Дисертації з теми "MiRNA target"

Vorhersagen von miRNA-Bindestellen enthalten oft einen hohen Prozentsatz an falsch positiven Ergebnissen (24-70%). Gleichzeitig ist es schwierig die biologischen Interaktionen von miRNAs und ihren Zieltranskripten auf experimentellem Wege und Genom weit zu messen. Daher wurde in der vorliegenden Arbeit die Frage beantwortet, ob ChIP-Sequenzierungsdaten, von denen es immer mehr gibt, verwendet werden können, um Vorhersagen von miRNA-Bindestellen zu filtern. Dabei wurde von einem Netzwerk aus miRNAs und Transkriptionsfaktoren gebraucht gemacht, die Zieltranskripte gemeinsam regulieren. Zunächst wurden verschiedene Methoden getestet, mit denen „Peaks“ aus der ChIP-Sequenzierung Zielgenen zugeordnet werden können. Zielgenlisten des transkriptionalen Repressors RE1-silencing transcription factor (REST/NRSF) wurden mithilfe von ChIP-Sequenzierungsdaten erzeugt. Ein Algorithmus zur Suche nach überrepräsentierten miRNA-Zielgenen in REST-Genlisten basierend auf Vorhersagen von TargetScanHuman wurde entwickelt und angewandt. Die detektierten „enrichment“-miRNAs waren Teil eines vielfältig regulierten REST-miRNA-Netzwerks. Mögliche Funktionen von miRNAs wurden vorgeschlagen und ihre Rolle im gemeinsamen Netzwerk mit REST und im damit gebildeten Netzwerkmotiv (Inkoherente Schleife zur Vorwärtskopplung Typ 2) wurde analysiert. Es stellte sich heraus, dass ein Filtern der Vorhersagen tatsächlich möglich ist, da Gene, die sowohl von REST als auch von einer oder mehreren „enrichment“-miRNAs reguliert werden, einen höheren Anteil an wahren miRNA-Transkript-Interaktionen haben.
Predictions of miRNA binding sites suffer from high false positive rates (24-70%) and measuring biological interactions of miRNAs and target transcripts on a genome wide scale remains challenging. In the thesis at hand the question was answered if the ever growing body of ChIP-sequencing data can be applied to filter miRNA target predictions by making use of the underlying regulatory network of miRNAs and transcription factors. First different methods for association of ChIP-sequencing peaks to target genes were tested. Target gene lists of the transcriptional repressor RE1-silencing transcription factor (REST/NRSF) were generated by means of ChIP-sequencing data. An enrichment analysis tool based on predictions from TargetScanHuman was developed and applied to find ‘enrichment’-miRNAs with over-represented targets in the REST gene lists. The detected miRNAs were shown to be part of a highly regulated REST-miRNA network. Possible functions could be assigned to them and their role in the regulatory network and special network motifs (incoherent feedforward loop of type 2) was analyzed. It turned out that miRNA target predictions of genes shared by enrichment-miRNAs and REST had a higher proportion of true positive associations than the TargetScanHuman background, thus the procedure made a filtering possible.

