Expertise

La plateforme de génomique fonctionnelle de Nice Sophia Antipolis existe depuis 1999. Initialement orientée vers la conception, la fabrication et l'analyse de puces à ADN, elle a contribué à ouvrir cette nouvelle technologie à une large communauté, mettant à cette occasion en place un système d'information performant (Mediante), capable de gérer de grandes masses de données, et fonctionnant en production depuis plus de 10 ans.

Tout en fournissant encore aujourd'hui un service d'analyse de puces à ADN s'appuyant sur la technologie développée par Agilent, son activité s'est principalement réorientée vers des services de séquencage à haut-débit (Illumina NextSeq500), offrant dans ce contexte de nombreux types d'analyses des acides nucléiques, et une capacité pour analyser de grandes collections d'échantillons, y compris au niveau de la cellule unique. L'activité de routine concerne des applications comme le RNA-seq, le smallRNA-seq, le CHiP-seq, le CLIP-seq, le reséquencage, mais des projets spécifiques peuvent aussi etre mis en place dans des domaines moins standards, comme le séquencage de novo de génomes, ou certains protocoles particuliers : riboSeq, capSeq,... La plateforme se compose de 4 ingénieurs wet lab et de 4 bio-informaticiens.

Equipements

1. Pré-séquencage : Nanodrop, Bioanalyzer, Qubit, CovarisS2, Ion Chef, NeoPrep, Blue pippin
2. Analyse Single Cell : 10x Genomics Chromium, Fluidigm C1, Fluidigm Biomark
3. Séquencage : NextSeq500 Illumina, MinION et PromethION Oxford Nanopore Technology, Chromium 10X Genomics
4. Puces à ADN : High-Resolution Microarray Scanner Agilent, Station Affymetrix

Les résultats sont stockés automatiquement sur le portail d'informations de la plateforme Mediante. Cela concerne notamment les fichiers .BAM d'alignement, les fichiers .BW de couverture et l'ensemble des fichiers de l'analyse secondaire et des analyses statistiques conduites en partenariat avec le collaborateur. Sur demande l'ensemble des données brutes sont également mises à disposition et une aide est fournit pour la soumission des données vers la base de données publiques GEO (Gene Expression Omnibus).

Related publications

Multiciliated cells (MCCs), which are present in specialized vertebrate tissues such as mucociliary epithelia, project hundreds of motile cilia from their apical membrane. Coordinated ciliary beating in MCCs contributes to fluid propulsion in several biological processes. In a previous work, we demonstrated that microRNAs of the miR-34/449 family act as new conserved regulators of MCC differentiation by specifically repressing cell cycle genes and the Notch pathway. Recently, we have shown that miR-34/449 also modulate small GTPase pathways to promote, in a later stage of differentiation, the assembly of the apical actin network, a prerequisite for proper anchoring of centrioles-derived neo-synthesized basal bodies. We characterized several miR-34/449 targets related to small GTPase pathways including R-Ras, which represents a key and conserved regulator during MCC differentiation. Direct RRAS repression by miR-34/449 is necessary for apical actin meshwork assembly, notably by allowing the apical relocalization of the actin binding protein Filamin-A near basal bodies. Our studies establish miR-34/449 as central players that orchestrate several steps of MCC differentiation program by regulating distinct signaling pathways.

