Submitted by Cláudia Bueno (claudiamoura18@gmail.com) on 2015-11-12T18:06:58Z No. of bitstreams: 2 Dissertação - Iane de Oliveira Pires Porto - 2014.pdf: 1352493 bytes, checksum: 3e4c1213c96035cbae32d6eeccdd14e5 (MD5) license_rdf: 23148 bytes, checksum: 9da0b6dfac957114c6a7714714b86306 (MD5) / Approved for entry into archive by Luciana Ferreira (lucgeral@gmail.com) on 2015-11-13T10:33:50Z (GMT) No. of bitstreams: 2 Dissertação - Iane de Oliveira Pires Porto - 2014.pdf: 1352493 bytes, checksum: 3e4c1213c96035cbae32d6eeccdd14e5 (MD5) license_rdf: 23148 bytes, checksum: 9da0b6dfac957114c6a7714714b86306 (MD5) / Made available in DSpace on 2015-11-13T10:33:50Z (GMT). No. of bitstreams: 2 Dissertação - Iane de Oliveira Pires Porto - 2014.pdf: 1352493 bytes, checksum: 3e4c1213c96035cbae32d6eeccdd14e5 (MD5) license_rdf: 23148 bytes, checksum: 9da0b6dfac957114c6a7714714b86306 (MD5) Previous issue date: 2014-02-19 / Conselho Nacional de Pesquisa e Desenvolvimento Científico e Tecnológico - CNPq / MicroRNAs (miRNAs) are small non-coding RNAs involved in post-transcriptional expression regulation by inducing mRNA degradation or translation inhibition. Some miRNAs are known to regulate HLA-G expression, an important immunemodulatory molecule that inhibits both Natural Killer and cytotoxic T cells through interaction with inhibitory receptors. The HLA-G is associated with maternal-fetal tolerance, tissue acceptance in transplants and the progression of tumors. The mechanisms underlying HLA-G expression control are not completely understood, however, its 3’untranslated region (3’UTR) is reported to play an important role on gene regulation influencing mRNA stability and interacting with miRNAs such as miR-148a-3p. In this study, we performed a systematic analysis of all miRNAs that are good candidates to act as HLA-G regulators. In order to determine the miRNAs with the highest potential to influence HLA-G expression, we compared the outputs of three distinct algorithms - miRanda, RNAhybrid and Pita. For this purpose, a method of miRNA inference was developed using Perl scripts to compare and filter results and a scoring system was created in order to evaluate both the binding stability of the miRNA/mRNA interaction and the miRNA specificity to its target sequence. Then, a panel of miRNAs with great potential of controlling HLA-G expression was generated. / MicroRNAs (miRNAs) são pequenos RNAs não codificantes envolvidos na regulação gênica pós-transcricional por meio da degradação da molécula de RNA mensageiro ou da inibição da tradução. Alguns miRNAs foram relatados como sendo responsáveis pela regulação da expressão do gene HLA-G, um importante imunomodulador que inibe a ação de células Natural Killer e células T citotóxicas ao interagir com receptores inibitórios. Este gene está associado à tolerância maternofetal, aceitação de tecidos após transplantes e progressão de tumores. Os mecanismos subjacentes à regulação da expressão de HLA-G não foram completamente elucidados, mas sabe-se que sua região 3’ não traduzida (3’NT) possui um papel importante na regulação gênica tanto por manter a estabilidade da molécula de mRNA quanto por interagir com miRNAs como miR-148a-3p. Neste estudo, foram inferidos miRNAs que são bons candidatos para atuarem como reguladores do gene HLA-G. Para determinar os miRNAs com o maior potencial de operarem no controle pós-transcricional dos níveis de HLA-G, comparamos os resultados de três algoritmos distintos – miRanda, RNAhybrid e Pita. Para tanto, foi desenvolvida uma estratégia de inferência de miRNAs que utiliza scripts em Perl para comparação e filtragem dos dados e um sistema de pontuação que permite avaliar tanto a estabilidade da interação miRNA/mRNA quanto a especificidade do miRNA à sua sequência alvo. Assim, um painel confiável de miRNAs com grande possibilidade de influenciar a expressão de HLA-G foi gerado considerando as regiões polimórficas e não polimórficas da região 3’NT do gene HLA-G individualmente.

