicated in tumorigenesis, the fact that microRNA expression can be monitored in a variety of human specimens, including biological fluids, supports studies aimed at characterizing microRNA signatures able to detect various cancers (diagnosis), predict their outcome (prognosis), monitor their treatment (theranosis), and adapt therapy to a patient (precision medicine). Here, we review and discuss pros and cons of microRNA-based approaches that can support their exploitation as cancer biomarkers.

Background: microRNAs (miRNAs) play crucial roles in major biological processes and their deregulations are often associated with human malignancies. As such, they represent appealing candidates as targets of innovative therapies. Another interesting aspect of their biology is that they are present in various biological fluids where, advantageously, they appear to be very stable. A plethora of studies have now reported their potential as biomarkers that can be used in diagnosis, prognosis and/or theranostic issues. However, the application of circulating miRNAs in clinical practices still requires the identification of highly efficient, robust and reproducible methods for their isolation from biological samples.In that context, we performed an independent cross-comparison of three commercially available RNA extraction kits for miRNAs isolation from human blood samples (Qiagen and Norgen kits as well as the new NucleoSpin miRNAs Plasma kit from Macherey-Nagel). miRNAs were further profiled using the Taqman Low Density Array technology., Results: We found that, although these 3 kits had equal performances in extracting miRNAs from peripheral blood mononuclear cells, the Macherey-Nagel kit presented several advantages when isolating miRNAs from sera. Besides, our results have indicated that, depending on the quantity of the biological samples used, the extraction procedure directly impacted on the G/C composition of the miRNAs detected., Conclusion: Overall, our study contributes to the definition of a reliable framework for profiling circulating miRNAs.

NK cell is an innate immune system lymphocyte lineage with natural cytotoxicity. Its optimal use in the clinic requires in vitro expansion and activation. Cytokines and encounter with target cells activate NK cells and induce proliferation, and this could depend on the presence of other immune cells. Here we activated PBMCs during 5 days with IL-2, with IL-2 plus the tumor cell line K562 and with the lymphoblastoid cell line R69 and perform integrated analyses of microRNA and mRNA expression profiles of purified NK cells. The samples cluster depending on the stimuli and not on the donor, indicating that the pattern of NK cell stimulation is acutely well conserved between individuals. Regulation of mRNA expression is tighter than that of miRNA expression. All stimuli induce a common preserved genetic remodeling. In addition, encounter with target cells mainly activates pathways related to metabolism. Different target cells induce different NK cell remodeling which affects cytokine response and cytotoxicity, supporting the notion that encounter with different target cells significantly changing the activation pattern. We validate our analysis by showing that activation down regulates miR-23a, which is a negative regulator of cathepsin C (CTSC) mRNA, a gene up regulated by all stimuli. The peptidase CTSC activates the granzymes, the main effector proteases involved in NK cell cytotoxicity. All-trans retinoic acid (ATRA), which induces miR-23a expression, decreases CTSC expression and granzyme B activity leading to impaired NK cell cytotoxicity in an in vivo mouse model., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

MicroRNAs orchestrate the expression of the genome and impact many, if not all, cellular processes. Their deregulation is thus often causative of human malignancies, including cancers. Numerous studies have implicated microRNAs in the different steps of tumorigenesis including initiation, progression, metastasis, and resistance to chemo/radiotherapies. Thus, microRNAs constitute appealing targets for novel anticancer therapeutic strategies aimed at restoring their expression or function. As microRNAs are present in a variety of human cancer types, microRNA profiles can be used as tumor-specific signatures to detect various cancers (diagnosis), to predict their outcome (prognosis), and to monitor their treatment (theranosis). In this review, we present the different aspects of microRNA biology that make them remarkable molecules in the emerging field of personalized medicine against cancers and provide several examples of their industrial exploitation.

Impact of female aging is an important issue in human reproduction. There was a need for an extensive analysis of age impact on transcriptome profile of cumulus cells (CCs) to link oocyte quality and developmental potential with patient's age. CCs from patients of three age groups were analyzed individually using microarrays. RT-qPCR validation was performed on independent CC cohorts. We focused here on pathways affected by aging in CCs that may explain the decline of oocyte quality with age. In CCs collected from patients >37 years, angiogenic genes including ANGPTL4, LEPR, TGFBR3, and FGF2 were significantly overexpressed compared to patients of the two younger groups. In contrast genes implicated in TGF-β signaling pathway such as AMH, TGFB1, inhibin, and activin receptor were underexpressed. CCs from patients whose ages are between 31 and 36 years showed an overexpression of genes related to insulin signaling pathway such as IGFBP3, PIK3R1, and IGFBP5. A bioinformatic analysis was performed to identify the microRNAs that are potential regulators of the differentially expressed genes of the study. It revealed that the pathways impacted by age were potential targets of specific miRNAs previously identified in our CCs small RNAs sequencing.

