Dysregulated microRNAs in osteoclasts could cause many skeletal diseases. The therapeutic manipulation of these pathogenic microRNAs necessitates novel, efficient delivery systems to facilitate microRNAs modulators targeting osteoclasts with minimal off-target effects. Bone resorption surfaces characterized by highly crystallized hydroxyapatite are dominantly occupied by osteoclasts. Considering that the eight repeating sequences of aspartate (D-Asp8) could preferably bind to highly crystallized hydroxyapatite, we developed a targeting system by conjugating D-Asp8 peptide with liposome for delivering microRNA modulators specifically to bone resorption surfaces and subsequently encapsulated antagomir-148a (a microRNA modulator suppressing the osteoclastogenic miR-148a), i.e. (D-Asp8)-liposome-antagomir-148a. Our results demonstrated that D-Asp8 could facilitate the enrichment of antagomir-148a and the subsequent down-regulation of miR-148a in osteoclasts in vivo, resulting in reduced bone resorption and attenuated deterioration of trabecular architecture in osteoporotic mice. Mechanistically, the osteoclast-targeting delivery depended on the interaction between bone resorption surfaces and D-Asp8. No detectable liver and kidney toxicity was found in mice after single/multiple dose(s) treatment of (D-Asp8)-liposome-antagomir-148a. These results indicated that (D-Asp8)-liposome as a promising osteoclast-targeting delivery system could facilitate clinical translation of microRNA modulators in treating those osteoclast-dysfunction-induced skeletal diseases.

Orientador: Iscia Lopes-Cendes / Tese (doutorado) - Universidade Estadual de Campinas, Faculdade de Ciencias Medicas / Made available in DSpace on 2018-08-15T13:20:17Z (GMT). No. of bitstreams: 1 Dogini_DanyellaBarbosa_D.pdf: 2328676 bytes, checksum: 73a9334f34715cbcfdd00b38ccd1a93f (MD5) Previous issue date: 2010 / Resumo: MicroRNAs são moléculas recém-descobertas de RNA não-codificadores que possuem de 21 a 24 nucleotídeos e que regulam a expressão após a transcrição dos genes alvo. Essa regulação pode ser realizada através da inibição da tradução ou da degradação do RNA mensageiro. Os miRNAs estão envolvidos em vários processo biológicos como, diferenciação celular e desenvolvimento embrionário, além de apresentarem expressão tecido e tempo-específica. Eles podem regular a expressão de pelo menos 1/3 de todos os genes humanos e estão envolvidos com a regulação do metabolismo e da apoptose. Os miRNAs são a chave como reguladores pós-transcricionais da neurogênese; estudos mostram que eles possuem a expressão associada com a transição entre proliferação e diferenciação e também tem expressão constitutiva em neurônios maduros, evidenciando o envolvimento dessas moléculas com o desenvolvimento do sistema nervoso central (SNC). Outros miRNAs estão sendo estudados e verifica-se que eles agem como reguladores de genes envolvidos em doenças como Alzheimer, Parkinson e, provavelmente, também devam possuir um papel na regulação das epilepsias. No primeiro trabalho, apresentado no segundo capítulo, investigamos o papel dos miRNAs no desenvolvimento do SNC através da quantificação de 104 miRNAs em cérebros em desenvolvimento de camundongos. No segundo trabalho, apresentado no terceiro capítulo, para analisarmos o papel dos miRNAs na epilepsia de lobo temporal, verificamos se havia presença de miRNAs com expressão diferenciada entre tecidos removidos de pacientes que se submeteram a cirurgia de hipocampectomia e tecidos normais provenientes de autópsias. Para ambos os experimentos, foram extraídos os RNAs dos tecidos e quantificados por PCR em tempo real com o kit MicroRNA Assay baseado em iniciadores com estrutura em stem loop. Nos camundongos, análises de bioinformática encontraram quatro cluster de acordo com a expressão dos miRNAs. Um cluster (C1) com 12 miRNAs (miR-9; miR-17- 5p; miR-124a; miR-125a; miR-125b;miR-130a; miR-140; miR-181a; miR-199a; miR-205; miR-214; miR-301) apresentou expressão com diferença significativa durante o desenvolvimento. Nos tecidos dos pacientes, após a análise de bioinformática, encontramos três miRNAs com expressão diferenciada entre pacientes e controle (miR-29b, miR-30d e let-7). Em ambos os experimentos analisamos os possíveis genes alvo desse miRNAs. Nos camundongos, nossos resultados sugerem a presença de um padrão específico de expressão no cluster C1, indicando que esses miRNAs possam ter um papel na regulação de genes envolvidos na neurogênese. Nos tecidos humanos, os genes alvo encontrados estão envolvidos, principalmente, em proliferação celular, neurogênese e apoptose, indicando uma provável atuação dos miRNAs na regulação de genes que estão envolvidos na epilepsia de lobo temporal / Abstract: MicroRNAs are a new class of small RNA molecules (21-24 nucleotide-long) that negatively regulate gene expression either by translational repression or target mRNA degradation. It is believed that about 30% of all human genes are targeted by these molecules. MiRNAs are involved in many important biological processes including cell differentiation, embryonic development and central nervous system formation, besides they showed specific temporal-space expression. They can regulate 1/3 of human genes and are involved in metabolism and apoptosis. miRNAs are the key as neurogenesis postranscriptional regulation; studies previous indicates miRNA expression associate with proliferation and differentiation in development of central nervous system (CNS) and housekeeping expression in mature neurons. They are involved in several diseases as Alzkeimer's and Parkinson and may have a role in epilepsy regulation. In second chapter, we analyze the miRNA expression in mouse brain during four stages of CNS development; in third chapter, we analyze hippocampal tissue of four patients who underwent selective resection of the mesial temporal structures for the treatment of clinically refractory seizures. In addition we used control samples from autopsy (n=4) for comparison. In both experiments, total RNA was isolated from tissues and used in real-time PCR reactions with TaqMan¿ microRNA assays (Applied Biosystems) to quantify 104 (mouse brain) or 157 (human tissue) different miRNAs. In mouse brain analysis, we were able to identified four different clusters (C1, C2, C3 and C4) of miRNAs expression. Significant differences in expression during development were observed only in miRNAs included in C1. Our results suggest the presence of a specific expression pattern in C1, indicating that these miRNAs could have an important role in gene regulation during neurogenesis. We found a significant decrease (p<0,05) in expression of 12 miRNAs (miR-9; miR-17-5p; miR-124a; miR-125a; miR-125b;miR-130a; miR-140; miR-181a; miR-199a; miR-205; miR-214; miR- 301) belonging to cluster C1 in latter stages of development. Computational target identification showed that 10 of the 12 miRNAs present in C1 could be involved in neurogenesis. In human tissues, bioinformatics analyzes identified three miRNAs species which were differently expressed in patients as compared to controls: let7a was over expressed in patients (4 fold increased), miR-29b and miR-30d were down-regulated in patients (2.5 fold and 0.5 fold decreased, respectively). Possible target genes for let-7a are NME6 and NCAM1 (which would be down-regulated in patients); for miR-29b is MCL-1 and for miR30d are CTNND2, LGI1 and SON (which would be up-regulated in patients). We have identified three different miRNA species differently expressed in hippocampal sclerosis. Gene functions related to the possible miRNA targets are involved mainly with cell proliferation, neurogenesis, cell adhesion and apoptosis. Our results indicate new molecular targets which should be explored in additional studies addressing miRNA regulation in hippocampal sclerosis / Doutorado / Neurociencias / Doutor em Fisiopatologia Medica

