Estima-se que 3% da população mundial esteja infectada pelo vírus da hepatite C (HCV). O HCV tem como alvo o tecido hepático e a maioria dos pacientes infectados desenvolvem infecção crônica. Nos últimos anos, estudos in vitro têm demonstrado interações entre o miRNA-122 (miR-122) da célula hospedeira e dois sítios localizados na região 5\' UTR do genoma do vírus da hepatite C (HCV), os quais são essenciais ao processo de replicação viral. O miR-122 é altamente expresso no fígado, onde atua na regulação do metabolismo de lipídios juntamente com outro miRNA, o miRNA-33a (miR-33a), porém, o mecanismo envolvido nesta regulação ainda é pouco conhecido. Sabe-se que a infecção pelo HCV altera a expressão de genes envolvidos na biossíntese e transporte de lipídios, resultando na estimulação do metabolismo de lipídios e criando um ambiente favorável para sua replicação. Neste contexto os objetivos deste trabalho foram avaliar a expressão de miR-33a e miR-122 em indivíduos cronicamente infectados pelo HCV-1 e HCV-3 em amostras obtidas antes do início da terapia. Os miRNAs foram isolados a partir de amostras de sangue periférico e de tecido hepático. A quantificação da expressão relativa de ambos miRNAs foi pela técnica de PCR em tempo real. Os níveis de miR-33a no sangue periférico foram mais elevados do que no tecido hepático em indivíduos infectados pelo HCV-1(p < 0,0001) e HCV-3 (p=0,0025). Observou-se uma correlação inversa entre os níveis de miR-33a no sangue periférico e tecido hepático dos indivíduos infectados pelo HCV-1 (r=-0,281, p=0,039) e correlação positiva para os indivíduos infectados pelo HCV-3 (r=0,9286, p < 0,0001). Correlação inversa entre os níveis hepáticos de miR-33a com o nível sérico de insulina (r=-0,371, p =0,005) nos indivíduos infectados pelo HCV-1 e correlação positiva entre os níveis no sangue periférico com os níveis séricos de GGT (r=0,553, p=0,049) foram observadas. Em relação ao miR-122, de maneira geral o nível hepático foi mais elevado do que o sérico (p < 0,0001). Entretanto, o nível hepático de miR-122 em indivíduos infectados pelo HCV-3 foi maior quando comparado aos infectados pelo HCV-1 (6,22 vezes, p < 0,001). Uma correlação inversa entre os níveis séricos de ApoA-II e os níveis de expressão de miR-122 no sangue (r=-0,330; p=0,014) e tecido hepático (r=-0,311; p=0,020) foi observada nos pacientes infectados pelo HCV-1. Os pacientes infectados pelo HCV- 3 mostraram correlação positiva entre os níveis hepáticos de miR-122 e os níveis de HDL (r=0,412, p=0,036) e insulina (r=0,478, p=0,044). O miR-33a e o miR-122 atuam regulando genes que controlam o metabolismo dos lipídios no fígado. Até o presente momento, não existem relatos que associem a expressão do miR-33a e do miR-122 com o perfil lipídico na infecção pelo HCV. Além disso, o acúmulo de lipídio (esteatose) intensamente descrito na infecção pelo HCV-3 pode sugerir interação diferenciada desse genótipo com os mecanismos envolvidos na regulação do metabolismo lipídico, envolvendo o miR-33a e miR-122 / The prevalence of infection by hepatitis C virus (HCV) is about 3% of the world population. HCV targets the liver tissue and the majority of infected patients develop chronic infection. In recent years, in vitro studies have demonstrated interactions between miRNA-122 (miR-122) the host cell to two places located in the 5\' untranslated region of the HCV genome which are essential for virus replication process. miR-122 is highly expressed in the liver, which has been implicated as a fatty acid metabolism regulator. Another mine has also been described as a key regulator of lipid metabolism, miRNA-33a (miR-33a), however, the mechanisms involved in this regulation are still little known. It is known that HCV infection changes the expression of genes involved in the biosynthesis and transport of lipids, resulting in stimulation of the lipid metabolism and creating a favorable environment for replication of the virus. To our knowledge, there are no reports linking the expression of miR-33a with lipid profile in HCV infection. In this context the objectives of