Stem-cell antigen 1-positive (Sca-1+) cardiac stem cells (CSCs), a vital kind of CSCs in humans, promote cardiac repair in vivo and can differentiate to cardiomyocytes with 5'-azacytizine treatment in vitro. However, the underlying molecular mechanisms are unknown. b-arrestin2 is an important scaffold protein and highly expressed in the heart. To explore the function of b-arrestin2 in Sca-1+ CSC differentiation, we used b-arrestin2-knockout mice and overexpression strategies. Real-time PCR revealed that b-arrestin2 promoted 5'-azacytizine-induced Sca-1+ CSC differentiation in vitro. Because the microRNA 155 (miR-155) may regulate b-arrestin2 expression, we detected its role and relationship with b-arrestin2 and glycogen synthase kinase 3 (GSK3β), another probable target of miR-155. Real-time PCR revealed that miR-155, inhibited by b-arrestin2, impaired 5'-azacytizine-induced Sca-1+ CSC differentiation. On luciferase report assay, miR-155 could inhibit the activity of b-arrestin2 and GSK3β, which suggests a loop pathway between miR-155 and b-arrestin2. Furthermore, b-arrestin2-knockout inhibited the activity of GSK3β. Akt, the upstream inhibitor of GSK3β, was inhibited in b-arrestin2-Knockout mice, so the activity of GSK3β was regulated by b-arrestin2 not Akt. We transplanted Sca-1+ CSCs from b-arrestin2-knockout mice to mice with myocardial infarction and found similar protective functions as in wild-type mice but impaired arterial elastance. Furthermore, low level of b-arrestin2 agreed with decreased phosphorylation of AKT and increased phophorylation of GSK3β, similar to in vitro findings. The β-arrestin2/miR-155/GSK3β pathway may be a new mechanism with implications for treatment of heart disease.

Diffuse large B-cell lymphoma (DLBCL) is a genetically heterogeneous disease with multiple distinct molecular subtypes. Increased NF-κB activity and expression of the microRNA miR-155 (product of the BIC gene) are associated with one subtype, called the activated B-cell (ABC) subtype. It is shown here that induction of NF-κB activity leads to increased miR-155 expression, the levels of miR-155 in a panel of B-lymphoma cell lines correlate with increased NF-κB activity, and the NF-κB p50/p65 heterodimer binds to a specific DNA site in the BIC promoter. Also described is a regulatory network wherein NF-κB-dependent up-regulation of miR-155 leads to reduced PU.1 transcription factor expression and consequently reduced PU.1-driven expression of B-lymphoma marker CD10 in the human B-lymphoma cell line BJAB. Genetic variation in DLBCL can be used to explain the response of individual patients to chemotherapy. One cancer therapeutic approach currently in clinical trials uses histone deacetylase inhibitors (HDACi's) as a monotherapy or in combination with other vi agents. It is shown here that two pan-HDACi's, trichostatin A and vorinostat, induce apoptosis in seven of eight human DLBCL cell lines. Ectopic over-expression of antiapoptotic proteins BCL-2 and BCL-XL or the pro-apoptotic protein BIM in select DLBCL cell lines can confer further resistance or sensitivity, respectively, to HDACi treatment. Additionally, the BCL-2 family antagonist ABT-737 can increase the sensitivity of several DLBCL cell lines to vorinostat-induced apoptosis, including the HDACi-resistant SUDHL6 cell line. Moreover, one vorinostat-resistant variant of the HDACi-sensitive cell line SUDHL4 has increased expression of anti-apoptotic proteins BCL-XL and MCL-1 and decreased sensitivity to ABT-737, and a second such variant cell line has increased expression of anti-apoptotic protein MCL-1. These results suggest that the balance of anti- to pro-apoptotic BCL-2 family protein expression is important in determining the sensitivity of DLBCL cell lines to HDACi-induced apoptosis. Thus, the sensitivity of DLBCL cell lines to treatment with HDACi's appears to depend on the complex regulation of BCL-2 family members, suggesting that the response of a subset of DLBCL patients to HDACi treatment may benefit from co-treatment with BCL-2 antagonists.