Insufficient maternal folate during pregnancy increases the risk of the baby being small for gestational age (SGA). Studies in teenagers, a population vulnerable to folate deficiency and SGA birth, have shown that low maternal folate status is associated with impaired placental cell turnover and reduced transport suggesting placental dysfunction underlies SGA in maternal folate deficiency. Mechanisms through which folate-depletion compromises placental function are currently unknown. In non-placental cells, folate modulates microRNAs (miRs), post-transcriptional regulators of cellular functions. Expression of miRs is altered in placentas of SGA compared to normally grown babies but there are no data on differential miR expression or regulation in placentas from folate deficient women. This PhD investigated the hypothesis that placental dysfunction observed in folate deficient women is mediated by altered miR expression. Three placental preparations were compared (villous tissue in explant culture, BeWo choriocarcinoma cells and isolated cytotrophoblast cells) to determine the optimum in vitro system to study the direct effects of folate deficiency. In cytotrophoblast cells, folate deficiency significantly elevated apoptosis and reduced the activity of the system A amino acid transporter, consistent with observations in the placentas of folate-deficient teenagers. The reduction in system A activity by low folate was not associated with altered mRNA expression for the isoforms of system A, implicating an effect of low folate on post-translational regulation of the nutrient transporter. Targeted examination of villous tissue from teenagers with low folate status identified up-regulation of miR-222-3p a folate-sensitive miR. An unbiased miR array identified up-regulation of a further 16 miRs suggesting that maternal folate deficiency in vivo results in aberrant placental miR expression. Bioinformatic analysis of the folate sensitive miRs predicted gene targets known to be altered in placentas from SGA pregnancy that were likely to alter placental function. Two miRs altered in placentas from women with low folate status, miR-30e-3p and miR-34b-5p, were also significantly altered in folate deficient cytotrophoblasts confirming a direct effect of folate on trophoblast miR expression. Inhibition of these miRs in vitro had no effects on placental functions that are altered in vivo in folate-deficient women. Gene array and in silico analysis identified functional endpoints affected by these folate sensitive miRs, including cell signalling for proliferation and survival and oxidative stress, which might contribute to placental dysfunction in folate deplete women. Overall, this study has demonstrated for the first time that folate deficient conditions can directly alter trophoblast system A transport and cell survival and thus could contribute to the increased susceptibility to SGA births in folate deficient women. It has also contributed to the knowledge that miR expression is differentially altered in placentas exposed to folate-deficient versus sufficient conditions in vivo and that miRs are directly altered by folate depletion in vitro. These studies provide the foundation for future research to define the functional consequences of altered expression of folate-sensitive miRs and their target genes to explain how altered miRs could be affecting placental function resulting in development of SGA.