this study were to evaluate the expression of miR-33a and miR-122 in chronically infected individuals with HCV-1 and HCV-3 in samples obtained prior to initiation of therapy. MiRNAs were isolated from peripheral blood samples and liver tissue. The quantification of relative expression of both miRNAs was by PCR in real time. MiR-33a levels in peripheral blood were higher than in liver tissue in patients infected with HCV-1 (p < 0.0001) and HCV-3 (p=0.0025). Levels in the peripheral blood of miR-33a were lower in patients infected with HCV-3 (p=0.0169). There was an inverse correlation between hepatic levels of miR-33a with serum insulin levels (p=0.005) in individuals infected with HCV-1 and a positive correlation between the levels in the peripheral blood serum levels of GGT (p=0.049). Hepatic levels of miR-122 were higher than the levels in the peripheral blood of individuals infected by HCV-1 and HCV-3 (p < 0.0001). Hepatic miR-122 levels were higher in patients infected with HCV-3 than those infected with HCV-1 (6.22 times, p < 0.001). There was a positive correlation between miR-122 levels in the blood and liver tissue of patients infected with HCV-1 (r=0.302, p=0.026). An inverse correlation between serum ApoA-II was observed in these patients the levels of expression of miR-122 in blood (r=-0.330; p =0.014) and liver tissue (r=-0.311; p=0.020). Patients infected with HCV-3 showed a positive correlation between hepatic miR-122 levels to HDL levels (r=0.412, p=0.036) and insulin levels (r=0.478, p=0.044). The miR-33a and miR-122 act by regulating genes that control lipid metabolism in the liver. The different interactions with lipid metabolism exerted by HCV-3 may explain why his relationship with the miR-33a and miR-122 was different when compared with HCV-1

MicroRNAs are emerging as key players in the regulation of the post-myocardial infarction (MI) environment. We previously identified that matrix-treated hearts had down-regulated expression of miR-92a, a miRNA with inflammatory and migratory effects that is normally up-regulated after MI. We have shown that type I collagen matrix treatment at 3h post-MI leads to less inflammation and improved cardiac function, but the underlying mechanisms remain to be better characterized. The goal of this study was to elucidate a possible role of miR-92a in the anti-inflammatory/pro-wound healing effect of matrix treatment post-MI. C57BL/6J mice underwent LAD ligation to induce MI. Hearts were removed at 4h, 1d, 3d, and 7d post-MI and RNA was extracted from infarct and peri-infarct tissue. PCR analysis revealed that hearts injected with matrix at 3h post-MI resulted in significantly decreased miR-92a at 4h, 1d, and 3d compared to non-injected animals at each time point (p<0.0001) and PBS injected animals at 4h and 7d (p<0.004). Several targets of miR-92a and regulators of macrophage polarization were found to be up-regulated (p<0.05) early in MI indicating early amelioration of inflammatory processes. In vitro, macrophages cultured on matrix also had decreased expression of miR-92a compared to cultures on tissue culture poly styrene (TCPS) (p<0.001). Integrins α5 (ITGAα5) and αV (ITGAαV), involved in cell-matrix interactions, as well as inflammatory regulators S1PR1 and SIRT1 were identified as putative miR-92a targets. When miR-92a is over-expressed in macrophages, ITGα5 (p=0.0002), ITGαV (p=0.02), and S1Pr1 (p<0.0001), and SIRT1 (p=0.03) all had decreased expression. STAT3 and IL-10 were found to be moderately down-regulated. In evaluating macrophage phenotypes, M2 macrophages had reduced miR-92a expression on matrix compared to M1 macrophages. The migration of M2 macrophages into the matrix is increased compared to M1 macrophages. We report that the beneficial effects of matrix treatment post-MI may be mediated, at least in part, through its ability to regulate miR-92a and pro-wound healing mechanisms in macrophages. These results present the matrix as a novel non-pharmacological approach to locally regulate miRNAs in vivo for reducing inflammation and protecting the myocardium post-MI.