L’objectif de mon projet principal de thèse est de déterminer la fonction biologique d’un lncARN conservés chez le zebrafish que nous avons appelé libra. La séquence de libra étant hautement homologue à la région 3’UTR de la protéine Nrep. Ces deux transcrits, libra et Nrep, contiennent en effet un site de liaison au miARN profondément conservé et inhabituellement complémentaire au miR-29. En utilisant à le modèle souris et les cellules murines, nous avons décrypté la relation régulatrice entre ce transcrit conservé dans l’évolution des vertébrés et la voie métabolique des miARN. Nous avons montré que Nrep limite le domaine d’expression de miR-29 au cervelet, et qu’il le déstabilise en rognant sa séquence. Notre travail révèle donc le premier exemple de dégradation endogène ciblée des miARN (ou TDMD). De plus, un ensemble d’expériences in vivo sur les modèles zebrafish et souris, nous a permis de démontrer que libra et Nrep contrôlent tout les deux le comportement animal. Via la perturbation génétique du site de liaison au miARN de Nrep murin, nous avons observé que ce gène régule le dosage du miR29 de part son site de liaison aux miARN, et que cette régulation est nécessaire à un comportement animal normal. Dans la seconde partie de ma thèse, je décris une stratégie exploré afin de déréguler les lncARN de la manière la moins invasive possible. Les lncARN sont actuellement neutralisés par des approches qui introduisent de vastes changements de séquence au niveau génomique. Nous avons donc développer une stratégie in vivo, appliquée au zebrafish, qui inactive les lncARN via l’insertion génomique d’une séquence ribozyme autoclivante ou d’un signal polyA prématuré
The goal of my main thesis project was to determine the biological function of a deeply conserved zebrafish long noncoding RNAs (lncRNA) which we called libra. libra shows sequence similarity with the 3'UTR of the NREP a protein coding transcript. Both libra and Nrep contain a deeply conserved and unusually complementary microRNA (miRNA) binding site for miR-29. Using both the mouse model and mouse cell lines, we deciphered the regulatory relationship between this conserved transcript and the miRNA pathway. We showed that Nrep restricts the spatial expression domain of miR-29 in the cerebellum and that it destabilizes miR-29 through 3' trimming. Until now, only viral transcripts and artificial reporters engineered to contain highly complementary miRNA binding sites have been shown to regulate miRNAs in this fashion. Thus, our work uncovers the first example of endogenous target-directed miRNA degradation (TDMD). In addition, through a set of in vivo experiments in zebrafish and mouse, we showed that both libra and Nrep control normal animal behavior. By genetically disrupting the miR-29 binding site in Nrep in mouse, we showed that Nrep regulates miR-29 dosage through its miR-29 site and controls animal behavioral. In a second part of my thesis I describe a strategy to genetically downregulate lncRNAs in a minimally invasive manner. Approaches to knock-out lncRNAs that do not introduce vast sequence changes at the genomic level have not been adequately developed yet. I present our in vivo strategy applied to the zebrafish model using a genomic knock-in of a self-cleaving ribozyme sequence and a premature poly(A) signal to knock-out lncRNAs

Pancreatic cancer (pancreatic ductal adenocarcinoma, PDAC) is one of the most lethal tumour types world-wide. The majority of patients present late with locally advanced or metastatic disease. Therefore, despite advances in operative techniques, perioperative management and oncological treatments, the overall 5-year survival remains <5%. Determining tumoural factors that contribute towards its aggressive nature may help in identifying novel molecular biomarkers and/or therapeutic targets. MicroRNAs (miRNAs) are small non-coding RNAs that negatively regulate target gene expression and are able to act as tumour suppressors or oncogenes. MiRNAs have been extensively profiled and implicated in the initiation and progression of PDAC. Furthermore, there is a possibility of translating miRNAs into clinically useful biomarkers. Here, I developed upon these initial observations and demonstrate that miRNAs can be used to differentiate low risk pancreatic benign cystic tumours (BCTs) from PDAC. We confirmed that these miRNAs regulate the expression of known PDAC oncogenes, and that miR-16, miR-126 and let-7d target BCL2, CRK and KRAS respectively. Next, in order to investigate the main contributors to tumourigenesis, an integrated molecular analysis (miRNA-mRNA) was performed in PDAC. By using a combination of network-based bioinformatics, miR-21, miR-23a and miR-27a were prioritised as important in PDAC progression. We demonstrated that the use of a combination of miRNA inhibitors (against miR-21, miR-23a and miR-27a) in a murine subcutaneous PDAC xenograft model was able to reduce tumour growth, better than oncomiR-21 inhibition alone. BTG2 and NEDD4L were found to be direct targets of the miRNA combination and were established as new candidate tumour suppressors in PDAC. The clinical relevance of this 3 miRNA signature was demonstrated, as high expressors of the combination have poor overall survival after surgical resection, independent of other clinicopathologic factors. Together, these studies identify specific miRNAs as important regulators of PDAC tumourigenesis and their possible use as biomarkers.