Les propriétés centrales des cellules souches sont la pluripotence et la capacité d'auto-renouvellement. Les cellules souches hématopoïétiques (CSHs) sont dotées de ces caractéristiques qui leur permettent de générer toutes les cellules du compartiment hématopoïétique, tout en maintenant en parallèle leur compartiment. Nous menons des approches visant à amplifier ex vivo les CSHs en les activant par HOXB4 exogène (CSHs humaines) ou via la signalisation Notch/DLL-4 (CSHs murines). Or deux analyses transcriptomiques indépendantes de ces deux modes d'activation ont de manière étonnante convergé sur une augmentation de l'expression de deux gènes jamais identifiés auparavant comme étant impliqués dans le maintien des CSHs : Pumilio1 (Pum1) et Pumilio2 (Pum2). Pum1 et Pum2 sont des régulateurs post-transcriptionnels appartenant à la famille Pumilio-FBF (PUF) des protéines liant l'ARN. Bien qu'il ait été établi que le rôle princeps de ces protéines PUF est de soutenir la prolifération des cellules souches chez les Invertébrés, jusqu'à présent on ne sait rien du rôle de Pum1 et Pum2 dans les CSH humaines et murines.Pour toutes ces raisons, nous avons étudié le rôle et les mécanismes d'action de Pum1 et Pum2 dans les CSH murines et humaines en utilisant l'interférence ARN (ARNi). L'invalidation de Pum1 ou de Pum2 dans les CSHs murines conduit à une réduction de l'expansion et du potentiel clonogénique ex vivo, associée à une apoptose accrue et l'arrêt du cycle cellulaire en phase G0/G1. L'invalidation concomitante de Pum1 et Pum2 majore ces effets ce qui suggère un effet coopératif entre les deux protéines. L'expansion et le potentiel clonogénique des CSH invalidées pour Pum1 sont restaurés suite à l'expression forcée de Pum1 (insensible au shRNA utilisé), validant ainsi la spécificité de nos shRNAs. Par contre la surexpression de Pum1 dans les CSHs invalidées pour Pum2 ne restaure pas leurs fonctions, soulignant le rôle non redondant de chaque protéine. En outre, lorsque les CSHs invalidées pour Pum1 ou Pum2 sont inoculées à des souris irradiées létalement de suivre le potentiel hématopoïétique à long terme, seules quelques rares cellules de la moelle osseuse issues des CSH KD pour Pum1 ou Pum2 sont mises en évidence après 4 mois de reconstitution, contrairement aux CSH contrôles. Des résultats identiques ont été obtenus en invalidant Pum1 ou Pum2 dans les CSH humaines.En conclusion, nos résultats démontrent l'implication des facteurs Pumilio dans le maintien du potentiel souche, l'expansion et la survie des CSHs murines et humaines. L'identification des facteurs Pumilio et de leurs cibles comme nouveaux régulateurs des CSHs permettra d'envisager de nouveaux outils en vue de perspectives thérapeutiques. / The central properties of stem cells are the pluripotency and the capacity of self-renewal. Hematopoietic stem cells (HSCs) posses such common features that allows them to generate all the cells of the hematopoietic compartments, maintaining in the same time the HSC pool. We develop approaches focused on ex vivo HSC expansion through activation by exogenous HOXB4 (human HSCs) or Notch/Dll-4 ligand (murine HSCs). Two independent transcriptomic analyses surprisingly converged toward an increased expression of two genes never identified sofar as crucial for HSC functions: Pumilio1 (Pum1) and Pumilio2 (Pum2). Pum1 and Pum2 are posttranscriptional regulators belonging to the Pumilio-FBF (PUF) family of RNA-binding proteins. Although it was established that the primordial role of PUF proteins is to sustain mitotic proliferation of stem cells in Invertebrates, so far nothing is known about the role of Pum1 and Pum2 in human and murine