The resistance of hypoxic cells to radiotherapy and chemotherapy is a major problem in the treatment of cancer. Recently, an additional mode of hypoxia-inducible factor (HIF)-dependent transcriptional regulation, involving modulation of a specific set of micro RNAs (miRNAs), including miR-210, has emerged. We have recently shown that HIF-1 induction of miR-210 also stabilizes HIF-1 through a positive regulatory loop. Therefore, we hypothesized that by stabilizing HIF-1 in normoxia, miR-210 may protect cancer cells from radiation. We developed a non-small cell lung carcinoma (NSCLC)-derived cell line (A549) stably expressing miR-210 (pmiR-210) or a control miRNA (pmiR-Ctl). The miR-210-expressing cells showed a significant stabilization of HIF-1 associated with mitochondrial defects and a glycolytic phenotype. Cells were subjected to radiation levels ranging from 0 to 10 Gy in normoxia and hypoxia. Cells expressing miR-210 in normoxia had the same level of radioresistance as control cells in hypoxia. Under hypoxia, pmiR-210 cells showed a low mortality rate owing to a decrease in apoptosis, with an ability to grow even at 10 Gy. This miR-210 phenotype was reproduced in another NSCLC cell line (H1975) and in HeLa cells. We have established that radioresistance was independent of p53 and cell cycle status. In addition, we have shown that genomic double-strand breaks (DSBs) foci disappear faster in pmiR-210 than in pmiR-Ctl cells, suggesting that miR-210 expression promotes a more efficient DSB repair. Finally, HIF-1 invalidation in pmiR-210 cells removed the radioresistant phenotype, showing that this mechanism is dependent on HIF-1. In conclusion, miR-210 appears to be a component of the radioresistance of hypoxic cancer cells. Given the high stability of most miRNAs, this advantage could be used by tumor cells in conditions where reoxygenation has occurred and suggests that strategies targeting miR-210 could enhance tumor radiosensitization.

Abstract Disruption of contact inhibition and serum afflux that occur after a tissue injury activate cell cycle, which then stops when confluence is reached again. Although the events involved in cell cycle entry have been widely documented, those managing cell cycle exit have remained so far ill defined. We have identified that the final stage of wound closure is preceded in keratinocytes by a strong accumulation of miR-483-3p, which acts as a mandatory signal triggering cell cycle arrest when confluence is reached. Blocking miR-483-3p accumulation strongly delays cell cycle exit, maintains cells into a proliferative state and retards their differentiation program. Using two models of cell cycle synchronization (i.e. mechanical injury and serum addition), we show that an ectopic upregulation of miR-483-3p blocks cell cycle progression in early G1 phase. This arrest results from a direct targeting of the CDC25A phosphatase by miR-483-3p, which can be impeded using an anti-miRNA against miR-483-3p or a protector that blocks the complex formation between miR-483-3p and the 3'-untranslated region (UTR) of CDC25A transcript. We show that the miRNA-induced silencing of CDC25A increases the tyrosine phosphorylation status of CDK4/6 cyclin-dependent kinases which, in turn, abolishes CDK4/6 capacity to associate with D-type cyclins. This prevents CDK4/6 kinases' activation, impairs downstream events such as cyclin E stimulation and sequesters cells in early G1. We propose this new regulatory process of cyclin-CDK association as a general mechanism coupling miRNA-mediated CDC25A invalidation to CDK post-transcriptional modifications and cell cycle control.

As miRNAs are associated with normal cellular processes, deregulation of miRNAs is thought to play a causative role in many complex diseases. Nevertheless, the precise contribution of miRNAs in fibrotic lung diseases, especially the idiopathic form (IPF), remains poorly understood. Given the poor response rate of IPF patients to current therapy, new insights into the pathogenic mechanisms controlling lung fibroblasts activation, the key cell type driving the fibrogenic process, are essential to develop new therapeutic strategies for this devastating disease. To identify miRNAs with potential roles in lung fibrogenesis, we performed a genome-wide assessment of miRNA expression in lungs from two different mouse strains known for their distinct susceptibility to develop lung fibrosis after bleomycin exposure. This led to the identification of miR-199a-5p as the best miRNA candidate associated with bleomycin response. Importantly, miR-199a-5p pulmonary expression was also significantly increased in IPF patients (94 IPF versus 83 controls). In particular, levels of miR-199a-5p were selectively increased in myofibroblasts from injured mouse lungs and fibroblastic foci, a histologic feature associated with IPF. Therefore, miR-199a-5p profibrotic effects were further investigated in cultured lung fibroblasts: miR-199a-5p expression was induced upon TGFβ exposure, and ectopic expression of miR-199a-5p was sufficient to promote the pathogenic activation of pulmonary fibroblasts including proliferation, migration, invasion, and differentiation into myofibroblasts. In addition, we demonstrated that miR-199a-5p is a key effector of TGFβ signaling in lung fibroblasts by regulating CAV1, a critical mediator of pulmonary fibrosis. Remarkably, aberrant expression of miR-199a-5p was also found in unilateral ureteral obstruction mouse model of kidney fibrosis, as well as in both bile duct ligation and CCl4-induced mouse models of liver fibrosis, suggesting that dysregulation of miR-199a-5p represents a general mechanism contributing to the fibrotic process. MiR-199a-5p thus behaves as a major regulator of tissue fibrosis with therapeutic potency to treat fibroproliferative diseases.