Thesis: Ph. D., Massachusetts Institute of Technology, Department of Biology, 2014.
Vita. Cataloged from PDF version of thesis.
Includes bibliographical references.
regulators of gene expression in a wide range of organisms and biological processes. Each miRNA guides Argonaute (Ago) protein complexes to target and repress hundreds of genes in a sequence-dependent manner. To identify all targets of miRNA regulation, we performed UV crosslinking and immunoprecipitation (CLIP) of Ago complexes in mouse embryonic (ESC) and mesenchymal (MSC) stem cell lines. We also captured the genome-wide miRNA-independent binding footprint of Ago by performing CLIP in cells that lack Dicer, an enzyme required for mature miRNA biogenesis. We surprisingly found that Ago bound a similar set of genes in the absence of Dicer, and this overlap in target genes was due partially to residual, unprocessed miRNAs in the Dicer KO cells. Other potential sites of miRNA-independent Ago interactions, such as histone transcripts and poly-A cleavage and polyadenylation sites, were also identified. One Ago CLIP dataset revealed the enrichment for a G-rich sequence motif at Ago target sites. We later demonstrated that the G-motif is not directly bound to Ago but rather is enriched near miRNA-guided Ago binding sites. Its presence near miRNA target sites is associated with stronger repression of Ago-miRNA targets. Fortuitously, the original Ago CLIP dataset that identified the G-motif was later shown to likely contain target sites of another co-immunoprecipitating RNA binding protein (RBP). Using mass spectroscopy of Ago antibody immunoprecipitations from Ago KO cells, we identified a list of interacting RBPs that could potentially augment Ago-miRNA activity through the G-motif. To investigate target competition in miRNA networks, we related our CLIP analysis of genome-wide, quantitative Ago binding to measurements of absolute miRNA and target RNA concentrations. We found that all miRNAs other than the miR-290 family in ESCs and let-7 family in MSCs were expressed at concentrations below their total target pool. However, 8-12 miRNA families were expressed at near or greater than equimolar ratios with their pool of high affinity targets, and this affinity-partitioned stoichiometry led to significant Ago accumulation and repression of high affinity target sites despite little consequential binding at low affinity sites. Single-cell reporter assays demonstrated that high expressed miRNAs are not susceptible to physiological inductions of competing target transcripts but targets of lower expressed miRNAs are derepressed in a weakly threshold-like manner upon increased target pool levels. In summary, we identify a network of confidently bound targets of miRNA regulation in ESCs and MSCs, reveal the extent of miRNA-independent binding in these two cell types, provide a list of potential miRNA enhancer RBPs, and create a quantitative context for evaluating target competition in miRNA networks.
by Andrew D. Bosson.
Ph. D.

The related genes lin28a and lin28b code for conserved RNA-binding proteins, which contain two key RNA-binding motifs that determine their functions. Previously, our laboratory identified lin28a as a putative downstream target of FGF signalling in the early Xenopus embryo. This was found to occur at gastrulation stage, during which key signalling pathways such as the FGF pathway are active in specifying germ layer development. lin28 is a heterochronic gene in C. elegans and controls the timing of developmental events. In vertebrates, the lin28a gene shows pluripotent-specific expression, and has come to particular interest as one of four factors used to successfully re-program differentiated somatic cells into induced pluripotent stem cells. Both lin28a and lin28b have a high prevalence of ectopic expression in cancer. A well characterised target of both lin28a and lin28b is the microRNA let-7, inhibiting biogenesis to the mature microRNA form. The lin28 proteins have also been shown to potentiate translation of numerous mRNA targets. The aim of this project was to identify if lin28 has an important developmental role in vertebrates. Work in the Xenopus tropicalis embryo found that both lin28a and lin28b were targets of FGF signalling at gastrulation, and were required for correct germ layer patterning at this stage. In order to explore conservation in humans, mesenchymal stem cells were used to model the effects of FGF signalling upon the mesodermal germ layer, and whether lin28 played a role in this. Additional pluripotent cell models were investigated for their suitability in which to study lin28 function. Analysis of lin28 targets in Xenopus revealed that the miR-17-92 cluster family of miRNA may be positively regulated by the lin28 proteins, with a direct interaction possible with a member of these: pre-mir-363. These miRNAs may have further important developmental roles in response to this regulation by the lin28 proteins.

RNA is a highly versatile molecule with functions that span from being a messenger in the transfer from DNA to protein, a catalytic molecule important for key processes in the cell to a regulator of gene expression. The post-genomic era and the use of new techniques to sequence RNAs have dramatically increased the number of regulatory RNAs during the last decade. Many of these are antisense RNAs, as for example the miRNA in eukaryotes and most sRNAs in bacteria. Antisense RNAs bind to specific targets by basepairing and thereby regulate their expression. A major step towards an understanding of the biological role of a miRNA or an sRNA is taken when one identifies which target it regulates. We have used RNA libraries to study the RNA interference pathway during development in the unicellular model organism Dictyostelium discoideum. We have also, by combining computational and experimental methods, discovered the first miRNAs in this organism and shown that they have different expression profiles during development. In parallel, we have developed a novel approach to predict targets for sRNAs in bacteria and used it to discover sRNA/target RNA interactions in the model organism Escherichia coli. We have found evidence for, and further characterized, three of these predicted sRNA/target interactions. For instance, the sRNA MicA is important for regulation of the outer membrane protein OmpA, the sRNAs OmrA and OmrB regulate the transcription factor CsgD, which is important in the sessile lifestyle of E. coli, and MicF regulates its own expression in a feed forward loop via the regulatory protein Lrp. In conclusion, we have discovered novel antisense RNAs, e.g. miRNAs in D. discoideum, developed an approach to identify targets for antisense RNAs, i.e. a target prediction program for sRNAs in bacteria, and verified and characterized some of the predicted antisense RNA interactions.