Multiple sclerosis (MS) is a debilitating disease in which demyelinated lesions form in the central nervous system (CNS). A specific microRNA, miR-145-5p, is dysregulated both in blood samples from RRMS patients and in chronic lesions from progressive MS patients. In the context of remyelination, miR-145-5p regulation may be important as it exhibits strong differential regulation in oligodendrocytes (OLs), the myelinating cells of the CNS, and is also expressed in other CNS glial cell types. Dysregulation of miR-145-5p may therefore play into pathologies observed in both relapsing-remitting (RRMS) and progressive MS. Using pre-clinical rodent models, we aimed to determine how altering normal expression of miR-145-5p specifically affects OL maturation, and how the dysregulation observed in MS may affect various aspects of disease. First using a miR-145 knockdown model in primary rat OLs, we found in vitro that miR-145-5p plays a role both in maintaining oligodendrocyte progenitor cells (OPCs) in their proliferative state and preventing premature differentiation to OLs and that knockdown of miR-145 in OLs enhanced their differentiation. These effects were due at least in part to miR-145-5p regulation of a critical myelin gene transcription factor. The effects of miR-145-5p were further assessed in a miR-145 knockout mouse model in vivo. Contrary to in vitro assays, enhanced myelination was not detectable during development in these animals, nor when remyelination was assessed using the cuprizone toxic model of acute demyelination. However, chronic cuprizone exposure resulted in striking remyelination and functional recovery in miR-145 deficient animals. Sparse remyelination in wild-type animals with chronic cuprizone exposure was concomitant with upregulation of miR-145-5p, which was not the case with acute exposure, identifying miR-145-5p dysregulation as a unique feature of chronic demyelination. Specific assessment of miR-145-5p overexpression in OLs in vitro resulted in severe differentiation deficits and eventual apoptosis, driven molecularly by altered expression of multiple pathways critical to successful OL differentiation and subsequent myelination. Finally, we induced an inflammatory model of demyelination, experimental autoimmune encephalomyelitis (EAE), in our miR-145 knockout mouse to assess the role of miR-145-5p in autoimmune-mediated myelin damage. The clinical severity of EAE in miR-145 deficient animals was reduced, and this was accompanied by reduced loss of myelin and lessened immune cell infiltration in miR-145 knockout spinal cords. Alterations in both astrocytic and microglial activation were detected with loss of miR-145, suggesting that improved clinical outcomes in this model may be underpinned by changes in EAE-mediated neuroinflammation. Collectively, these data suggest that miR-145-5p plays differing roles in both progressive and inflammatory MS, affecting multiple glial cell types in the CNS. Excitingly, loss of miR-145 expression in our mouse model of chronic demyelination allowed extensive remyelination and functional recovery following chronic demyelination, and in EAE improved clinical outcomes driven by underlying improvements in myelin retention and altered neuroinflammatory reactions. Thus, miR-145-5p merits further investigation as a potential therapeutic target to help overcome both remyelination failure in all forms of progressive MS and inflammation-driven demyelination in RRMS and early secondary progressive MS (SPMS).

Myocardial infarction (MI), a late-stage event of many cardiovascular diseases (CVD), results in cardiomyocyte death, myeloid cell recruitment to promote cellular debris removal and excessive cardiac remodeling affecting architecture and function, which can ultimately lead to heart failure. Currently, the use of biomaterials to intervene on the hostile post-MI environment and promote myocardial healing is being investigated to restore cardiac function. It has been shown that an injectable collagen matrix improves cardiac repair by altering macrophage polarization, reducing cell death and enhancing angiogenesis, leading to a reduction in infarct size and improved cardiac function when delivered at 3 hours post-MI. MicroRNAs (miRNA) “fine tune” gene expression by negatively regulating the translational output of target messenger RNA (mRNA). As such, miRNAs present interesting therapeutic opportunities for the treatment of MI. However, the delivery of miRNA mimics and/or inhibitors can be complicated by degradation and off target effects. The objectives of this thesis were to determine how the matrix may regulate endogenous miRNAs and to explore the biomaterial’s ability to deliver therapeutic miRNAs. It was shown that matrix treatment of MI mouse hearts resulted in altered expression of 119 miRNAs, some of which had functions linked to the beneficial effects of matrix treatment. Of particular interest, miR-92a was down-regulated within the infarct and peri-infarct cardiac tissue 2 days after matrix treatment (delivered at 3-hours post-MI) compared to PBS treatment. In in vitro cultures, the matrix down-regulated miR-92a levels in macrophages but did not significantly alter miR-92a expression in endothelial cells, circulating angiogenic cells or fibroblasts. In addition, using an in vitro model system, it was shown that the matrix may have the potential to deliver functional therapeutic miRNAs to cells; however further experimental optimisation is required to confirm these results. Therefore, collagen matrix treatment may be a promising approach to regulate and/or deliver miRNAs for protecting the myocardial environment and improving function of the infarcted heart.