While FMR1 is silenced in Fragile X syndrome (FXS) patients carrying the full mutation, its expression is elevated (2-8 fold) in premutated individuals. These people may develop the Fragile X-associated Tremor/Ataxia syndrome (FXTAS), a late onset neurodegenerative disorder characterized by ataxia and parkinsonism. In addition, people carrying the premutation can be affected by a set of neurological and behavioral disorders during young age. Problems of memory have been detected in these patients as well as in the mouse models for FXTAS. To date little is known concerning the metabolism of FMR1 mRNA, notwithstanding the importance of the finely tuned regulation of the expression of this gene. In the present study, we identified three microRNAs that specifically target the 3' UTR of FMR1 and can modulate its expression throughout the brain particularly at the synapse where their expression is very high. The expression level of miR-221 is reduced in synaptosomal preparations of young FXTAS mice suggesting a general deregulation of transcripts located at the synapse of these mice. By transcriptome analysis, we show here a robust deregulation of the expression levels of genes involved in learning, memory and autistic behavior, Parkinson disease and neurodegeneration. These findings suggest the presence of a synaptopathy in these animals. Interestingly, many of those deregulated mRNAs are target of the same microRNAs that modulate the expression of FMR1 at the synapse.

Hypoxia in the tumor microenvironment plays a central role in the evolution of immune escape mechanisms by tumor cells. In this study, we report the definition of miR-210 as a miRNA regulated by hypoxia in lung cancer and melanoma, documenting its involvement in blunting the susceptibility of tumor cells to lysis by antigen-specific cytotoxic T lymphocytes (CTL). miR-210 was induced in hypoxic zones of human tumor tissues. Its attenuation in hypoxic cells significantly restored susceptibility to autologous CTL-mediated lysis, independent of tumor cell recognition and CTL reactivity. A comprehensive approach using transcriptome analysis, argonaute protein immunoprecipitation, and luciferase reporter assay revealed that the genes PTPN1, HOXA1, and TP53I11 were miR-210 target genes regulated in hypoxic cells. In support of their primary importance in mediating the immunosuppressive effects of miR-210, coordinate silencing of PTPN1, HOXA1, and TP53I11 dramatically decreased tumor cell susceptibility to CTL-mediated lysis. Our findings show how miR-210 induction links hypoxia to immune escape from CTL-mediated lysis, by providing a mechanistic understanding of how this miRNA mediates immunosuppression in oxygen-deprived regions of tumors where cancer stem-like cells and metastatic cellular behaviors are known to evolve.