La principale contribution de cette thèse est le développement d'une méthode fiable, robuste, et rapide pour la prédiction des pré-miARNs. Deux objectifs avaient été assignés : efficacité et flexibilité. L'efficacité a été rendue possible au moyen d'un algorithme quadratique. La flexibilité repose sur deux aspects, la nature des données expérimentales et la position taxonomique de l'organisme (en particulier plantes ou animaux). Mirinho accepte en entrée des séquences de génomes complets mais aussi les très nombreuses séquences résultant d'un séquençage massif de type NGS de “RNAseq”. “L'universalité” taxonomique est obtenu par la possibilité de modifier les contraintes sur les tailles de la tige (double hélice) et de la boule terminale. Dans le cas de la prédiction des miARN de plantes la plus grande longueur de leur pré-miARN conduit à des méthodes d'extraction de la structure secondaire en tige-boule moins précises. Mirinho prend en compte ce problème lui permettant de fournir des structures secondaires de pré-miARN plus semblables à celles de miRBase que les autres méthodes disponibles. Mirinho a été utilisé dans le cadre de deux questions biologiques précises l'une concernant des RNAseq l'autre de l'ADN génomique. La première question a conduit au traitement et l'analyse des données RNAseq de Acyrthosiphon pisum, le puceron du pois. L'objectif était d'identifier les miARN qui sont différentiellement exprimés au cours des quatre stades de développement de cette espèce et sont donc des candidats à la régulation des gènes au cours du développement. Pour cette analyse, nous avons développé un pipeline, appelé MirinhoPipe. La deuxieme question a permis d'aborder les problèmes liés à la prévision et l'analyse des ARN non-codants (ARNnc) dans la bactérie Mycoplasma hyopneumoniae. Alvinho a été développé pour la prédiction de cibles des miRNA autour d'une segmentation d'une séquence numérique et de la détection de la conservation des séquences entre ncRNA utilisant un graphe k-partite. Nous avons finalement abordé un problème lié à la recherche de motifs conservés dans un ensemble de séquences et pouvant ainsi correspondre à des éléments fonctionnels
The main contribution of this thesis is the development of a reliable, robust, and much faster method for the prediction of pre-miRNAs. With this method, we aimed mainly at two goals: efficiency and flexibility. Efficiency was made possible by means of a quadratic algorithm. Flexibility relies on two aspects, the input type and the organism clade. Mirinho can receive as input both a genome sequence and small RNA sequencing (sRNA-seq) data of both animal and plant species. To change from one clade to another, it suffices to change the lengths of the stem-arms and of the terminal loop. Concerning the prediction of plant miRNAs, because their pre-miRNAs are longer, the methods for extracting the hairpin secondary structure are not as accurate as for shorter sequences. With Mirinho, we also addressed this problem, which enabled to provide pre-miRNA secondary structures more similar to the ones in miRBase than the other available methods. Mirinho served as the basis to two other issues we addressed. The first issue led to the treatment and analysis of sRNA-seq data of Acyrthosiphon pisum, the pea aphid. The goal was to identify the miRNAs that are expressed during the four developmental stages of this species, allowing further biological conclusions concerning the regulatory system of such an organism. For this analysis, we developed a whole pipeline, called MirinhoPipe, at the end of which Mirinho was aggregated. We then moved on to the second issue, that involved problems related to the prediction and analysis of non-coding RNAs (ncRNAs) in the bacterium Mycoplasma hyopneumoniae. A method, called Alvinho, was thus developed for the prediction of targets in this bacterium, together with a pipeline for the segmentation of a numerical sequence and detection of conservation among ncRNA sequences using a kpartite graph. We finally addressed a problem related to motifs, that is to patterns, that may be composed of one or more parts, that appear conserved in a set of sequences and may correspond to functional elements