Inorganic phosphate (Pi) is an essential component for structure and metabolism. PiT1 (SLC20A1) and PiT2 (SLC20A2) are members of the mammalian type-III inorganic phosphate transporters. SLC20A2 missense variants are associated with primary brain calcification. MicroRNAs (miRNAs) are endogenous noncoding regulatory RNAs, which play important roles in post-transcriptional gene regulation. MicroRNA-9 (miR-9) acts at different stages of neurogenesis, is deeply rooted in gene networks controlling the regulation of neural progenitor proliferation, and is also linked with cancers outside the nervous system. We evaluated possible interactions between miR-9 and the phosphate transporters (PiT1 and PiT2). SLC20A2, platelet-derived growth factor receptor beta (PDGFRB) and Fibrillin-2 (FBN2) showed binding sites with high affinity for mir-9, in silico. miR-9 mimic was transfected into HEK293 cells and expression confirmed by RT-qPCR. Overexpression of miR-9 in these cells caused a significant reduction in PiT2 and FBN2. PDGFRB appeared to be decreased, but was not significantly down-regulated in our hands. PiT1 showed no significant difference relative to controls. The down-regulation of PiT2 protein by miR-9 was confirmed by western blotting. In conclusion, we showed miR-9 can down-regulate PiT2, in HEK293 cells.

No description available.

BACKGROUND:Colony stimulating factor-1 (CSF-1) plays an important role in ovarian cancer biology and as a prognostic factor in ovarian cancer. Elevated levels of CSF-1 promote progression of ovarian cancer, by binding to CSF-1R (the tyrosine kinase receptor encoded by c-fms proto-oncogene).Post-transcriptional regulation of CSF-1 mRNA by its 3' untranslated region (3'UTR) has been studied previously. Several cis-acting elements in 3'UTR are involved in post-transcriptional regulation of CSF-1 mRNA. These include conserved protein-binding motifs as well as miRNA targets. miRNAs are 21-23nt single strand RNA which bind the complementary sequences in mRNAs, suppressing translation and enhancing mRNA degradation.RESULTS:In this report, we investigate the effect of miRNAs on post-transcriptional regulation of CSF-1 mRNA in human ovarian cancer. Bioinformatics analysis predicts at least 14 miRNAs targeting CSF-1 mRNA 3'UTR. By mutations in putative miRNA targets in CSF-1 mRNA 3'UTR, we identified a common target for both miR-128 and miR-152. We have also found that both miR-128 and miR-152 down-regulate CSF-1 mRNA and protein expression in ovarian cancer cells leading to decreased cell motility and adhesion in vitro, two major aspects of the metastatic potential of cancer cells.CONCLUSION:The major CSF-1 mRNA 3'UTR contains a common miRNA target which is involved in post-transcriptional regulation of CSF-1. Our results provide the evidence for a mechanism by which miR-128 and miR-152 down-regulate CSF-1, an important regulator of ovarian cancer.

Mémoire numérisé par la Direction des bibliothèques de l'Université de Montréal.