Specificity of interaction between a microRNA (miRNA) and its targets crucially depends on the seed region located in its 5'-end. It is often implicitly considered that two miRNAs sharing the same biological activity should display similarity beyond the strict six nucleotide region that forms the seed, in order to form specific complexes with the same mRNA targets. We have found that expression of hsa-miR-147b and hsa-miR-210, though triggered by different stimuli (i.e. lipopolysaccharides and hypoxia, respectively), induce very similar cellular effects in term of proliferation, migration and apoptosis. Hsa-miR-147b only shares a "minimal" 6-nucleotides seed sequence with hsa-miR-210, but is identical with hsa-miR-147a over 20 nucleotides, except for one base located in the seed region. Phenotypic changes induced after heterologous expression of miR-147a strikingly differ from those induced by miR-147b or miR-210. In particular, miR-147a behaves as a potent inhibitor of cell proliferation and migration. These data fit well with the gene expression profiles observed for miR-147b and miR-210, which are very similar, and the gene expression profile of miR-147a, which is distinct from the two others. Bioinformatics analysis of all human miRNA sequences indicates multiple cases of miRNAs from distinct families exhibiting the same kind of similarity that would need to be further characterized in terms of putative functional redundancy. Besides, it implies that functional impact of some miRNAs can be masked by robust expression of miRNAs belonging to distinct families.

In contrast to expectations in the past that tumor starvation or unselective inhibition of proteolytic activity would cure cancer, there is accumulating evidence that microenvironmental stress, such as hypoxia or broad-spectrum inhibition of metalloproteinases can promote metastasis. In fact, malignant tumor cells, due to their genetic and epigenetic instability, are predisposed to react to stress by adaptation and, if the stress persists, by escape and formation of metastasis. Recent recognition of the concepts of dynamic evolution as well as population and systems biology is extremely helpful to understand the disappointments of clinical trials with new drugs and may lead to paradigm-shifts in therapy strategies. This must be complemented by an increased understanding of molecular mechanism involved in stress response. Here, we review new roles of Hypoxia-inducible factor-1 (HIF-1), one transcription factor regulating stress response-related gene expression: HIF-1 is crucial for invasion and metastasis, independent from its pro-survival function. In addition, HIF-1 mediates pro-metastatic microenvironmental changes of the proteolytic balance as triggered by high systemic levels of tissue inhibitor of metalloproteinases-1 (TIMP-1), typical for many aggressive cancers, and regulates the metabolic switch to glycolysis, notably via activation of the microRNA miR-210. There is preliminary evidence that TIMP-1 also induces miR-210. Such positive-regulatory co-operation of HIF-1α, miR-210, and TIMP-1, all described to correlate with bad prognosis of cancer patients, opens new perspectives of gaining insight into molecular mechanisms of metastasis-inducing evasion of tumor cells from stress.

In the postnatal and adult mouse forebrain, a mosaic of spatially separated neural stem cells along the lateral wall of the ventricles generates defined types of olfactory bulb neurons. To understand the mechanisms underlying the regionalization of the stem cell pool, we focused on the transcription factor Pax6, a determinant of the dopaminergic phenotype in this system. We found that, although Pax6 mRNA was transcribed widely along the ventricular walls, Pax6 protein was restricted to the dorsal aspect. This dorsal restriction was a result of inhibition of protein expression by miR-7a, a microRNA (miRNA) that was expressed in a gradient opposing Pax6. In vivo inhibition of miR-7a in Pax6-negative regions of the lateral wall induced Pax6 protein expression and increased dopaminergic neurons in the olfactory bulb. These findings establish miRNA-mediated fine-tuning of protein expression as a mechanism for controlling neuronal stem cell diversity and, consequently, neuronal phenotype.

B-cell acute lymphoblastic leukemia (B-ALL) is often associated with chromosomal translocations leading to the deregulation of proto-oncogenes. MicroRNAs can also be affected by chromosomal alterations and thus contribute to carcinogenesis. The microRNA miR-125b-1 is over-expressed in B-ALL cases with the t(11;14)(q24;q34) translocation, therefore we sought to determine the role of this microRNA in B-cell fate. We used murine pre-BI cells alongside murine and human leukemic B-cell lines to show that miR-125b expression enhances proliferation by targeting Bright/ARID3a, an activator of immunoglobulin heavy-chain transcription. Accordingly, this target gene was down-regulated in B-ALL patients with the t(11;14)(q24;q34) translocation. Repression of Bright/ARID3A blocked differentiation and conferred a survival advantage to Ba/F3 cells under IL3 starvation. In addition, over-expression of miR-125b protected pre-BI and leukemic B-cell lines from apoptosis through blockade of caspase activation via a mechanism that was independent of p53 and BAK1. In summary, miR-125b can act as an oncogene in B-ALL by targeting ARID3a and mediating its repression, thus leading to a blockage in differentiation, increased proliferation and inhibition of apoptosis.Leukemia accepted article preview online, 3 April 2012; doi:10.1038/leu.2012.95.