Neurotrophins and their receptors are key molecules in the development of the
nervous system. Neurotrophin-3 binds preferentially to its high-affinity receptor
NTRK3, which exists in two major isoforms in humans, the full-length kinaseactive
form (150 kDa) and a truncated non-catalytic form (50 kDa). The two
variants show different 3'UTR regions, indicating that they might be differentially
regulated at the post-transcriptional level. In this work we explore how
microRNAs take part in the regulation of full-length and truncated NTRK3,
demonstrating that the two isoforms are targeted by different sets of microRNAs.
We analyze the physiological consequences of the overexpression of some of the
regulating microRNAs in human neuroblastoma cells. Finally, we provide
preliminary evidence for a possible involvement of miR-124 - a microRNA with no
putative target site in either NTRK3 isoform - in the control of the alternative
spicing of NTRK3 through the downregulation of the splicing repressor PTBP1.
Las neurotrofinas y sus receptores constituyen una familia de factores cruciales
para el desarrollo del sistema nervioso. La neurotrofina 3 ejerce su función
principalmente a través de una unión de gran afinidad al receptor NTRK3, del cual
se conocen dos isoformas principales, una larga de 150KDa con actividad de tipo
tirosina kinasa y una truncada de 50KDa sin dicha actividad. Estas dos isoformas
no comparten la misma región 3'UTR, lo que sugiere la existencia de una
regulación postranscripcional diferente. En el presente trabajo se ha explorado
como los microRNAs intervienen en la regulación de NTRK3, demostrando que las
dos isoformas son reguladas por diferentes miRNAs. Se han analizado las
consecuencias fisiológicas de la sobrexpresión de dichos microRNAs utilizando
células de neuroblastoma. Finalmente, se ha estudiado la posible implicación del
microRNA miR-124 en el control del splicing alternativo de NTRK3 a través de la
regulación de represor de splicing PTBP1.

Stem cell research, especially the one dealing with human embryonic stem cells, is a major topic nowadays. In the last few years studies about human embryonic stem cell derived cardiomyocytes highlighted the importance of those, as their characteristics are almost identical as of the cardiomyocytes in the heart (i.e. the contraction of those cells). The studies concentrate on the ability of using cardiomyocytes in the drug development for cardiac diseases or in regenerative medicine and cell replacement therapies. In contrast some researchers concentrate on microRNAs (miRNAs) as regulators in the development of cardiomyocytes. This study combines both research topics as it deals with stem cells and miRNAs (as well as their target mRNAs). A main objective is to find differentially expressed genes by using Significance Analysis of Microarrays (SAM) as method. Furthermore miRNA target prediction is applied and the identified targets are compared with the ones found by SAM. With an intersection approach we derived 41 targets of up-regulated miRNAs and 25 targets of down-regulated miRNAs, which can be the basis for further studies (i.e. knock-out experiments).

Philosophiae Doctor - PhD
Ovarian cancer (OC) is the most fatal gynaecologic malignancy that is generally diagnosed in the advanced stages, resulting in a low survival rate of about 40%. This emphasizes the need to identify a biomarker that can allow for accurate diagnosis at stage I. MicroRNAs (miRNAs) are appealing as biomarkers due to their stability, non-invasiveness, and differential expression in tumour tissue compared to healthy tissue. Since they are non-coding, their biological functions can be uncovered by examining their target genes and thus identifying their regulatory pathways and processes. This study aimed to identify miRNAs and genes as candidate biomarkers for early stage OC diagnosis, through two distinct in silico approaches. The first pipeline was based on sequence similarity between miRNAs with a proven mechanism in OC and miRNAs with no known role. This resulted in 9 candidate miRNAs, that have not been previously implicated in OC, that showed 90-99% similarity to a miRNA involved in OC. Following a series of in silico experimentations, it was uncovered that these miRNAs share 12 gene targets that are expressed in the ovary and also have proven implications in the disease. Since the miRNAs target genes contribute to OC onset and progression, it strengthens the notion that the miRNAs may be dysregulated as well. Using TCGA, the second pipeline involved analysing patient clinical data along with implementing statistical measures to isolate miRNAs and genes with high expression in OC. This resulted in 26 miRNAs and 25 genes being shortlisted as the potential candidates for OC management. It was also noted that targeting interactions occur between 15 miRNAs and 16 genes identified through this pipeline. In total, 35 miRNAs and 37 genes were identified from both pipelines.

