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L’infarctus du myocarde (IDM), responsable du remodelage ventriculaire, peut conduire, s’il est délétère, à l’insuffisance cardiaque (IC), principale cause de mortalité à travers le monde. Les récentes découvertes ont montré l’implication des ARNs non codants, microARNs (miARNs) et les longs ARNs non codants (lncARNs), dans les processus physiologiques et pathologiques et notamment dans les maladies cardiovasculaires. L’objectif de ce travail a été d’étudier le potentiel des miARNs et des lncARNs en tant que biomarqueurs pronostiques et diagnostiques ainsi qu’en tant que cibles thérapeutiques dans l’IDM et le remodelage ventriculaire. Dans un premier temps, nous avons évalué le pouvoir diagnostique des miARNs sur une cohorte de patients présentant des douleurs thoraciques. Le miR-208b et le miR-499 ont montré une bonne capacité diagnostique de l’IDM, ne dépassant toutefois pas celle des troponines. Nous avons ensuite observé que le miR-150 présente une plus faible concentration dans le sang de patients avec un remodelage ventriculaire post-IDM par rapport aux patients sans remodelage, le positionnant comme un biomarqueur intéressant dans le pronostic de l’IC. Enfin, nous avons montré une régulation importante de plusieurs lncARNs dans le cœur de souris, 24 heures après IDM et 2 lncARNs, MIRT1 et MIRT2, ont été mis en avant pour leur association avec le remodelage. En conclusion, nos études ont montré l’utilité des ARNs non codants pour améliorer l’identification des patients à risque de développer une IC après IDM et ont permis également de mettre en évidence de nouvelles cibles thérapeutiques qui pourraient prévenir le remodelage ventriculaire post-IDM / Myocardial infarction (MI responsible for left ventricular remodeling which can be deleterious and the development of heart failure (HF). HF is one of the leading causes of mortality worldwide and despite many improvements, it remains a major challenge in clinical practice. Recent discoveries in genomics have showed the involvement of non-coding RNAs, including microRNAs (miRNAs) and long non-coding RNAs (lncRNAs), in physiological and pathological processes and notably linked to cardiovascular diseases. The goal of this work was to study the potential of miRNAs and lncRNAs as prognostic and diagnostic biomarkers and as therapeutic targets in MI and left ventricular remodeling leading to HF. First, we evaluated the diagnostic value of miRNAs in patients with chest pain. MiRNA-208b and miR-499 have shown a good diagnostic capacity for MI. However, these miARNs failed to improve the diagnosis of MI by troponins. MiRNA-208b could also predict patient mortality after MI but this capacity was modest. Then, we observed that miR-150 was present at a low level in the blood of patients with left ventricular remodeling post-MI compared to patients without remodeling. Therefore, miRNA-150 is an interesting prognostic biomarker. Finally, we have shown a significant regulation of several lncRNAs in mouse heart, 24 hours after MI, and 2 lncRNAs, MIRT1 and MIRT2, have been demonstrated for their association with left ventricular remodeling. In conclusion, our studies have shown the utility of non-coding RNAs to improve the identification of patients at risk of developing HF after MI and also allowed to identify potential therapeutic targets to prevent left ventricular remodeling

Hypoxia pathways are associated with the pathogenesis of both ischaemic and neoplastic diseases. In response to hypoxia the transcription factor hypoxia‐inducible factor (HIF) induces the expression of hundreds of genes with diverse functions. These enable cells to adapt to low oxygen availability. To date, pan-genomic analyses of these transcriptional responses have focussed on protein-coding genes and microRNAs. However, the role of other classes of non-coding RNAs, in particular lncRNAs, in the hypoxia response is largely uncharacterised. My thesis aimed at improving understanding of the transcriptional regulation of the non-coding transcriptome in hypoxia. I performed an integrated genomic analysis of both non-coding and coding transcripts by massively parallel sequencing. This was interfaced with pan-genomic analyses of DNAse hypersensitivity and HIF, H3k4me3 