The molecular mechanism responsible for the antitumor activity of histone deacetylase inhibitors (HDACi) remains elusive. As HDACi have been described to alter miRNA expression, the aim of this study was to characterize HDACi-induced miRNAs and to determine their functional importance in the induction of cell death alone or in combination with other cancer drugs. Two HDACi, trichostatin A and vorinostat, induced miR-129-5p overexpression, histone acetylation and cell death in BCPAP, TPC-1, 8505C, and CAL62 cell lines and in primary cultures of papillary thyroid cancer (PTC) cells. In addition, miR-129-5p alone was sufficient to induce cell death and knockdown experiments showed that expression of this miRNA was required for HDACi-induced cell death. Moreover, miR-129-5p accentuated the anti-proliferative effects of other cancer drugs such as etoposide or human α-lactalbumin made lethal for tumor cells (HAMLET). Taken together, our data show that miR-129-5p is involved in the antitumor activity of HDACi and highlight a miRNA-driven cell death mechanism.

The term 'thyroid tumors of uncertain malignant potential' (TT-UMP) was coined by surgical pathologists to define well-differentiated tumors (WDT) showing inconclusive morphological evidence of malignancy or benignity. We have analyzed the expression of microRNA (miRNA) in a training set of 42 WDT of different histological subtypes: seven follicular tumors of UMP (FT-UMP), six WDT-UMP, seven follicular thyroid adenomas (FTA), 11 conventional papillary thyroid carcinomas (C-PTC), five follicular variants of PTC (FV-PTC), and six follicular thyroid carcinomas (FTC), which led to the identification of about 40 deregulated miRNAs. A subset of these altered miRNAs was independently validated by qRT-PCR, which included 18 supplementary TT-UMP (eight WDT-UMP and ten FT-UMP). Supervised clustering techniques were used to predict the first 42 samples. Based on the four possible outcomes (FTA, C-PTC, FV-PTC, and FTC), about 80% of FTA and C-PTC and 50% of FV-PTC and FTC samples were correctly assigned. Analysis of the independent set of 18 WDT-UMP by quantitative RT-PCR for the selection of the six most discriminating miRNAs was unable to separate FT-UMP from WDT-UMP, suggesting that the miRNA signature is insufficient in characterizing these two clinical entities. We conclude that considering FT-UMP and WDT-UMP as distinct and specific clinical entities may improve the diagnosis of WDT of the thyroid gland. In this context, a small set of miRNAs (i.e. miR-7, miR-146a, miR-146b, miR-200b, miR-221, and miR-222) appears to be useful, though not sufficient per se, in distinguishing TT-UMP from other WDT of the thyroid gland.

The mechanisms that regulate keratinocyte migration and proliferation in wound healing remain largely unraveled, notably regarding possible involvements of microRNAs (miRNAs). Here we disclose up-regulation of miR-483-3p in 2 distinct models of wound healing: scratch-injured cultures of human keratinocytes and wounded skin in mice. miR-483-3p accumulation peaks at the final stage of the wound closure process, consistent with a role in the arrest of "healing" progression. Using an in vitro wound-healing model, videomicroscopy, and 5-bromo-2'-uridine incorporation, we observed that overexpression of miR-483-3p inhibits keratinocyte migration and proliferation, whereas delivery of anti-miR-483-3p oligonucleotides sustains keratinocyte proliferation beyond the closure of the wound, compared with irrelevant anti-miR treatment. Expression profiling of keratinocytes transfected with miR-483-3p identified 39 transcripts that were both predicted targets of miR-483-3p and down-regulated after miR-483-3p overexpression. Luciferase reporter assays, Western blot analyses, and silencing by specific siRNAs finally established that kinase MK2, cell proliferation marker MKI67, and transcription factor YAP1 are direct targets of miR-483-3p that control keratinocyte proliferation. miR-483-3p-mediated down-regulation of MK2, MKI67, and YAP1 thus represents a novel mechanism controlling keratinocyte growth arrest at the final steps of reepithelialization.