HSCs.For these reasons, we have investigated the roles and mechanisms of action of Pum1 and Pum2 in murine and human HSCs through shRNA strategy. Pum1 and Pum2 knockdown (KD) in murine HSCs led to a decreased HSC expansion and clonogenic potential ex vivo, associated with an increased apoptosis and a cell cycle arrest in G0/G1 phase. KD of both Pum1 and Pum2 enhanced these effects, suggesting a cooperative effect. Expansion and clonogenic potential of KD Pum1 HSCs were rescued by enforced expression of Pum1 (insensitive to our shRNA), thus validating the specificity of our shRNA. Enforced expression of Pum1 could not rescue the functions of Pum2 KD HSCs, highlighting the non-redundant role of these proteins. Furthermore, when Pum1 or Pum2 KD HSCs were inoculated into lethally irradiated mice to follow the long-term hematopoietic potential, only rare bone marrow cells derived from Pum1 and Pum2 KD HSCs were evidenced after 4 months, contrary to control HSCs. Identical results were obtained with human Pum1 or Pum2 KD HSCs.In conclusion, our results demonstrate the involvement of Pumilio factors in stemness maintenance, expansion and survival of murine and human HSCs. Identification of Pumilio factors and their targets as new regulators of HSCs expansion will allow consider them as new tools for therapeutic perspectives.

Abstract Type 1 neurofibromatosis (NF1) syndrome is caused by a mutation of the NF1 gene. NF1 protein (neurofibromin) contains a domain which is related to the GTPase activating protein (GAP) and accelerates the switch of active Ras-GTP to inactive Ras-GDP. The clinical symptoms of NF1 patients include e.g. the formation of benign neurofibroma tumors and hyperpigmented lesions of the skin. The NF1 protein has been referred to as a tumor suppressor since cells of malignant schwannomas of NF1 patients may display loss of heterozygosity of the NF1 gene. In the present study, the expression of the NF1 gene was investigated during tissue repair in human skin. Elevated NF1 protein levels were seen in a fibroblastic cell population of healing wounds. In vitro studies were designed to investigate NF1 expression in dermal fibroblasts under the influence of growth factors that are operative during wound healing. Platelet-derived growth factor (PDGF) isoforms AB and BB and transforming growth factor β1 (TGFβ1) elevated NF1 mRNA levels in cultured dermal fibroblasts. In further studies, histological examination on apparently healthy skin of NF1 patients revealed frequently small masses of neurofibromatous tissue at the vicinity of hair follicles. Thus, action of the NF1 gene appears to be an integral part of normal tissue repair. Enhanced NF1 tumor suppressor expression may serve to limit excessive fibrosis in wound healing. As Ras proteins play a role in the regulation of cell differentiation and formation of cell junctions, the functional expression of NF1 protein was elucidated using differentiating keratinocytes as an in vitro model system. The results demonstrate that an intense NF1 tumor suppressor signal on intermediate filaments was temporally limited to the period in which the formation of desmosomes takes place. In analogy to NF1 protein, a rapid elevation of NF1 mRNA level was detected following initiation of differentiation. Interestingly, NF1 mRNA hybridization signal polarized towards cell-cell contact zones. This finding recognizes a potential way for post-transcriptional modification of NF1 expression and targeting of translation to subplasmalemmal location. The results demonstrate that the function of NF1 protein is associated with the formation of cell junctions, and thus to cellular communication.