and RNApol2 binding in hypoxic cells. These analyses have revealed that hypoxia profoundly regulated all RNA classes. snRNAs and tRNAs are globally downregulated in hypoxia, whilst miRNAs, mRNAs and lncRNAs are both up- and downregulated with an overall trend towards slight upregulation. In addition, a significant number of previously non-annotated (and largely hypoxia upregulated) transcripts were identified, including novel intergenic transcripts and natural antisense transcripts. HIF bound close to genes for mRNAs, miRNAs and lncRNAs that were upregulated by hypoxia, but was excluded from binding at genes for RNA classes that showed global downregulation. This suggests that HIF acts as a transcriptional activator (but not repressor), of lncRNAs as well as mRNAs and miRNAs. Consistent with direct regulation by HIF, many of these hypoxia-inducible, HIF-binding lncRNAs were downregulated following HIF knockdown. Analysis of RNApol2 binding and DNAse HSS signals at HIF transcriptional target genes indicated that HIF-dependent transcriptional activation occurs through release of RNApol2 that is pre-bound to open promoters of lncRNAs as well as mRNAs. In these datasets, NEAT1 was the most hypoxia-upregulated, HIF-targeted lncRNA in MCF-7 cells and, despite binding of both HIF-1 and HIF-2 isoforms at its promoter, was selectively regulated by HIF-2 alone. Furthermore, NEAT1 was induced by hypoxia in a wide range of breast cancer cell lines and in hypoxic xenograft models. Functionally, NEAT1 is required for the assembly of nuclear paraspeckle structures. Increased nuclear paraspeckle formation was observed in hypoxia and was dependent on both NEAT1 and HIF-2. Knockdown of hypoxia-induced NEAT1 significantly reduced cell proliferation and survival and induced apoptosis. Finally, high expression of NEAT1 correlated with poor clinical outcome in a large cohort of breast cancer patients. These findings extend the role of the hypoxic transcriptional response in cancer into the spectrum of non-coding transcripts and provide new insights into molecular roles of hypoxia-regulated lncRNAs, which may provide the basis for novel therapeutic targets in the future.

Kürzlich wurde mithilfe von Ribo-seq Experimenten die Translation hunderter Mikroproteine in menschlichen Herzen entdeckt. Diese blieben zuvor aufgrund ihrer geringen Größe (< 100 Aminosäuren) unentdeckt, und ihre physiologische Rolle ist noch weitgehend unbekannt. Ziel dieser Promotionsarbeit ist es, potentielle Funktionen dieser neuartigen Mikroproteine zu entschlüsseln. Dabei sollen insbesondere die Aufklärung ihrer evolutionären Konservierungssignatur, subzellulären Lokalisierung und ihres Proteininteraktoms helfen. Die Konservierungsanalyse ergab, dass fast 90% der Mikroproteine nur in Primaten konserviert ist. Weiterhin konnte ich die Produktion von Mikroproteine in vitro und in vivo nachweisen, die subzelluläre Lokalisierung von 92 Mikroproteinen definieren, und Interaktionspartner für 60 Mikroproteine identifizieren. Dutzende dieser Mikroproteine lokalisieren in Mitochondrien. Dazu gehörte ein im Herzen angereichertes Mikroprotein, das aufgrund der Interaktions- und Lokalisationsdaten einen neuartigen Modulator der mitochondrialen Proteintranslation darstellen könnte. Der Interaktom-Screen zeigte außerdem, dass evolutionär junge Mikroproteine ähnliche Interaktionsfähigkeiten wie konservierte Kandidaten haben. Schließlich wurden kurze Sequenzmotive identifiziert, die Mikroprotein-Protein-Wechselwirkungen vermitteln, wodurch junge Mikroproteine mit zellulären Prozessen – wie z.B. Endozytose und Spleißen – in Verbindung gebracht werden konnten. Zusammenfassend wurde die Produktion vieler kleiner Proteine im menschlichen Herzen bestätigt, von denen die meisten lediglich in Primaten konserviert sind. Zusätzlich verknüpften umfangreiche Lokalisierungs- und Interaktionsdaten mehrere Mikroproteine mit Prozessen wie Spleißen, Endozytose und mitochondrialer Translation. Weitere Untersuchungen dieses zuvor verborgenen Teils des Herzproteoms werden zu einem besseren Verständnis von evolutionär jungen Proteinen und kardiologischen Prozessen beitragen. / Recently, the active translation of hundreds of previously unknown microproteins was detected using ribosome profiling on tissues of human