(EN) Chronic lymphocytic leukemia (B-CLL) represents a disease of mature-like B-cells. Due to failed apoptosis but also due to enhanced proliferative signals, the leukemic B-cells accumulate in the peripheral blood, bone marrow, lymph nodes and spleen. The clinical course of B-CLL is very heterogeneous; in some patients B-CLL progresses very rapidly into an aggressive form. Such patients need therapy sooner while in other patients with indolent B-CLL the onset of therapy takes years. Several standard prognostic and disease progression markers are used for disease staging and monitoring, however a reliable marker that will suggest when to start therapy is unknown. Expression of small, non-coding microRNAs is often deregulated and represent important prognostic markers in variety of cancers including leukemia. Hence in our study we concentrated to miR-155, an important molecule regulating differentiation of hematopoietic cells, inflammation process and antibody production. Its aberrant expression was described in Hodgkin`s as well as in non-Hodgkin`s lymphoma, including indolent lymphoproliferations like B- CLL. Our results confirmed elevated levels of both, primary miR-155 transcript and mature form of miR-155 in our B-CLL patient samples (N=239). The aberrant expression of miR-155 in B-CLL samples...

T cells chronically stimulated by their antigen often become dysfunctional and lose effector functions and proliferative capacity. This state of unresponsiveness is referred as T cell exhaustion. In order to investigate this, we developed a laboratory model, which allowed us to stimulate chronically in vivo a monoclonal population of CD4+ T cells. This model is based on the adoptive transfer of TCR transgenic CD4+ T cells specific for the male mHAg into male recipient mice. We found that systemic exposure to the male antigen modified deeply anti-male TcR-transgenic CD4+ T cells, plunging them into a state of functional unresponsiveness. Microarray analysis revealed that, in comparison with naive T cells, transferred T cells displayed a gene expression profile very similar to that of virus-specific exhausted CD8+ T cells. Moreover, like exhausted CD8+ T cells, exhausted CD4+ T cells lost their capacity to secrete IFN-ã as well as to proliferate in response to antigen stimulation, and T cell unresponsiveness was controlled by the engagement of programmed death receptor 1 (PD-1) present at the surface of T cells. <p>MicroRNAs are key molecules in shaping T cell function. In order to explore the possibility that chronic antigenic stimulation could shape the pool of microRNAs in exhausted anti-male CD4+ T cells that would account for specific changes in protein synthesis, we compared by microarray analysis the specific expression of microRNAs in naive CD4+ T cells and exhausted CD4+ T cells. Ninety five of them were found differentially expressed, among which, microRNA-155 (miR-155) displayed one of the highest changes. To identify the importance of miR-155 in T cell exhaustion, we analyzed miR-155-deficient CD4+ T cells after chronic exposure to systemic antigen. We found that, chronically-stimulated miR-155-/- CD4+ T cells were retained in a deeper state of unresponsiveness than miR-155+/+ CD4+ T cells. Furthermore, inhibition of PD-1/PD-L1 interaction did not promote antigen-dependent expansion of miR-155-deficient CD4+ T cells, nor did it stimulate T cell inflammation of several organs, contrary to what was observed in mice that received miR-155-sufficient CD4+ T cells. Thus, our observations demonstrated that miR-155 deficiency played a dominant role over PD-1-mediated inhibition of T cells and that miR-155 was required for restoring function in exhausted CD4+ T cells.<p>Next, we explored the mechanism by which exhausted miR-155-/- CD4+ T cells were kept in a deeper unresponsiveness state than miR-155+/+ counterparts. By comparative microarray analysis of gene expression between exhausted miR-155+/+ CD4+ T cells and miR-155-/- CD4+ T cells, heme oxygenase 1 (HO-1) was identified as a specific target of miR-155. Finally, inhibition of HO-1 activity restored the capacity of exhausted miR-155-/- CD4+ T cells to promote autoimmune inflammation in adoptively-transferred recipients. <p>Taken together, our study identified miR-155-mediated regulation of protein expression as a critical factor for restoring function in exhausted CD4+ T cells. Our results also present regulation of HO-1 expression in T cells as one of the mechanisms by which miR-155 promote T cell-driven inflammation. / Doctorat en Sciences biomédicales et pharmaceutiques / info:eu-repo/semantics/nonPublished