Dans son environnement, la bactérie Escherichia coli (E. coli) fait face à d’importantes fluctuations des ressources carbonées. Une capacité d’adaptation métabolique lui permet de coloniser ou de subsister en fonction des substrats disponibles. Cette adaptation est régie par un réseau complexe de régulations de l’expression génique. Le régulateur global post-transcriptionnel CSR (Carbon Storage Regulator) régule la stabilité et/ou l’initiation de la traduction d’ARNm par l’intermédiaire de la protéine CsrA. Ce système, essentiel en présence de glucose et est également supposé être impliqué dans la régulation d’une transition métabolique glycolyse vers gluconéogenèse. Le caractère essentiel de CSR est à ce jour inexploré, tout comme son implication dans la régulation d’une adaptation métabolique. Une approche de biologie intégrative menée pour différents mutants du système CSR a permis d’avancer pour la première fois, une explication de l’essentialité de CSR lors d’une croissance exponentielle sur glucose et de caractériser son implication dans la régulation de la transition métabolique glucose-acétate. Des approches de transcriptomique et de stabilomique utilisées pour une souche sauvage au cours d’une adaptation métabolique ont mis en évidence l’importance des régulations de la stabilité des ARNm au cours de l’adaptation. En conclusion, ces travaux approfondissent grandement les connaissances concernant le système CSR et son implication dans la régulation du métabolisme d’E. coli. Ce système, indispensable à la régulation du métabolisme durant une phase de croissance sur glucose s’ajoute de façon indéniable au réseau déjà complexe de régulations du métabolisme d’E. coli / In its natural environment, Escherichia coli (E. coli) faces strong fluctuations of the nutrient availability. A complex gene regulatory network makes the bacterium able to switch between a state of growth in the presence of an appropriate carbon source and a non-growth state in its absence. Within this network, the global post-transcriptional regulator CSR (Carbon Storage Regulator) modifies mRNA stability and/or translation initiation by the CsrA protein. This system has been shown to be essential for cells to grow on glucose and is hypothesized to be involved in the regulation of metabolic transitions. However both observations remained unexplored so far. An integrative approach has been used to investigate for the first time the essentiality of CSR on glucose as well as its involvement in the regulation of the glucose-acetate transition. Molecular and phenotypic data for different mutants of the CSR system have been produced and integrated into mathematical models. Transcriptomic and Stabilomic approaches have been used eventually to characterize the importance of the control of mRNA stability during the metabolic adaptation. mRNA stability regulations appear to be of particular importance in gene expression regulation during metabolic adaptation. To conclude, this work shed a new light upon CSR’s involvement in the regulation of E. coli’s metabolism. CSR is definitely essential to regulate glycolysis and thus constitutes another regulator to be integrated into the already complex regulations network of E.coli’s metabolism

Dbp2p, a member of the large family of DEAD-box proteins and a yeast homolog of human p68, was shown to interact with Upf1p, an essential component of the nonsense-mediated mRNA decay pathway. Dbp2p:Upf1p interaction occurs within a large conserved region in the middle of Upf1p that is largely distinct from its Nmd2p and Sup35/45p interaction domains. Deletion of DBP2, or point mutations within its highly conserved DEAD-box motifs, increased the abundance of nonsense-containing transcripts, leading us to conclude that Dbp2p also functions in the nonsense-mediated mRNA decay pathway. Dbp2p, like Upf1p, acts before or at decapping, is predominantly cytoplasmic, and associates with polyribosomes. Interestingly, Dbp2p also plays an important role in rRNA processing. In dbp2Δ cells, polyribosome profiles are deficient in free 60S subunits and the mature 25S rRNA is greatly reduced. The ribosome biogenesis phenotype, but not the mRNA decay function, of dbp2Δ cells can be complemented by the human p68 gene. We propose a unifying model in which Dbp2p affects both nonsense-mediated mRNA decay and rRNA processing by altering rRNA structure, allowing specific processing events in one instance and facilitating dissociation of the translation termination complex in the other.

No description available.

No description available.