Multiciliated cells lining the surface of some vertebrate epithelia are essential for various physiological processes, such as airway cleansing. Their apical surface is constituted by hundreds of motile cilia, which beat in a coordinated manner to generate directional fluid flow. We recently reported the identification of microRNAs of the miR-449 family as evolutionary conserved key regulators of vertebrate multiciliogenesis. This novel function of miR-449 was established using in vivo and in vitro antisense approaches in two distinct experimental models. miR-449 strongly accumulated in multiciliated cells in human airway epithelium and Xenopus laevis embryonic epidermis, where it triggered centriole multiplication and multiciliogenesis by directly repressing the Delta/Notch pathway. Our data complement previous reports that showed the blocking action of miR-449 on the cell cycle, and unraveled a novel conserved mechanism whereby Notch signaling must undergo microRNA-mediated inhibition to permit differentiation of ciliated cell progenitors. We review here several important questions regarding the links between microRNAs and the Notch pathway in the control of cell fate.

Article in French

BACKGROUND: In severe obesity, as well as in normal development, the growth of adipose tissue is the result of an increase in adipocyte size and numbers, which is underlain by the stimulation of adipogenic differentiation of precursor cells. A better knowledge of the pathways that regulate adipogenesis is therefore essential for an improved understanding of adipose tissue expansion. As microRNAs (miRNAs) have a critical role in many differentiation processes, our study aimed to identify the role of miRNA-mediated gene silencing in the regulation of adipogenic differentiation. RESULTS: We used deep sequencing to identify small RNAs that are differentially expressed during adipogenesis of adipose tissue-derived stem cells. This approach revealed the un-annotated miR-642a-3p as a highly adipocyte-specific miRNA. We then focused our study on the miR-30 family, which was also up-regulated during adipogenic differentiation and for which the role in adipogenesis had not yet been elucidated. Inhibition of the miR-30 family blocked adipogenesis, whilst over-expression of miR-30a and miR-30d stimulated this process. We additionally showed that both miR-30a and miR-30d target the transcription factor RUNX2, and stimulate adipogenesis via the modulation of this major regulator of osteogenesis. CONCLUSIONS: Overall, our data suggest that the miR-30 family plays a central role in adipocyte development. Moreover, as adipose tissue-derived stem cells can differentiate into either adipocytes or osteoblasts, the down-regulation of the osteogenesis regulator RUNX2 represents a plausible mechanism by which miR-30 miRNAs may contribute to adipogenic differentiation of adipose tissue-derived stem cells.

Multiciliated cells lining the surface of some vertebrate epithelia are essential for various physiological processes, such as airway cleansing. However, the mechanisms governing motile cilia biosynthesis remain poorly elucidated. We identify miR-449 microRNAs as evolutionarily conserved key regulators of vertebrate multiciliogenesis. In human airway epithelium and Xenopus laevis embryonic epidermis, miR-449 microRNAs strongly accumulated in multiciliated cells. In both models, we show that miR-449 microRNAs promote centriole multiplication and multiciliogenesis by directly repressing the Delta/Notch pathway. We established Notch1 and its ligand Delta-like 1(DLL1) as miR-449 bona fide targets. Human DLL1 and NOTCH1 protein levels were lower in multiciliated cells than in surrounding cells, decreased after miR-449 overexpression and increased after miR-449 inhibition. In frog, miR-449 silencing led to increased Dll1 expression. Consistently, overexpression of Dll1 mRNA lacking miR-449 target sites repressed multiciliogenesis, whereas both Dll1 and Notch1 knockdown rescued multiciliogenesis in miR-449-deficient cells. Antisense-mediated protection of miR-449-binding sites of endogenous human Notch1 or frog Dll1 strongly repressed multiciliogenesis. Our results unravel a conserved mechanism whereby Notch signalling must undergo miR-449-mediated inhibition to permit differentiation of ciliated cell progenitors.