Mast cells are resident immune cells abundantly found in the tissue at the host-environment interface, where they play a critical role in inflammatory allergic responses. Mast cell responses may be regulated by the cytokine milieu at the site of inflammation. Recent studies have revealed microRNAs to be important in altering cytokine signaling in immune cells. Here, we demonstrate for the first time that IL-10 and IL-33 induce miR-155 and miR-146a, respectively, to alter mast cell functions. We report that IL-10 enhanced IgE induced activation of mast cells. IL-10 effects are dependent on Stat3 activation, which elicits miR-155 expression, resulting in a loss of suppressor of cytokine signaling-1 (SOCS-1). The importance of miR-155 was demonstrated by the inability of IL-10 to enhance anaphylaxis in miR-155–deficient mice. Additionally, we show that IL-33 treatment greatly enhances miR-146a expression in mast cells and in mast cell derived exosomes. miR-146a induction is dependent on MyD88 and NFκB and seems to negatively regulate ST2 signaling, which is demonstrated by the hyperresponsiveness of miR-146a knockout BMMC in response to IL-33. Our preliminary data suggest that miR-146a serves as a feedback negative regulator of IL-33 signaling by targeting IRAK proteins. miR-155 and miR-146a are key microRNAs that regulate a range of immune functions. Taken together, our results reveal two novel microRNA pathways that regulate mast cell IgE and IL-33 induced responses.

Les douleurs neuropathiques ayant une origine à la suite de blessures traumatiques du SNC ou du SNP sont particulièrement difficiles à traiter en utilisant les moyens thérapeutiques actuellement disponibles. Il est donc nécessaire d'identifier de nouvelles stratégies thérapeutiques. Notre objectif était donc de définir les mécanismes impliqués dans ces douleurs neuropathiques. LIMK1 est l'un des acteurs possibles de la réorganisation épinière qui caractérise les lésions nerveuses. Une fonction très caractérisé de cette protéine, est la phosphorylation d'une famille de protéines appelées « cofilines ». Sa phosphorylation, ce qui induit la réorganisation du cytosquelette d'actine. Récemment, il a été montré qu’un microARN (miARNs) nomé miR-134 régule l'expression de LIMK1 en se liant au messager de LIMK1 (ARNm), inhibant sa traduction en protéine physiologiquement active. Notre hypothèse était que la régulation de LIMK1 par miR-134 pourrait jouer un rôle essentiel dans la sensibilisation à la douleur. Cette régulation pourrait ainsi être liée non seulement à la modulation neurochimique neuronale mais aussi à la plasticité fonctionnelle associée. Au cours de cette thèse, l’HIS a montré une diminution de miR-134 chez des rats SNL (neuropathique), cette sous-expression était concomitante à une augmentation de LIMK1 illustrée par l’IHC. Il est important de noter ici que l'ISH est une méthode de détection connue récemment et qui a été identifiée pour visualiser les miARNs. Des différents protocoles de l’HIS ont également été discutés dans le cadre de cette thèse. Ce résultat a été confirmé par Le qRT-PCR . Par la suite, afin de vérifier les changements comportementaux douloureux induits par miR-134 et LIMK1. Nous avons effectués des injections intrathécales de siRNA anti-LIMK1 pour inhiber l'expression endogène de LIMK1 chez les SNL. C’était intéressant de ne pas avoir trouvé aucun changement comportemenal chez les SNL après ce type d’injection. Une surexpression artificielle de miR-134 en utilisant un précurseur de miR-134 (premiR-134) chez les SNL a montré le même effet. Ensuite, nous avons essayé d'effectuer les mêmes injections chez les Sham (control), et c’était plus intéressant de trouver que ces injections (siRNA LIMK1 et premiR-134) ont provoqué une hypersensibilité douleureuse chez les sham. Cela a été illustré au moyen de deux tests de comportement; le Von Frey (VF) et la distribution pondérale dynamique (DWB). Pour etudier l'effet inverse, nous avons inhibé miR-134 en utilisant une sonde spécifique KD (Knock-Down); une diminution significative inattendue dans le seuil de retrait a été observée avec VF et DWB. qRT-PCR dans la plupart de ces cas, a confirmé la corrélation in vivo entre miR-134 et LIMK1. Enfin, nous avons cherché un mécanisme d'action possible qui pourrait réguler cette modulation. Des données récentes publiées ont montré une implication de l'ADF/cofiline sur le trafic des récepteurs AMPA (AMPAR). En accord avec les résultats mentionnés ci-dessus, la