MiRNAs são pequenas moléculas de RNAs endógenos não codificantes com aproximadamente 23nt que atuam na regulação da expressão gênica. A sua função é inibir a tradução de genes transcritos através de um mecanismo que viabiliza a ligação do miRNA com o mRNA alvo levando à inibição da tradução ou a degradação do RNA mensageiro. Estudos evidenciam a relação dos miRNAs com diversos processos biológicos como proliferação celular, diferenciação, desenvolvimento e doenças. Uma vez que estão envolvidos na regulação gênica, também alteram as vias metabólicas. Atualmente, as ferramentas computacionais disponíveis para o estudo dos miRNAs são o miRBase, microCosm, o miRGen e o miRNAmap. Elas possuem informações sobre as sequências dos miRNAs, genes alvos e sobre elementos que estão próximos à região dos miRNAs. Embora o avanço até o momento, não existia que relacionasse os miRNAs com as vias metabólicas, para isso foi construída a plataforma miRNApath que auxilia no estudo da função dos miRNAs por meio da análise do seus alvos dentro vias metabólicas. De modo semelhante, também não existia uma abordagem que relacione dados de expressão miRNAs e seus alvos dentro de um mesmo experimento. Para tanto, neste trabalho foi feita uma abordagem utilizando bibliotecas de SAGE (Serial Analysis of Gene Expression) que será incorporada no miRNApath. O miRNApath encontra-se disponível em http://lgmb.fmrp.usp.br/mirnapath.
MiRNAs are small molecules of endogenous non-coding RNAs with approximately 23nt in length that acts over gene expression regulation. Its function is inhibit the translation of gene transcripts through a mechanism that links the miRNA with its mRNA target leading to a translational repression or degradation. Studies show the relation of RNAs in many biological processes like cell proliferation, dierentiation and development of diseases. Since they are involved in gene regulation, they also change the metabolic pathways. Currently, the available computational tools for the study of miRNAs are miRBase, microCosm, miRGen and miRNAmap. They have information about miRNAs sequences, targets and features. Despite the the advances, until now, there is no tool that correlates the miRNAs with metabolic pathways, therefore we developed the miRNApath platform that helps in the analysis of miRNAs function through the study of its targets that are into the metabolic pathway. In the same way, there is no approach that put together information of expression of miRNAs and its targets in the same experiment. In this work we develop an approach with SAGE (Serial Analysis of Gene Expression ) libraries that will be integrated to miRNApath. The plataform is avaible at http://lgmb.fmrp.usp.br/mirnapath.

Transposable elements (TEs) are acknowledged sources of genetic change within organisms. The effects of transposition can range from the disruption or creation of a single gene to large-scale genome rearrangements. Transposition events can result in beneficial mutations which allow an organism to adapt to a new environment. In the last three years, several studies have reported that some miRNAs, small RNAs involved in post-transcriptional gene regulation, have evolved from TEs. miRNAs play an important role in the stress responses of many organisms. Interestingly, TEs are derepressed under the same stress conditions that miRNAs are known to ameliorate. The observation that miRNAs are known to evolve from TEs and that TEs are derepressed under stress conditions lead me to question whether TEs play a role in environmental adaptation through the creation of small RNA networks. To test this idea, Chlamydomonas reinhardtii cultures were grown under low carbon, nitrate enriched conditions and the small RNA pool was analyzed. I found that these conditions do stimulate the expression of novel small RNAs and that some of these RNAs and their targets are derived from transposition events.

Mestrado em Biologia Funcional - Instituto Superior de Agronomia
Portugal is one of the largest producers of pulp and paper derived from Eucalyptus globulus, which makes this a valuable species for the country. Wood is a complex and variable material, and its posttranscriptional regulation knowledge is only beginning. MicroRNAs (miRNA) are small size (21-24nt), endogenous non-coding RNAs, involved in post-transcriptional regulation. MiRBase v20 database encloses thousands of entries, however none from Eucalyptus. In this study we aim to validate E. globulus miRNAs candidates; to predict in silico and validate experimentally the miRNAs targets; and analyze the gene expression of validated targets. Four miRCa-02, miRCa-04, miRCa-08 and miRCa-09 candidates were validated by Northern blot and there in silico prediction revealed 42 target genes. Fourteen predicted target genes were tested through the RLM 5’-RACE methodology, but only three predicted targets were validated (Eucgr.E01509, Eucgr.C01382 and Eucgr.J02113 predicted target genes for miR171, miRCa-04 and miRCa-08, respectively). Expression of these three target genes analyzed by RT-qPCR suggests that the distinct expression levels found may be related with to wood formation in Eucalyptus globulus. For the first time, four Eucalytus miRNAs and their target genes were disclosed and validated by bioinformatic and molecular tools.

