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

Fourre Sandra

Incidence of cutaneous squamous cell carcinomas (cSCCs) constantly increases in the Caucasian population. Developing preferentially on precancerous lesions such as actinic keratoses due to chronic sunlight exposure, cSCCs result from the malignant transformation of keratinocytes. Although a resection of the primary tumor is usually curative, a subset of aggressive cSCCs shows a high risk of recurrence and metastases. The characterization of the molecular dysfunctions involved in cSCC development should help to identify new relevant targets against these aggressive cSCCs. In that context, we have used small RNA sequencing to identify 100 microRNAs (miRNAs) whose expression was altered during chemically induced mouse skin tumorigenesis. The decreased expression of the miR-193b/365a cluster during tumor progression suggests a tumor suppressor role. Ectopic expression of these miRNAs in tumor cells indeed inhibited their proliferation, clonogenic potential and migration, which were stimulated in normal keratinocytes when these miRNAs were blocked with antisense oligonucleotides. A combination of in silico predictions and transcriptome analyses identified several target genes of interest. We validated KRAS and MAX as direct targets of miR-193b and miR-365a. Repression of these targets using siRNAs mimicked the effects of miR-193b and miR-365a, suggesting that these genes might mediate, at least in part, the tumor-suppressive action of these miRNAs.

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.

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.