Extensive sequencing efforts, combined with ad hoc bioinformatics developments, have now led to the identification of 1222 distinct miRNAs in human (derived from 1368 distinct genomic loci) and of many miRNAs in other multicellular organisms. The present chapter is aimed at describing a general experimental strategy to identify specific miRNA expression profiles and to highlight the functional networks operating between them and their mRNA targets, including several miRNAs deregulated in cystic fibrosis and during differentiation of airway epithelial cells.

Susceptibility to Crohn's disease, a complex inflammatory disease, is influenced by common variants at many loci. The common exonic synonymous SNP (c.313C>T) in IRGM, found in strong linkage disequilibrium with a deletion polymorphism, has been classified as non-causative because of the absence of an alteration in the IRGM protein sequence or splice sites. Here we show that a family of microRNAs (miRNAs), miR-196, is overexpressed in the inflammatory intestinal epithelia of individuals with Crohn's disease and downregulates the IRGM protective variant (c.313C) but not the risk-associated allele (c.313T). Subsequent loss of tight regulation of IRGM expression compromises control of intracellular replication of Crohn's disease-associated adherent invasive Escherichia coli by autophagy. These results suggest that the association of IRGM with Crohn's disease arises from a miRNA-based alteration in IRGM regulation that affects the efficacy of autophagy, thereby implicating a synonymous polymorphism as a likely causal variant.

SUMMARY: Current challenges in microRNA (miRNA) research are to improve the identification of in vivo mRNA targets and clarify the complex interplay existing between a specific miRNA and multiple biological networks. MiRonTop is an online java web tool that integrates DNA microarrays or high-throughput sequencing data to identify the potential implication of miRNAs on a specific biological system. It allows a rapid characterization of the most pertinent mRNA targets according to several existing miRNA target prediction approaches. It also provides useful representations of the enrichment scores according to the position of the target site along the 3'- UTR, where the contribution of the sites located in the vicinity of the stop codon and of the polyA tail can be clearly highlighted. It provides different graphs of miRNA enrichment associated with up- or down-regulated transcripts and different summary tables about selections of mRNA targets and their functional annotations by Gene Ontology. AVAILABILITY: http://www.microarray.fr:8080/miRonTop/index

Following the identification of a set of hypoxia-regulated microRNAs (miRNAs), recent studies have highlighted the importance of miR-210 and of its transcriptional regulation by the transcription factor hypoxia-inducible factor-1 (HIF-1). We report here that miR-210 is overexpressed at late stages of non-small cell lung cancer. Expression of miR-210 in lung adenocarcinoma A549 cells caused an alteration of cell viability associated with induction of caspase-3/7 activity. miR-210 induced a loss of mitochondrial membrane potential and the apparition of an aberrant mitochondrial phenotype. The expression profiling of cells overexpressing miR-210 revealed a specific signature characterized by enrichment for transcripts related to 'cell death' and 'mitochondrial dysfunction', including several subunits of the electron transport chain (ETC) complexes I and II. The transcript coding for one of these ETC components, SDHD, subunit D of succinate dehydrogenase complex (SDH), was validated as a bona fide miR-210 target. Moreover, SDHD knockdown mimicked miR-210-mediated mitochondrial alterations. Finally, miR-210-dependent targeting of SDHD was able to activate HIF-1, in line with previous studies linking loss-of-function SDH mutations to HIF-1 activation. miR-210 can thus regulate mitochondrial function by targeting key ETC component genes with important consequences on cell metabolism, survival and modulation of HIF-1 activity. These observations help explain contradictory data regarding miR-210 expression and its putative function in solid tumors.