The E2F family of transcription factors regulate cellular growth, death and differentiation, but their role in cardiac biology remains to be fully explored. We hypothesized that the balance of the E2F pathway would determine cardiac development and function. We provide evidence for this via modulation of the E2F6 repressor, in a transgenic (Tg) mouse model. Targeted expression of E2F6 in the heart led to dilated cardiomyopathy (DCM) and death. Microarray analysis revealed that E2F responsive pathways were activated in Tg mice. Furthermore, we found that E2F6 and YY1 (E2F-co-factor) were translocated to the nucleus in Tg mice, providing a potential mechanism for the observed transcriptional activation. We also observed a marked decrease of Connexin43 protein in the myocardium, and reduced atrial conductivity in Tg mice which may lead to reduced cardiac function. The data demonstrates a novel role for E2F pathway outside of cell cycle control in the heart.

Cultures of peripheral blood mononuclear cells (MNCs) give rise to at least two different variants of endothelial progenitor cells (EPCs), early and late outgrowth EPCs. We investigated whether microRNAs in early and late EPCs could serve as markers of internal processes that can be exploited to distinguish cell identity and functional capacity. We hypothesized that as MNCs give rise to early and late EPCs, there is a gradual change in total microRNA profile, reflecting a total change in processes within the predominant cell population. Using a candidate microRNA array, early and late EPCs showed vastly different microRNA expression profiles. MiR-146a expression increased progressively as early EPCs emerged around 5-7 days (p<0.05). Through targeting TRAF6 and IRAK1, miR-146a conferred inflammatory tolerance in early EPCs, likely contributing to their purported ability to suppress inflammation. MiR-146a knock down (KD) in endotoxin-stimulated early EPCs reduced anti-inflammatory cytokine IL-1RA (p<0.001), and increased expression of pro-inflammatory cytokines IL-1 (p<0.001) and IL-8 (p<0.01). Interestingly, the microRNA expression profile of late EPCs was highly congruent to mature endothelial cells, with 100-fold greater miR-126 expression than monocytes and early EPCs (p<0.01). MiR-126KD in late EPCs abolished matrigel-network formation (p<0.05); while overexpression (OE) in early EPC augmented network formation (p<0.05) and chemotactic migration (p<0.001). We also found that the melanoma cell adhesion molecule or MCAM (CD146) identified late EPC precursors. Only MCAM+MNCs from adult blood (<5% of total MNCs) yielded late EPC-like colonies. Robust miR-126 expression in these cells predicted the generation of late EPCs. Overall, our results suggest that miR-146a in early EPCs likely contributes to repair by suppressing inflammation during cardiovascular injury; while in late EPCs, miR-126 directly promotes angiogenesis and vascular repair. Finally, we highlight a unique method for the efficient generation of late EPCs by using MCAM selection and screening for miR-126.

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.