MicroRNAs control the expression of their target genes by translational repression.

They are involved in various biological processes including cancer progression.

To uncover the biological role of microRNAs it is necessary to identify their

target genes. The small number of experimentally validated target genes makes

computer prediction methods very important. However, state of the art prediction

tools result in a great number of putative targets. The number of false positives

among those putative targets is unknown. This report proposes, investigates and

analyses two ways of ranking the biological relevance of putative targets of miRNAs

which are associated with breast cancer.

One approach characterises values of network properties of the putative microRNA

targets in the human Protein-Protein Interaction network and compares

them to network property values of validated microRNA targets. Using these results

we suggest a simple approach for ranking the relevance of putative targets.

The approach consists of testing if a network property value of a putative target

differs from the mean value of the network. In addition we study which network

property contributes most to ranking using this approach.

The second approach identifies commonly overrepresented Gene Ontology categories

among putative microRNA targets, validated targets and known breast

cancer genes. We investigate possibilities to use the occurrence of a putative target

in these categories to rank its biological relevance.

Finally we present a number of genes with interesting features considering both

approaches. These genes might play a role in breast cancer progression and might

be worth investigating further.

Le mélanome est le cancer de la peau le plus agressif de par sa grande plasticité phénotypique, son potentiel métastatique et sa résistance aux traitements. Malgré la percée des thérapies ciblant la voie oncogénique MAP kinase, la résistance du mélanome a ces traitements demeure un obstacle majeur qui limite le bénéfice pour les patients porteurs de la mutation BRAFV600E. Les cellules de mélanome peuvent transiter vers un état de type mésenchymateux dédifférencie en fonction des pressions du microenvironnement et des traitements. Cette plasticité cellulaire phénotypique adaptative a été décrite comme un facteur essentiel de résistance aux thérapies ciblées. Mon équipe de recherche travaille sur ce type de résistance non-génétique définie comme ≪ mésenchymateuse ≫, dans lequel les cellules tumorales présentent un comportement invasif et acquièrent des caractéristiques observées typiquement dans les fibroses telles que la capacité à accumuler et à remodeler la matrice extracellulaire et activer les voies de mécanotransduction. Dans ce contexte, mon projet a consiste à caractériser un cluster compose de deux ≪ FibromiRs ≫, microARN impliques dans les mécanismes de fibrogènes et qui sont fortement exprimes dans les mélanomes résistants. Mes résultats obtenus à l’aide d’approches in vitro et in vivo démontrent le rôle du locus miR-143/-145 dans la régulation de la résistance non-génétique en raison de sa capacite à remodeler la matrice et façonner une niche de protection et de tolérance pour la tumeur face aux inhibiteurs de la voie MAP kinase. MiR-143 et miR-145 contribuent également au passage d’un phénotype cellulaire différentié prolifératif a un phénotype mésenchymal plus invasif et résistant. Au niveau moléculaire, j’ai identifié parmi les nombreuses cibles potentielles du cluster, la FSCN1 comme un gène clé cible de miR-143 et -145. Ces travaux ont permis de dévoiler le rôle du cluster miR143/-145 dans le comportement agressif des cellules de mélanome dédifférenciées résistantes et de proposer miR-143 et miR-145 comme nouvelles cibles thérapeutiques pour vaincre la résistance mésenchymateuse et mieux combattre la maladie métastatique réfractaire
Because of its intrinsic plasticity and resistance to treatment, melanoma is one of the most aggressive cancers. Due to the MAPK pathway hyperactivation, targeted therapies counteracting this signaling cascade are efficient in most patients harboring BRAFV600E metastatic melanoma. However, innate and acquired resistances constitute major therapeutic challenges. Acquired resistance to MAPK-targeted therapies arises from de novo genetic lesions and non-genetic events such as transcriptional reprogramming and epigenetic changes. Upon MAPK inhibitors exposure, melanoma cells assume functionally different phenotypic states defined by master transcription factors differential activity and fixed by epigenetic events. Among them, the emergence of a poorly differentiated cell state