BACKGROUND: Epithelial-mesenchymal interactions are critical in regulating many aspects of vertebrate embryo development, and for the maintenance of homeostatic equilibrium in adult tissues. The interactions between epithelium and mesenchyme are believed to be mediated by paracrine signals such as cytokines and extracellular matrix components secreted from fibroblasts that affect adjacent epithelia. In this study, we sought to identify the repertoire of microRNAs (miRNAs) in normal lung human fibroblasts and their potential regulation by the cytokines TNF-alpha, IL-1beta and TGF-beta. METHODOLOGY/PRINCIPAL FINDINGS: MiR-155 was significantly induced by inflammatory cytokines TNF-alpha and IL-1beta while it was down-regulated by TGF-beta. Ectopic expression of miR-155 in human fibroblasts induced modulation of a large set of genes related to "cell to cell signalling", "cell morphology" and "cellular movement". This was consistent with an induction of caspase-3 activity and with an increase in cell migration in fibroblasts tranfected with miR-155. Using different miRNA bioinformatic target prediction tools, we found a specific enrichment for miR-155 predicted targets among the population of down-regulated transcripts. Among fibroblast-selective targets, one interesting hit was keratinocyte growth factor (KGF, FGF-7), a member of the fibroblast growth factor (FGF) family, which owns two potential binding sites for miR-155 in its 3'-UTR. Luciferase assays experimentally validated that miR-155 can efficiently target KGF 3'-UTR. Site-directed mutagenesis revealed that only one out of the 2 potential sites was truly functional. Functional in vitro assays experimentally validated that miR-155 can efficiently target KGF 3'-UTR. Furthermore, in vivo experiments using a mouse model of lung fibrosis showed that miR-155 expression level was correlated with the degree of lung fibrosis. CONCLUSIONS/SIGNIFICANCE: Our results strongly suggest a physiological function of miR-155 in lung fibroblasts. Altogether, this study implicates this miRNA in the regulation by mesenchymal cells of surrounding lung epithelium, making it a potential key player during tissue injury.

MicroRNAs (miRNAs) are small non-protein-coding RNA that negatively control mRNA expression at a post-transcriptional level. They regulate various cellular functions and bioinformatic data suggest that they collectively control about 30% of human mRNAs. MiRNAs have been recently implicated in several carcinogenic processes, where they can act either as oncogenes or as tumor suppressors. This is the case in lung cancer, i.e. the leading cause of cancer deaths in Western countries, in which about 40-45 miRNAs have been found to be aberrantly expressed, thereby constituting a specific miRNA signature. Some of these miRNAs can play an important role in lung carcinogenesis. Indeed, some transcripts of the let-7 family that are significantly down-regulated in lung tumors have been identified as tumor suppressors through their ability to control several oncogenic pathways, including the RAS pathway. Identification of a growing number of other potential oncogenic or tumor suppressor miRNAs in lung cancers is in constant progress. Recent evidence supports the use of specific miRNA signatures to predict clinical outcome. This review aims to report the current knowledge about the role of miRNAs in lung cancer carcinogenesis, their potential for improving diagnosis and prognosis and their impact on future therapeutic strategies.

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.

MicroRNAs (miRNAs) are single-stranded noncoding RNAs of 19 to 25 nucleotides that function as gene regulators and as a host cell defense against both RNA and DNA viruses. We provide evidence for a physiological role of the miRNA-silencing machinery in controlling HIV-1 replication. Type III RNAses Dicer and Drosha, responsible for miRNA processing, inhibited virus replication both in peripheral blood mononuclear cells from HIV-1-infected donors and in latently infected cells. In turn, HIV-1 actively suppressed the expression of the polycistronic miRNA cluster miR-17/92. This suppression was found to be required for efficient viral replication and was dependent on the histone acetyltransferase Tat cofactor PCAF. Our results highlight the involvement of the miRNA-silencing pathway in HIV-1 replication and latency.