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MicroRNAs (miRNAs) sind eine Klasse von ca. 22 nt langen, nicht-kodierenden RNAs, welche mittels Basenpaarung die Translationsrate und Stabilität von mRNAs herabsetzen. Die vorliegende Studie untersucht mittels Hochdurchsatz-Sequenzierung Expressionsveränderungen von miRNAs nach Infektion von kultivierten Wirtszellen mit dem mikrobiellen Modellpathogen Salmonella enterica serovar Typhimurium. In Makrophagen, welche eine Schlüsselfunktion in der Orchestrierung der angeborenen Immunität spielen, wurde im Zuge der Infektion eine Induktion der miRNAs miR-21, miR-146 und miR-155 beobachtet. Darüberhinaus stellten sich alle Mitglieder der evolutionär konservierten let-7 miRNA Familie in infizierten Makrophagen als herab reguliert heraus. Es konnte gezeigt werden, dass let-7 miRNAs die zentralen Makrophagen-Zytokine IL6 und IL10 post-transkriptional reprimieren. Konsequenterweise bewirkt eine Reduktion der let-7 Expression in mikrobiell aktivierten Makrophagen eine Erhöhung der IL6 und IL10 Produktion. Weiterhin konnten den miRNAs miR-146 und miR-155 wichtige Funktionen in der Steuerung der Sensitivität und Aktivität von Makrophagen gegenüber mikrobiellen Stimuli nachgewiesen werden: während miR-146 primär die Aktivität des plasmamembranständigen Lipopolysaccharid-Rezeptors TLR4 herabsetzte und damit einer vorzeitigen inflammatorischen Makrophagenantwort vorbeugte, blieb miR-155 strikt an letztere gekoppelt, um die Aktivität diverser pro-inflammatorischer Signalwege zu begrenzen. Es konnte gezeigt werden, dass eine Stimulation des cytosolischen Immunrezeptors NOD2 eine inflammatorische Makrophagenantwort und die resultierende miR-155 Induktion begünstigt und der negativen Kontrolle durch miR-146 entzieht. Dies verhindert möglicherweise eine Hyposensitivität gegenüber zellinvasiven Pathogenen. Zusammenfassend legen diese Beobachtungen nahe, dass miRNAs zentrale Funktionen in der post-transkriptionalen Steuerung der Wirtszellantwort auf mikrobielle Pathogene ausüben. / MicroRNAs (miRNAs) are a class of approx. 22 nt long non-coding RNAs that interfere with mRNA translation and stability. Using high-throughput sequencing the present study investigated miRNA expression changes after infection of cultured host cells with the microbial model pathogen Salmonella enterica serovar Typhimurium. In macrophages, which play a key role in the orchestration of innate immunity, infection caused the induction of miRNAs miR-21, miR-146 and miR-155. Moreover, all members of the evolutionarily conserved let-7 miRNA family were down-regulated in infected macrophages. This work reports let-7 miRNAs to function in the macrophage inflammatory response by repressing the major cytokines IL6 and IL10 post-transcriptionally. Consequently a reduction of let-7 expression in microbially activated macrophages results in a specific increase in IL6 and IL10 production. Furthermore, miR-146 and miR-155 could be assigned important functions in the control of the sensitivity and activity of macrophages to microbial stimuli: while miR-146 primarily reduced the activity of the plasma membrane associated lipopolysaccharide receptor TLR4, thereby preventing a premature macrophage inflammatory response, miR-155 stayed strictly coupled to inflammation in order to limit the activity of various pro-inflammatory signaling pathways. Interestingly, it could be shown that stimulation of the cytosolic immune receptor NOD2 favors the macrophage inflammatory response and the concomitant induction of miR-155, while bypassing the negative control by miR-146. This may prevent hyposensitivity to cell-invasive pathogens. In summary, these observations suggest that miRNAs exert key functions in the post-transcriptional control of the host cell response to microbial pathogens.

La sénescence se caractérise par un arrêt en phase G1/S du cycle cellulaire et peut être induit par une variété de stress tels que des télomères trop courts, l’activation d’oncogène ou encore à cause de stress oxydatifs. Cette réponse cellulaire s’accompagne de profonds changements au niveau de l’expression génique et les ARN non codants sont d’importants acteurs de ceux-ci. Bien que cette catégorie d’ARN ait longtemps été considérée comme un sous-produit non fonctionnel de la transcription, on sait maintenant qu’ils sont impliqués dans une pléthore de fonctions essentielles à l’homéostasie de la cellule. Les microARN (miR), de petits ARN non codants d’une vingtaine de nucléotides, sont souvent diminués ou surexprimés dans les maladies, soulignant leurs rôles importants dans le développement de celles-ci. C’est le cas notamment de deux oncomirs, miR-19 et miR-155, qui s’accumulent de manière aberrante dans les cancers hématopoïétiques. En condition normale, STAT5A, qui est souvent dérégulé dans ces cancers, induit SOCS1 qui agit comme un frein sur cette voie de signalisation afin de prévenir une prolifération incontrôlée. Des travaux conduits dans notre laboratoire montrent que SOCS1 est aussi impliqué dans la sénescence, car il est capable d’activer p53, un important suppresseur tumoral. SOCS1 peut être ciblé par les deux oncomirs et nos résultats montrent qu’une inhibition de ces derniers à l’aide d’éponges artificielles favorisait l’accumulation