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Functional characterization of long non-coding RNAs associated with the epithelial-to-mesenchymal transition

Abstract : In the last decade, new high-throughput sequencing techniques have revealed the complexity of the human transcriptome, allowing the characterization of long non-coding (lnc)RNAs. These transcripts have been reported as very specific to tissues, developmental stages and pathological variations such as cancer. However, mechanisms through which they may act in the promotion of cancer are still poorly characterized. In my PhD project, I investigated the role of lncRNAs and their association to the epithelial to mesenchymal transition (EMT), a biological process which has been linked to metastasis and cancer progression.Using transcriptomic approaches from total and subcellular-fractionated RNA extracts from a cell system that models EMT (Castro-Vega et al, 2013), I identified over a thousand differentially expressed yet unannotated lncRNAs. Then I validated their expression, subcellular localization and the chromatin marks associated with their regulation.In order to assess whether these new lncRNA are functional in the EMT, I designed a CRISPR-activating (CRISPRa) screen using a dead Cas9 fused to transcription activating proteins (in collaboration with the lab of Neville Sanjana, NYU). This screen is based on two main phenotypes associated with the EMT: invasion capacity of the cells, and expression of an epithelial surface marker (EpCAM).In addition to the CRISPRa screening, I identified a novel lncRNA in mesenchymal cells which leads to a loss of epithelial markers and an increase in migration capacities when overexpressed in epithelial cells.Altogether, I used a combination of high-throughput methods to characterize the non-coding transcriptome associated to the EMT and identify yet unannotated transcripts which are functional in its regulation.
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Julien Jarroux. Functional characterization of long non-coding RNAs associated with the epithelial-to-mesenchymal transition. Genomics [q-bio.GN]. Université Paris sciences et lettres, 2019. English. ⟨NNT : 2019PSLET021⟩. ⟨tel-02882448⟩

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