This thesis was presented by Aaron Webber on the 4th December 2013 for the degree of Doctor of Philosophy from the University of Manchester. The title of this thesis is ‘Transcriptional co-regulation of microRNAs and protein-coding genes’. The thesis relates to gene expression regulation within humans and closely related primate species. We have investigated the binding site distributions from publically available ChIP-seq data of 117 transcription regulatory factors (TRFs) within the human genome. These were mapped to cis-regulatory regions of two major classes of genes,  20,000 genes encoding proteins and  1500 genes encoding microRNAs. MicroRNAs are short 20 - 24 nt noncoding RNAs which bind complementary regions within target mRNAs to repress translation. The complete collection of ChIP-seq binding site data is related to genomic associations between protein-coding and microRNA genes, and to the expression patterns and functions of both gene types across human tissues. We show that microRNA genes are associated with highly regulated protein-coding gene regions, and show rigorously that transcriptional regulation is greater than expected, given properties of these protein-coding genes. We find enrichment in developmental proteins among protein-coding genes hosting microRNA sequences. Novel subclasses of microRNAs are identified that lie outside of protein-coding genes yet may still be expressed from a shared promoter region with their protein-coding neighbours. We show that such microRNAs are more likely to form regulatory feedback loops with the transcriptional regulators lying in the upstream protein-coding promoter region. We show that when a microRNA and a TRF regulate one another, the TRF is more likely to sometimes function as a repressor. As in many studies, the data show that microRNAs lying downstream of particular TRFs target significantly many genes in common with these TRFs. We then demonstrate that the prevalence of such TRF/microRNA regulatory partnerships relates directly to the variation in mRNA expression across human tissues, with the least variable mRNAs having the most significant enrichment in such partnerships. This result is connected to theory describing the buffering of gene expression variation by microRNAs. Taken together, our study has demonstrated significant novel linkages between the transcriptional TRF and post-transcriptional microRNA-mediated regulatory layers. We finally consider transcriptional regulators alone, by mapping these to genes clustered on the basis of their expression patterns through time, within the context of CD4+ T cells from African green monkeys and Rhesus macaques infected with Simian immunodeficiency virus (SIV). African green monkeys maintain a functioning immune system despite never clearing the virus, while in rhesus macaques, the immune system becomes chronically stimulated leading to pathogenesis. Gene expression clusters were identified characterizing the natural and pathogenic host systems. We map transcriptional regulators to these expression clusters and demonstrate significant yet unexpected co-binding by two heterodimers (STAT1:STAT2 and BATF:IRF4) over key viral response genes. From 34 structural families of TRFs, we demonstrate that bZIPs, STATs and IRFs are the most frequently perturbed upon SIV infection. Our work therefore contributes to the characterization of both natural and pathogenic SIV infections, with longer term implications for HIV therapeutics.

Genetic dissection of loci important in the control of neurogenesis has improved our understanding of both the evolutionarily conserved and divergent processes in neurodevelopment. These loci include not only protein coding genes [1, 2], but also noncoding RNAs [3-5]. One important family of non-coding RNAs is miRNAs, which control gene expression fundamental in developmental regulation and mature cell maintenance [3, 5-9]. Here, we will first focus our efforts by surveying miRNA regulation in the developing brain. We hypothesize a strong regulatory role of miRNAs during proliferation, cell death, migration and differentiation in the developing mammalian forebrain that has yet to be adequately described in the literature. Second, we will assess miRNA's role in the evolutionary divergence of brain-related gene expression. We hypothesize that a human specific single nucleotide change(s) in the miRNA recognition element of transcription factors 3' untranslated regions contributes to species-specific differences in transcription factor expression and ultimately alters regulatory function.

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.

Macrophages are central players during atherosclerosis. Especially, macrophage cholesterol efflux, which promote the removal of free cholesterol from foam cells, are crucial to prevent lipid accumulation and reverse atherogenesis. microRNAs (miRNAs) are important regulators of various pathways involved in atherosclerosis. During inflammation, macrophages secrete extracellular vesicles (EVs) carrying miRNAs to communicate signals to nearby cells. However, the role of macrophage-derived EVs in atherogenesis is not known. In the first study, we find that EVs derived from cholesterol-loaded macrophages can inhibit macrophage migration in vitro and in vivo. This effect appears to be mediated by the transfer of several miRNAs, including miR-146a, to recipient macrophages where they repress the expression of specific pro-migratory target genes Igf2bp1 and HuR. Our studies suggest that EV-derived miRNAs secreted from atherogenic macrophages may accelerate the development of atherosclerosis by decreasing cell migration and promoting macrophage entrapment in the vessel wall. Understanding macrophage communication via EVs provided the rationale for the design of nanoparticles (NPs) that mimic macrophage EVs to deliver beneficial miRNAs to the atherosclerotic plaque. While cationic lipid/polymer-based NPs have been employed as systemic delivery vehicles of siRNA, none of these have been used to deliver miRNAs to macrophages in vivo. In the second study, we developed a chitosan NP platform for effective delivery of miRNAs to alter macrophage function in vivo. We showed that our NPs made using a cross-linked chitosan polymer can protect as well as transfer miR-33 to naïve macrophages and regulate the expression of its target gene (Abca1) as well as cholesterol efflux in vitro and in vivo. Finally, almost all miRNAs that have been characterized are efflux-repressing miRNA, thereby accelerating atherosclerosis. miR-223 is one of a few miRNAs whose overexpression can promote cholesterol efflux, modulate the inflammatory response, and thus, be antiatherogenic. However, its contribution to the pathogenesis of atherosclerosis in vivo and the mechanism underlying its effects has not been thoroughly characterized. We herein find that miR-223 is capable of suppressing plaque development via modulating cholesterol efflux and inflammatory responses, thus may serve as a potential therapeutic to reduce atherosclerosis. These effects of miR-223 appear to be dependent on the inhibition of its target gene, the transcription factor Sp3. Overall, this thesis highlights the importance of both endogenous and extracellular miRNAs in controlling different aspects of atherogenic response.