d’un p53 actif. De plus, en intégrant le ribozyme à tête de marteau dans la conception des éponges, nous avons créé une nouvelle génération d’outils (éponges catalytiques) qui sont plus efficaces. Effectivement, l’utilisation de ces éponges contre miR-155 résultait en une diminution de la prolifération, de formation de colonie ainsi que de la migration de cellule de myélome multiple. En second lieu, nous nous sommes penchés sur l’étude d’éponges naturelles dans le contexte de la sénescence. Il existe en effet quelques exemples de lARNnc (Long Non-Coding RNA) qui peuvent agir de la sorte pour un miR donné. Nous pensons que c’est par ce mécanisme de régulation que miR-146a, un miR impliqué dans la réponse anti-inflammatoire, peut s’accumuler dans la sénescence induite par RAS sans toutefois sembler être pleinement actif. Effectivement, les cellules sénescentes sécrètent une variété de facteurs pro-inflammatoires. À l’aide d’une nouvelle technique nommée miR-CLIP, nous avons pu étudier l’interactome de miR-146a et avons identifié plusieurs lARNnc qui selon des outils de prédiction, semblent s’hybrider de manière extensive en région 3’ du miR. Ceci est requis pour l’initiation d’un TDMD (Target-Directed miR Degradation) et nous avons donc investigué la possibilité d’un tel évènement dans la régulation de miR-146a. Nos résultats montrent que la surexpression de XXBAC-B444P24.13 mène à une diminution des niveaux de miR-146a qui n’est pas due à une baisse de sa transcription. Bien que le premier article illustre les avantages d’une éponge catalytique artificielle, le second article suggère que cette stratégie pourrait déjà être en place dans les systèmes biologiques, et ce, de manière naturelle. En effet, une fois miR-146a lié à XXBAC-B444P24.13, ce dernier induirait la dégradation du miR par un TDMD. Ceci ouvre donc la porte au développement d’outils qui pourraient être plus performants à des niveaux d’expression plus bas. / Cellular senescence is characterized by a cell cycle arrest in the G1/S phase and can be induced by a variety of stresses which include telomere shortening, oncogene activation or oxidative stress. Its establishment is known to require changes in the genetic expression program and non-coding RNA play an important part in this phenomenon. For a long time, this RNA subtype was considered to only be a transcriptional byproduct, but we now know that they are involved in a plethora of functions which are essential to cell homeostasis. Various diseases display aberrant expression of microRNA (miR), small non-coding RNA of 18-22 nucleotides, suggesting they are involved in their development. Such is the case for miR-19 and miR-155, two oncomirs which are found to be overexpressed in hematopoietic cancers. In normal conditions, STAT5A, which is often found dysregulated in those cancers, induces SOCS1 which acts as a retro-inhibitor of this signaling pathway, preventing uncontrolled proliferation. Furthermore, our lab has shown that SOCS1 can also be involved in senescence by facilitating p53 activation. SOCS1 can be targeted by both oncomirs and our results show that artificial sponges, that inhibit miR-19 or miR-155’s functions, lead to the activation of p53. Also, we have incorporated the hammerhead ribozyme in the miR binding sites in the sponge, creating a sponge 2.0 (catalytic sponges). Expressing the latter in a multiple myeloma cell line (RPMI8226) resulted in less proliferation, colony formation and migration. Secondly, we aimed at studying natural sponges in the context of senescence. Indeed, there are quite a few examples of lncRNA (Long Non-Coding RNA) acting as a miRNA inhibitor by quenching them. We think that this mode of regulation could provide an explanation as to how an anti-inflammatory miR, miR-146a, can accumulate in senescence even though it is a pro-inflammatory response. Using a novel technique called miR-CLIP, we were able to study specifically miR-146a’s interactome and have found that it can interact with many lncRNAs. Interestingly, using computational tools, we noticed that miR-146a was predicted to interact with extensive 3’ end hybridization with a number of these lncRNA. This characteristic is known to be required to induce TDMD (Target-Directed miR Degradation). Indeed, when we overexpressed XXBAC-B444P24.13, miR-146a levels went down and this is not caused by a decrease in transcription of the miR. In the first part of this thesis, we show that artificial catalytic sponges have an advantage over a more “classical” design. This is further supported by the fact that this strategy seems to be employed in nature. Indeed, we might have uncovered a lncRNA that when bound to by miR-146a would lead to its degradation using TDMD. This could be taken advantage of in the development of new tools for miR inhibition that would be more powerful and could be potentially used at lower levels of expression.

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