Study Question: What is the expression pattern of microRNAs (miRNAs) in human cumulus-oocyte complexes (COCs)?, Summary Answer: Several miRNAs are enriched in cumulus cells (CCs) or oocytes, and are predicted to target genes involved in biological functions of the COC., What Is Known Already: The transcriptional profiles of human MII oocytes and the surrounding CCs are known. However, very limited data are available about post-transcriptional regulators, such as miRNAs. This is the first study focussing on the identification and quantification of small RNAs, including miRNAs, in human oocytes and CCs using a deep-sequencing approach., Study Design, Size, Duration: MII oocytes and CCs were collected from women who underwent IVF., Participants/materials, Setting, Methods: Using the Illumina/deep-sequencing technology, we analyzed the small RNAome of pooled MII oocytes (n = 24) and CC samples (n = 20). The mRNA targets of CC and MII oocyte miRNAs were identified using in silico prediction algorithms. Using oligonucleotide microarrays, genome-wide gene expression was studied in oocytes (10 pools of 19 ± 3 oocytes/each) and 10 individual CC samples. TaqMan miRNA assays were used to confirm the sequencing results in independent pools of MII oocytes (3 pools of 8 ± 3 oocytes/each) and CC samples (3 pools of 7 ± 3 CCs/each). The functional role of one miRNA, MIR23a, was assessed in primary cultures of human CCs., Main Results and the Role of Chance: Deep sequencing of small RNAs yielded more than 1 million raw reads. By mapping reads with a single location to the human genome, known miRNAs that were abundant in MII oocytes (MIR184, MIR100 and MIR10A) or CCs (MIR29a, MIR30d, MIR21, MIR93, MIR320a, MIR125a and the LET7 family) were identified. Predicted target genes of the oocyte miRNAs were associated with the regulation of transcription and cell cycle, whereas genes targeted by CC miRNAs were involved in extracellular matrix and apoptosis. Comparison of the predicted miRNA target genes and mRNA microarray data resulted in a list of 224 target genes that were differentially expressed in MII oocytes and CCs, including PTGS2, CTGF and BMPR1B that are important for cumulus-oocyte communication. Functional analysis using primary CC cultures revealed that BCL2 and CYP19A1 mRNA levels were decreased upon MIR23a overexpression., Limitations, Reasons for Caution: Only known miRNAs were investigated in the present study on COCs. Moreover, the source of the material is MII oocytes that failed to fertilize., Wider Implications of the Findings: The present findings suggest that miRNA could play a role in the regulation of the oocyte and CC crosstalk., Study Funding/competing Interest(s): This work was partially supported by a grant from Ferring Pharmaceuticals. The authors of the study have no conflict of interest to report., Trial Registration Number: Not applicable.

In addition to estrogen receptor modulators, retinoic acid and other retinoids are promising agents to prevent breast cancer. Retinoic acid and estrogen exert antagonistic regulations on the transcription of coding genes and we evaluated here whether these two compounds have similar effects on microRNAs. Using an integrative approach based on several bioinformatics resources together with experimental validations, we indeed found that retinoic acid positively regulates miR-210 and miR-23a/24-2 expressions and is counteracted by estrogen. Conversely, estrogen increased miR-17/92 and miR-424/450b expressions and is inhibited by retinoic acid. In silico functional enrichment further revealed that this combination of transcriptional/post-transcriptional regulations fully impacts on the molecular effects of estrogen and retinoic acid. Besides, we unveiled a novel effect of retinoic acid on aerobic glycolysis. We specifically showed that it increases extracellular lactate production, an effect counteracted by the miR-210 and the miR-23a/24-2, which simultaneously target lactate dehydrogenase A and B mRNAs. Together our results provide a new framework to better understand the estrogen/retinoic acid antagonism in breast cancer cells.