transfection du KD de miR-134 a montré une diminution dans AMPAR adressés à la membrane plasmique. Tout ensemble ces données suggèrent que l'effet antinociceptif de KD de miR-134 et la surexpression de LIMK1 sont indirectement régulé par l'insertion des AMPAR à la membrane plasmique.Il semble que miR-134 exerce un effet différent sur la douleur neuropathique que miR-103, discuté aussi dans le cadre de cette thèse. Il était demontré comme un régulateur de plusieurs cibles, les trois sous-unités formant les canaux calciques de type-L « Cav1.2 LTC ». MiR-103 a été trouvé également réprimés chez les SNL. La surexpression de miR-103 soulage la douleur neuropathique. Contrairement au miR-134, miR-103 exerce un rôle pronociceptive pendant la douleur neuropathique. / Pains having a neuropathic origin following CNS or PNS traumatic injury are particularly difficult to treat using the actually available therapeutic means. It is thus necessary to identify new therapeutic strategies. Hence, our aim was to define the mechanisms implicated in these neuropathic pains. Nervous lesions are characterised by an anatomical reorganization of the neuronal network of the dorsal horn. Neurochemical alterations are also involved. Some of the molecular mechanisms underlying the neuronal plasticity (a main feature of neuropathic pain) have been emphasized here by a variety of complementary technical approaches. LIMK1 is one of the possible actors of this reorganization. Among this protein’s known functions, and the most characterized is the phosphorylation of a family of proteins known as cofilins. Their phosphorylation induces the reorganization of actin cytoskeleton. Recently, it has been shown that a miR-134 miRNA regulates LIMK1 expression by binding to the LIMK1 messenger, inhibiting its translation into physiologically active protein. Our hypothesis is that LIMK1 regulation by miR-134 might play an essential role in pain sensitization by modulating neuron neurochemical reorganization and the associated functional neuronal plasticity. Firstly, by means of IHC and ISH, we studied miR-134/LIMK1 distribution within the dorsal horn of the spinal cord in sham animal (control group) and in neuropathic pain model (SNL model). Important to note here that ISH is a known detection method recently identified to visualize miRNA. Different protocols of ISH were discussed in a part of this thesis. ISH showed a decrease in miR-134 expression in SNL rats concomitantly with an increase in LIMK1 illustrated by IHC. This finding has been confirmed by qRT-PCR techniques. Afterward, in order to check for the possible behavioural-induced changes of miR-134 and LIMK1. We intrathecally injected an anti-LIMK1 siRNA to inhibit endogenous LIMK1 expression in SNL rats. Interestingly no significant changes in pain behaviour have been observed. Artificial overexpression of miR-134 using a PremiR-134, showed the same effect. Then we tried to perform the same injections on sham rats, and more interestingly, siRNA LIMK1 and premiR-134 evoked pain hypersensitivity in shams rats. This was illustrated by means of two behaviour tests; Von Frey (VF) and the Dynamic Weight bearing (DWB). To explore the reverse effect, we inhibited miR-134 using a specific KD probe in SNL rats; unexpectedly a significant decrease in pain withdrawal threshold was observed with VF and DWB. qRT-PCR in most cases confirmed the in vivo correlation between miR-134 and LIMK1. Finally, we searched for the possible mechanism of action that could regulate this modulation. Recent published data showed an involvement of ADF/cofilin on AMPAR trafficking. In line with the above mentioned findings, miR-134 KD transfection showed a decrease in AMPAR addressed to the plasma membrane. Altogether suggest that the antinociceptive effect of miR-134 KD and LIMK1 overexpression are mediated by AMPAR insertion at the plasma membrane. It seems that miR-134 exerts a different effect on neuropathic pain than miR-103 another miRNA discussed within the frame of this thesis. MiR-103 has been proved to regulate multiple targets, the three subunits forming Cav1.2 LTC. Pain sensitization involves Cav1.2 activation which consequently alters gene expression during this form of plasticity. MiR-103 was found downregulated also in the SNL model. Conversely to miR-134, overexpression of miR-103 partially alleviates pain. It decreases pain withdrawal threshold of the Von Frey test. Unlike miR-134, miR-103 exerts a pronociceptive role during neuropathic pain.