In evolution alternative genetic trajectories can potentially lead to similar phenotypic outcomes. However, certain trajectories are preferred over others. These preferences bias the genomes of living organisms and the underlying processes can be observed in ongoing evolution. We have studied a variety of biases that can be found in bacterial chromosomes and determined the selective causes and functional consequences for the cell. We have quantified codon usage bias in highly expressed genes and shown that it is selected to optimise translational speed. We further demonstrated that the resulting differences in decoding speed can be used to regulate gene expression, and that the use of ‘non-optimal’ codons can be detrimental to reading frame maintenance. Biased gene location on the chromosome favours recombination between genes within gene families and leads to co-evolution. We have shown that such recombinational events can protect these gene families from inactivation by mobile genetic elements, and that chromosome organization can be selectively maintained because inversions can lead to the formation of unstable hybrid operons. We have used the development of antibiotic resistance to study how different bacterial lifestyles influence evolutionary trajectories. For this we used two distinct pairs of antibiotics and disease-causing bacteria, namely (i) Mycobacterium tuberculosis that is treated with rifampicin and (ii) Escherichia coli that is treated with ciprofloxacin. We have shown that in the slow-growing Mycobacterium tuberculosis, resistance mutations are selected for high-level resistance. Fitness is initially less important, and over time fitness costs can be ameliorated by compensatory mutations. The need for rapid growth causes the selection of ciprofloxacin resistance in Escherichia coli not only to be selected on the basis of high-level resistance but also on high fitness. Compensatory evolution is therefore not required and is not observed. Taken together, our results show that the evolution of a phenotype is the product of multiple steps and that many factors influence which trajectory is the most likely to occur and be most beneficial. Over time, selection will favour this particular trajectory and lead to biased evolution, affecting genome sequence and organization.

La quantité de données générée dans le cadre d'étude à grande échelle du réseau d'interaction protéine-protéine dépasse notre capacité à les analyser et à comprendre leur sens; d'une part, par leur complexité et leur volume, et d'un autre part, par la qualité du jeu de donnée produit qui semble bondé de faux positifs et de faux négatifs. Cette dissertation décrit une nouvelle méthode de criblage des interactions physique entre protéines à haut débit chez Saccharomyces cerevisiae, la complémentation de fragments protéiques (PCA). Cette approche est accomplie dans des cellules intactes dans les conditions natives des protéines; sous leur promoteur endogène et dans le respect des contextes de modifications post-traductionnelles et de localisations subcellulaires. Une application biologique de cette méthode a permis de démontrer la capacité de ce système rapporteur à répondre aux questions d'adaptation cellulaire à des stress, comme la famine en nutriments et un traitement à une drogue. Dans le premier chapitre de cette dissertation, nous avons présenté un criblage des paires d'interactions entre les protéines résultant des quelques 6000 cadres de lecture de Saccharomyces cerevisiae. Nous avons identifié 2770 interactions entre 1124 protéines. Nous avons estimé la qualité de notre criblage en le comparant à d'autres banques d'interaction. Nous avons réalisé que la majorité de nos interactions sont nouvelles, alors que le chevauchement avec les données des autres méthodes est large. Nous avons pris cette opportunité pour caractériser les facteurs déterminants dans la détection d'une interaction par PCA. Nous avons remarqué que notre approche est sous une contrainte stérique provenant de la nécessité des fragments rapporteurs à pouvoir se rejoindre dans l'espace cellulaire afin de récupérer l'activité observable de la sonde d'interaction. L'intégration de nos résultats aux connaissances des dynamiques de régulations génétiques et des modifications protéiques nous dirigera vers une meilleure compréhension des processus cellulaires complexes orchestrés aux niveaux moléculaires et structuraux dans les cellules vivantes. Nous avons appliqué notre méthode aux réarrangements dynamiques opérant durant l'adaptation de la cellule à des stress, comme la famine en nutriments et le traitement à une drogue. Cette investigation fait le détail de notre second chapitre. Nous avons déterminé de cette manière que l'équilibre entre les formes phosphorylées et déphosphorylées de l'arginine méthyltransférase de Saccharomyces cerevisiae, Hmt1, régulait du même coup sont assemblage en hexamère et son activité enzymatique. L'activité d'Hmt1 a directement un impact dans la progression du cycle cellulaire durant un