Cancer cells have elevated aerobic glycolysis that is termed the Warburg effect. But several tumor cells, including leukemic cells, also increase glutamine metabolism, which is initiated by glutaminase (GLS). The microRNA (miRNA) miR-23 targets GLS mRNA and inhibits expression of GLS protein. Here we show that in human leukemic Jurkat cells the NF-κB p65 subunit binds to miR-23a promoter and inhibits miR-23a expression. Histone deacetylase (HDAC) inhibitors release p65-induced inhibition. Jurkat cells growing in glutamine decrease proliferation due to cell accumulation in G0/G1 phase. Nevertheless, cells get used to this new source of energy by increasing GLS expression, which correlates with an increase in p65 expression and its translocation to the nucleus, leading to a higher basal NF-κB activity. Jurkat cells overexpressing p65 show increase basal GLS expression and proliferate faster than control cells in glutamine medium. Overexpressing miR-23a in leukemic cells impaired glutamine use and induces mitochondrial dysfunction leading to cell death. Therefore, p65 activation decreases miR-23a expression, which facilitates glutamine consumption allowing leukemic cells to use this alternative source of carbon and favoring their adaptation to the metabolic environment., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

Cellular micro(mi)RNAs are able to recognize viral RNAs through imperfect micro-homologies. Similar to the miRNA-mediated repression of cellular translation, this recognition is thought to tether the RNAi machinery, in particular Argonaute 2 (AGO2) on viral messengers and eventually to modulate virus replication. Here, we unveil another pathway by which AGO2 can interact with retroviral mRNAs. We show that AGO2 interacts with the retroviral Group Specific Antigen (GAG) core proteins and preferentially binds unspliced RNAs through the RNA packaging sequences without affecting RNA stability or eliciting translation repression. Using RNAi experiments, we provide evidences that these interactions, observed with both the human immunodeficiency virus 1 (HIV-1) and the primate foamy virus 1 (PFV-1), are required for retroviral replication. Taken together, our results place AGO2 at the core of the retroviral life cycle and reveal original AGO2 functions that are not related to miRNAs and translation repression.

Suspicion towards technological advances has progressively grown during the xx(th) century. However, in the XXI(st) century, reading the NBIC (nanotechnology, biotechnology, information technology and cognitive science) report of the National Science Foundation, we can note that science has caught up with science fiction. These changes in public mentality on one side and in scientific capacities on the other argue for an evolution of the debate on sciences. The recent example of the national debate on nanotechnology in France has clearly shown that the public is no longer waiting for additional sources of scientific knowledge but rather waiting for the recognition of its authority to participate in the definition of the national R&D priority and associated scientific strategies. This is all the more legitimate that these strategies will have profound impact on the future of our societies and therefore cannot be decided only by scientists. Hence, it is crucial to identify innovative tools promoting debate on sciences and their technological spin-off. Here, we contend that science fiction has major assets that could face this challenge and facilitate the dialogue between sciences and society.

Epithelial to mesenchymal transition (EMT) is a key step toward metastasis. MCF7 breast cancer cells conditionally expressing the EMT master regulator SNAI1 were used to identify early expressed microRNAs (miRNAs) and their targets that may contribute to the EMT process. Potential targets of miRNAs were identified by matching lists of in silico predicted targets and of inversely expressed mRNAs. MiRNAs were ranked based on the number of predicted hits, highlighting miR-661, a miRNA with so far no reported role in EMT. MiR-661 was found required for efficient invasion of breast cancer cells by destabilizing two of its predicted mRNA targets, the cell-cell adhesion protein Nectin-1 and the lipid transferase StarD10, resulting, in turn, in the downregulation of epithelial markers. Reexpression of Nectin-1 or StarD10 lacking the 3'-untranslated region counteracted SNAI1-induced invasion. Importantly, analysis of public transcriptomic data from a cohort of 295 well-characterized breast tumor specimen revealed that expression of StarD10 is highly associated with markers of luminal subtypes whereas its loss negatively correlated with the EMT-related, basal-like subtype. Collectively, our non-a priori approach revealed a nonpredicted link between SNAI1-triggered EMT and the down-regulation of Nectin-1 and StarD10 through the up-regulation of miR-661, which may contribute to the invasion of breast cancer cells and poor disease outcome.