hearts. They had remained undetected due to their small size (< 100 amino acids), and their physiological roles are still largely unknown. This dissertation aims to investigate these novel microproteins and validate their translation by independent methods. Particularly, elucidating their conservation signature, subcellular localization, and protein interactome shall aid in deciphering their potential biological role. Conservation analysis revealed that sequence conservation of almost 90% of microproteins was restricted to primates. I next confirmed microprotein production in vitro and in vivo by in vitro translation assays and mass spectrometry-based approaches, defined the subcellular localization of 92 microproteins, and identified significant interaction partners for 60 candidates. Dozens of these microproteins localized to the mitochondrion. These included a novel cardiac-enriched microprotein that may present a novel modulator of mitochondrial protein translation based on its interaction profile and subcellular localization. The interactome screen further revealed that evolutionarily young microproteins have similar interaction capacities to conserved candidates. Finally, it allowed identifying short linear motifs that may mediate microprotein-protein interactions and implicated several young microproteins in distinct cellular processes such as endocytosis and splicing. I conclude that many novel small proteins are produced in the human heart, most of which exhibit poor sequence conservation. I provide a substantial resource of microprotein localization and interaction data that links several to cellular processes such as splicing, endocytosis, and mitochondrial translation. Further investigation into this hidden part of the cardiac proteome will contribute to our understanding of recently evolved proteins and heart biology.

Serous epithelial ovarian cancer (EOC) is a potent suppressor of the immune defense. Here, we studied interactions between EOC and the immune system that lead to escape from tumor immune surveillance. We explored: 1) tumor escape from cytotoxicity by exosome-mediated modulation of the NK-cell receptors NKG2D and DNAM-1; 2) cytokine mRNA profiles in the EOC microenvironment and peripheral blood and their role in the suppression of the anti-tumor immune responses; 3) expression of long non-coding (lnc) RNAs in EOC tumors and exosomes. We found that EOC-secreted exosomes carried MICA/B and ULBP1-3, ligands of NKG2D, and could downregulate the NKG2D receptor and impair NKG2D-mediated cytotoxicity. In contrast, the DNAM-1 receptor ligands PVR and nectin-2 were seldom found in exosomes and were not associated with the exosomal membrane leaving the DNAM-1 receptor-mediated cytotoxicity intact. We compared cytokine mRNA expression in the tumor microenvironment and in immune cells of peripheral blood in EOC patients and patients with benign ovarian conditions. EOC patients were unable to mount an IFN-gamma mRNA response needed for tumor cell elimination. Instead, there was a significant up-regulation of inflammation and immune suppression i.e. responses promoting tumorigenesis and T-regulatory cell priming that suppress anti-tumor immunity. In addition, we studied lncRNAs in tissues and sera exosomes from EOC and benign ovarian conditions aiming to assess the lncRNA(s) expression profile and look for lncRNA(s) as possible marker(s) for early diagnosis. We found a deregulated lncRNAs expression in EOC tissues that correlated well with the lncRNAs expression in exosomes. Candidate lncRNAs with the highest expression and abundance were suggested for evaluation as EOC diagnostic markers in a future large cohort study. Our studies of EOC tissue and EOC exosomes highlight the immunosuppressive tumor microenvironment and the complex tumor exosome-mediated network of immunosuppressive mechanisms, and provide a mechanistic explanation of the observation that NKG2D-mediated cytotoxicity does not function in EOC patients and is partially replaced by the accessory DNAM-1 dependent cytotoxic pathway. The deregulated lncRNAs expression in EOC tissues and exosomes might serve for diagnostic purposes but could also be a potential risk of spreading tumor-derived lncRNAs in EOC exosomes to recipient cells throughout the body.