stress, stabilisant les transcrits de CLB2 et permettant la synthèse de Cln3p. Nous avons utilisé notre criblage afin de déterminer les régulateurs de la phosphorylation d'Hmt1 dans un contexte de traitement à la rapamycin, un inhibiteur de la kinase cible de la rapamycin (TOR). Nous avons identifié la sous-unité catalytique de la phosphatase PP2a, Pph22, activé par l'inhibition de la kinase TOR et la kinase Dbf2, activé durant l'entrée en mitose de la cellule, comme la phosphatase et la kinase responsable de la modification d'Hmt1 et de ses fonctions de régulations dans le cycle cellulaire. Cette approche peut être généralisée afin d'identifier et de lier mécanistiquement les gènes, incluant ceux n'ayant aucune fonction connue, à tout processus cellulaire, comme les mécanismes régulant l'ARNm. / The quantity of data generated within the framework of protein-protein interaction network large-scale studies exceeds our capacity to analyze them and to understand their meaning; on one hand, by their complexity and their number, and on the other hand, by the quality of the produced data, which are populated with spurious interactions. This dissertation describes new applications of a protein-fragments complementation assay (PCA) to screen for interactions among all proteins in the budding yeast Saccharomyces cerevisiae. This approach is carried out in intact cells, with proteins expressed in their native contexts and under their endogenous promoter, thus assuring correct post-translational modifications and subcellular localization. A further novel application of PCA is described for investigating proteome wide changes in response to cellular adaptation to stresses, such as nutrient starvations and drug treatments. Finally, as a result of the latter strategy applied to characterizing proteome-wide response to the immunosuppressant drug, rapamycin, I describe the discovery of an unforeseen mechanism of modulating cell cycle progression through control of cyclin mRNA stability. In the first chapter of this dissertation, I present a pairwise screen of interactions among proteins resulting from the ~6000 open reading frames in Saccharomyces cerevisiae. We identified 2770 interactions among 1124 proteins. We estimated the quality of our screen by comparing our results to curated gold standard data and coverage of known interactions to all previous studies. The majority of our interactions were novel, but overlap with data from previous studies was as high as 40%. PCA is based on refolding of the reporter protein from complementary N- and C- terminal fragments following interaction of the two proteins to which they are fused. Thus, reporter activity is sterrically limited to interactions in which the termini of the proteins to which the complementary reporter fragments are fused are sufficiently close in space. In the case of our reporter, this limit was 8 nm. Thus PCA is a molecular ruler, providing information on both direct protein-protein interactions and sterrically restricted distances between proteins in complexes. We benchmarked and demonstrated correct topological relationships for a number of known complexes, including the proteasome, RNA polymerase II and the nuclear pore complex. Thus our study provided, for the first time, a topological map of complex organization in a living cell. The integration of the results from such efforts with those of gene regulation dynamics and protein modifications will lead to a fuller understanding of how complex cellular processes are orchestrated at a molecular and structural level in the living cell. In chapter 2, I describe the results of an application of PCA to study the dynamic rearrangement of the proteome under a specific stress; treatment of cells with rapamycin. The results of these efforts were the identification of a novel mechanism of cell cycle control at the level of cyclin mRNA. Specifically, we discovered that the balance between the phosphorylated and dephosphorylated forms of the Saccharomyces cerevisiae arginine methyltransferase, Hmt1, regulates both its assembly into a hexamer and its enzymatic activity. The Hmt1 activity modulates cell cycle progression through stabilizing the B cyclin CLB2 mRNA. We then used PCA to identify the Hmt1 regulators under rapamycin treatment. We identified the catalytic subunit of the PP2a phosphatase, Pph22, activated by the inhibition of TOR, and the kinase Dbf2, activated during entry into mitosis, as the phosphatase and the kinase responsible for the modification of Hmt1 and for its regulatory functions in the cell cycle. I thus, in the end close the circle I began in this summary, going from large-scale discovery of protein-protein interactions, to mapping dynamics of proteome changes during an adaptation and finally to mechanistic insight into a primordial control mechanism in cellular dynamics. The strategies that we devised to discover this mechanism can be generalized to identify and mechanistically link genes together, including those of unknown function, to any cellular process.