Metastasis and drug resistance are major problems in cancer chemotherapy. The purpose of this work was to analyze the molecular mechanisms underlying the invasive potential of drug-resistant colon carcinoma cells. Cellular models included the parental HT-29 cell line and its drug-resistant derivatives selected after chronic treatment with either 5-fluorouracil, methotrexate, doxorubicin, or oxaliplatin. Drug-resistant invasive cells were compared with noninvasive cells using cDNA microarray, quantitative reverse transcription-PCR, flow cytometry, immunoblots, and ELISA. Functional and cellular signaling analyses were undertaken using pharmacologic inhibitors, function-blocking antibodies, and silencing by retrovirus-mediated RNA interference. 5-Fluorouracil- and methotrexate-resistant HT-29 cells expressing an invasive phenotype in collagen type I and a metastatic behavior in immunodeficient mice exhibited high expression of the chemokine receptor CXCR4. Macrophage migration-inhibitory factor (MIF) was identified as the critical autocrine CXCR4 ligand promoting invasion in drug-resistant colon carcinoma HT-29 cells. Silencing of CXCR4 and impairing the MIF-CXCR4 signaling pathways by ISO-1, pAb FL-115, AMD-3100, monoclonal antibody 12G5, and BIM-46187 abolished this aggressive phenotype. Induction of CXCR4 was associated with the upregulation of two genes encoding transcription factors previously shown to control CXCR4 expression (HIF-2alpha and ASCL2) and maintenance of intestinal stem cells (ASCL2). Enhanced CXCR4 expression was detected in liver metastases resected from patients with colon cancer treated by the standard FOLFOX regimen. Combination therapies targeting the CXCR4-MIF axis could potentially counteract the emergence of the invasive metastatic behavior in clonal derivatives of drug-resistant colon cancer cells., (Copyright 2010 AACR.)

Micro(mi)RNAs are small noncoding RNAs that orchestrate many key aspects of cell physiology and their deregulation is often linked to distinct diseases including cancer. Here, we studied the contribution of miRNAs in a well-characterized human myeloid leukemia, acute promyelocytic leukemia (APL), targeted by retinoic acid and trioxide arsenic therapy. We identified several miRNAs transcriptionally repressed by the APL-associated PML-RAR oncogene which are released after treatment with all-trans retinoic acid. These coregulated miRNAs were found to control, in a coordinated manner, crucial pathways linked to leukemogenesis, such as HOX proteins and cell adhesion molecules whose expressions are thereby repressed by the chemotherapy. Thus, APL appears linked to transcriptional perturbation of miRNA genes, and clinical protocols able to successfully eradicate cancer cells may do so by restoring miRNA expression. The identification of abnormal miRNA biogenesis in cancer may therefore provide novel biomarkers and therapeutic targets in myeloid leukemias.

In eukaryotes, 21- to 24-nucleotide-long RNAs engage in sequence-specific interactions that inhibit gene expression by RNA silencing. This process has regulatory roles involving microRNAs and, in plants and insects, it also forms the basis of a defense mechanism directed by small interfering RNAs that derive from replicative or integrated viral genomes. We show that a cellular microRNA effectively restricts the accumulation of the retrovirus primate foamy virus type 1 (PFV-1) in human cells. PFV-1 also encodes a protein, Tas, that suppresses microRNA-directed functions in mammalian cells and displays cross-kingdom antisilencing activities. Therefore, through fortuitous recognition of foreign nucleic acids, cellular microRNAs have direct antiviral effects in addition to their regulatory functions.

We report that human T cells persistently infected with primate foamy virus type 1 (PFV-1) display an increased capacity to bind human immunodeficiency virus type 1 (HIV-1), resulting in increased cell permissiveness to HIV-1 infection and enhanced cell-to-cell virus transmission. This phenomenon is independent of HIV-1 receptor, CD4, and it is not related to PFV-1 Bet protein expression. Increased virus attachment is specifically inhibited by heparin, indicating that it should be mediated by interactions with heparan sulfate glycosaminoglycans expressed on the target cells. Given that both viruses infect similar animal species, the issue of whether coinfection with primate foamy viruses interferes with the natural course of lentivirus infections in nonhuman primates should be considered.

In plants, small interfering RNAs (siRNAs) and microRNAs (miRNAs) are effectors of RNA silencing, a process involved in defense through RNA interference (RNAi) and in development. Plant viruses are natural targets of RNA silencing, and as a counterdefensive strategy, they have evolved highly diverse silencing suppressor proteins. Although viral suppressors are usually thought to act at distinct steps of the silencing machinery, there had been no consensus system so far that allowed a strict side-by-side analysis of those factors. We have set up such a system in Arabidopsis thaliana and used it to compare the effects of five unrelated viral silencing suppressors on the siRNA and miRNA pathways. Although all the suppressors inhibited RNAi, only three of them induced developmental defects, indicating that the two pathways are only partially overlapping. These developmental defects were remarkably similar, and their penetrance correlated with inhibition of miRNA-guided cleavage of endogenous transcripts and not with altered miRNA accumulation per se. Among the suppressors investigated, the tombusviral P19 protein coimmunoprecipitated with siRNA duplexes and miRNA duplexes corresponding to the primary cleavage products of miRNA precursors. Thus, it is likely that P19 prevents RNA silencing by sequestering both classes of small RNAs. Moreover, the finding here that P19 binds siRNAs and suppresses RNAi in Hela cells also suggests that this factor may be useful to dissect the RNA silencing pathways in animals. Finally, the differential effects of the silencing suppressors tested here upon other types of Arabidopsis silencing-related small RNAs revealed a surprising variety of biosynthetic and, presumably, functional pathways for those molecules. Therefore, silencing suppressors are valuable probes of the complexity of RNA silencing.

'RNA silencing' is a highly conserved mechanism leading to suppression of gene expression through nucleotide sequence-specific interactions that are mediated by 21-24 nucleotide-long RNAs. This process was first discovered as an unexpected consequence of transgenesis in plants, and similarly, it was subsequently identified in animals as an anomaly of antisense gene inhibition. We summarize the progressive steps that paved the way to our current understanding of the molecular basis and fundamental biological roles of RNA silencing in both plants and animals. In particular, we describe the general antiviral function of this mechanism in higher plants where it forms the basis of a highly elaborate immune system. All defense systems show some level of fallibility, and RNA silencing is no exception to this rule, as plant viruses have developed sophisticated ways to counteract various steps of the process. Recent work indicates that viruses are also engaged into a similar arms race in insects, but it remains unclear if RNA silencing plays a defensive role against virus infection of higher vertebrates. We also discuss some biotechnological applications of RNA silencing in mammalian cells that have fueled optimism that this mechanism may hold a promising future in antiviral human therapy.

Foamy viruses (FVs) are nonpathogenic, widely spread complex retroviruses which have been isolated in nonhuman primates, cattle, cats, and more recently in horses. The equine foamy virus (EFV) was isolated from healthy horses and was characterized by molecular cloning and nucleotide sequence analysis. Here, to further characterize this new FV isolate, the location of the transcriptional cap and poly(A) addition sites as well as the main splice donor and acceptor sites were determined, demonstrating the existence of the specific subgenomic pol mRNA, one specific feature of FVs. Moreover, similar to what has been described for the human foamy virus (HFV), the prototype of FVs, a replication-defective EFV genome was identified during persistent infection. At the protein level, the use of specific antibodies allowed us to determine the size and the subcellular localization of EFV Gag, Env, and Tas, the viral transactivators. While EFV Gag was detected in both the cytoplasm and the nucleus, EFV Env mainly localized in the Golgi complex, in contrast to HFV Env, which is sequestered in the endoplasmic reticulum. In addition, electron microscopy analysis demonstrated that EFV budding occurs at the plasma membrane and not intracellularly, as is the case for primate FVs. Interestingly, EFV Tas was detected both in the nucleus and the cytoplasm of Tas-transfected cells, in contrast to the strict nuclear localization of other FV Tas but similar to the equine infectious anemia virus Tat gene product. Taken together, our results reveal that this new FV isolate exhibits remarkable features among FVs, bringing new insights into the biology of these unconventional retroviruses.

The Bet protein of foamy viruses (FVs) is an auxiliary protein encoded by the 3' end of the viral genome. Although its function during the viral replication cycle is still unknown, Bet seems to play a key role in the establishment and/or maintenance of viral persistence, representing the predominant viral protein detected during chronic infection. To clarify the function of this viral protein, the subcellular distribution of Bet from the prototypic human foamy virus (HFV) was examined. We report here that this protein is distributed in both the cytoplasm and the nucleus of HFV-infected or Bet-transfected cells. The nuclear targeting results from the presence of a bipartite nuclear localization signal at the C-terminal region, sufficient to direct heterologous reporter proteins to the nucleus. Since HFV Bet spreads between cells, we show here that the secreted protein targets the nuclei of recipient cells. HFV Bet follows an unconventional route to exit the cell since its secretion is not affected by brefeldin A, a drug which disrupts the trafficking between the endoplasmic reticulum and the Golgi complex. Finally, these inter- and intracellular movements were also observed for the equine foamy virus Bet protein, strongly suggesting that these remarkable features are conserved among FVs.

Foamy viruses (FVs) are complex retroviruses which have been isolated from different animal species including nonhuman primates, cattle, and cats. Here, we report the isolation and characterization of a new FV isolated from blood samples of horses. Similar to other FVs, the equine foamy virus (EFV) exhibits a highly characteristic ultrastructure and induces syncytium formation and subsequent cell lysis on a large number of cell lines. Molecular cloning of EFV reveals that the general organization is that of other known FVs, whereas sequence similarity with its bovine FV counterpart is only 40%. Interestingly, EFV buds exclusively from the plasma membrane and not from the endoplasmic reticulum (ER), as previously shown for other FVs. The absence of the ER retrieval dilysine motif in EFV Env is likely responsible for this unexpected sorting pathway.