L'inflammation est considérée comme un promoteur de la cancérogenèse. La cyclooxygénase-2 (COX-2), la forme inductible de la famille des cyclooxygénases est un médiateur important de l'inflammation. Cette enzyme est constitutivement exprimée dans un grand nombre de cancers tels que les cancers du sein, du colon ou de la prostate. De nombreuses études mettent en évidence que COX-2 est surexprimée lors des étapes pré-néoplasiques. La COX-2 représente de ce fait une cible thérapeutique potentielle en chimioprévention et également pour le traitement des cancers. L'utilisation d'inhibiteurs synthétiques de COX-2 qui ciblent l'activité enzymatique est le seul traitement clinique actuellement disponible pour réduire l'activité de COX-2. Cependant, ces agents présentent des effets secondaires sévères, ce qui limite leur prise chronique chimiopréventives ou au cours des traitements anti-cancéreux. Une stratégie alternative pour cibler la fonction de COX-2 est d'inhiber son expression. Un grand nombre d'études montrent que certains produits naturels (la curcumine, le resveratrol ou l'apigénine par exemple) inhibent, préférentiellement l'expression de COX-2, sans être toxique. Notre projet analyse l'effet de l'acide cinnamique, un produit naturel extrait de la plante Cinnamonium cassia, sur l'expression de COX-2 au cours de la cancérogenèse dans le but d'évaluer son intérêt en chimioprévention. Nous avons utilisé comme modèle les cellules mammaires non carcinogènes, MCF10A stimulées avec un ester de phorbol, le 12-phorbol myristate 13-acetate (PMA), qui induit l'expression de COX-2. Nous avons observé une diminution de l'expression de COX-2 au niveau de l'ARNm et de la protéine après le traitement avec différentes concentrations d'acide cinnamique (1 et 10μM). L'analyse des mécanismes impliqués dans la diminution de l'expression de COX-2 a mis en évidence que l'acide cinnamique régule l'expression de COX-2 de façon post-transcriptionnelle en réduisant la stabilité de son ARNm. Cet effet est associé à une prévention de la diminution de l'expression de microARN (miR)-16 et une inhibition de l'expression de p38 induite par l'acide cinnamique en réponse au traitement avec le PMA. / Inflammation is considered a cancer-promoting factor. Cyclooxygenase-2 (COX-2), the inducible form of the family of cyclooxygenases is an important mediator of inflammation, which has been found constitutively expressed in many forms of cancer including breast, colon or prostate. A number of studies show that COX-2 is stably expressed since the early pre-neoplastic stages. This encourages us to consider COX-2 as a potential target in chemoprevention as well as in the treatment of cancer. Synthetic inhibitors of COX-2, which target enzymatic activity, are the only clinical strategy to counteract COX-2. However, these compounds present severe side effects, a fact that limits their prolonged intake, like requested in chemoprevention or during anti-cancer treatment.An alternative strategy to target COX-2 is at the level of its expression. A number of studies show that several natural compounds including curcumin, resveratrol or apigenin preferentially target COX-2 expression without showing toxicity.Our study analyses the effect of cinnamic acid, a natural compound derived from Cinnamonium cassia on COX-2 expression during carcinogenesis, with the final aim to evaluate its potential in chemoprevention/therapy.For our chemopreventive purposes, we used the non-carcinogenic breast cell line MCF10A, stimulated by the phorbol ester 12-phorbol myristate 13-acetate (PMA), which typically induces COX-2. We show a reduction of induced COX-2 expression after treatment with different concentrations of cinnamic acid (1 and 10μM). The analysis of the mechanisms involved in COX-2 protein expression decrease shows that cinnamic acid regulated COX-2 expression at the post-transcriptional level by reducing COX-2 mRNA stability. This effect is associated with the ability of cinnamic acid to prevent downregulation of miR-16 expression and p38 activation in response to PMA treatment.