is strongly associated with resistance acquisition and tumor recurrence. Our team has previously shown that melanoma cells switching to a dedifferentiated phenotype in response to MAPK-targeted therapies display features of cancer-associated fibroblasts (CAFs) like extracellular matrix (ECM) remodeling and markers observed in fibrotic diseases, allowing them to generate a drug tolerant microenvironment.This fibrotic state is characterized in vitro and in vivo by increased deposition and altered ECM organization associated with a mechanophenotype regulated by the mechanotransducers YAP and MRTFA. However, post-transcriptional signaling networks that underpin this mesenchymal-like phenotype are still unknown and effective therapeutic treatments to overcome MAPK-targeted therapy resistance are missing. Given the tumorigenic role of ECM in cancer progression and resistance, therapies aimed at “normalizing” the tumorigenic ECM represent promising strategies to overcome non-genetic resistance to MAPK inhibitors. Based on the role of miRNAs in post-transcriptional regulation, I focused on the characterization of a pool of miRNAs, defined as “FibromiRs,” which have been shown to participate in the onset and progression of fibrotic diseases. Their crucial role in the fibrogenic process and the possibility to therapeutically manipulate them make them promising druggable targets to prevent the onset of resistance to MAPK-targeted therapies in melanoma. Starting from a screening designed to compare the expression of “FibromiRs” in MAPK inhibitors resistant mesenchymal melanoma cells compared to therapy-naive parental cells, we have identified the profibrotic miR-143/145 cluster as overexpressed in mesenchymal resistant cells. We then explored the profibrotic function of miR-143/145 cluster in the mesenchymal-like resistant cell state and melanoma phenotypic plasticity. First, we analysed the regulation of miR-143 and miR-145 in melanoma, identifying a negative regulation of the MAPK pathway on its expression and the involvement of signaling pathways typical of the mesenchymal resistant state, such as TGFβ and PDGF signaling, in the activation of their expression. Next, we investigated the function of the cluster in the context of adaptive and acquired resistance, showing its contribution in ECM reprogramming, activation of mechanotransduction pathways, and in driving the switch from a differentiated proliferative phenotype to a dedifferentiated invasive one with decreased sensitivity to MAPK inhibition. We characterized its mechanism of action, identifying FSCN1 as a key target gene of both mature miR-143 and miR-145 in the acquisition of the mesenchymal invasive phenotype. Finally, we tested the cluster as a potential therapeutic target in vitro and in vivo through antisense oligonucleotide-mediated inhibition of its expression or pharmacological modulation combined with MAPK inhibitors administration. Overall, this work highlights the importance of a FibromiR cluster in the acquisition of a dedifferentiated phenotype resistant to MAPK-targeted therapies and proposes new therapeutic strategies based on the inhibition of FibromiRs to overcome such resistance mechanism

Le virus associé au sarcome de Kaposi (KSHV) code pour un cluster de 12 précurseurs de micro (mi)ARN abondamment exprimés pendant les phases lytiques et latentes de l’infection. Des études précédentes ont rapporté que KSHV est capable d’inhiber l’apoptose pendant l’infection latente ; nous avons donc testé si les miARN du virus étaient impliqués dans ce processus. Nous avons trouvé que des cellules HEK293 et DG-75 exprimant de manière stable les miARN de KSHV étaient protégées de l’apoptose. Les cibles cellulaires potentielles qui étaient significativement négativement régulées lors de l’expression des miARNs de KSHV ont été identifiées par analyse transcriptomique par microarray. Parmi celles-ci, nous avons validé par tests rapporteurs luciférase, PCR quantitative, et western blot, Caspase 3 (CASP3), un facteur jouant un rôle critique dans le contrôle de l’apoptose. Via le biais de mutagenèse dirigée, nous avons montré que trois miARN de KSHV, miR- 12-1, 3 et 4-3p, étaient responsables du ciblage de CASP3. L’inhibition spécifique de ces miARN dans des cellules infectées par KSHV a résulté en une augmentation des niveaux d’expression de CASP3 endogène, et en une apoptose plus accrue. Vus dans leur ensemble, nos résultats suggèrent que les miARN de KSHV participent directement à l’inhibition précédemment rapportée de l’apoptose par le virus, et donc qu’ils jouent probablement un rôle dans l’oncogenèse induite par KSHV
Kaposi’s sarcoma herpesvirus (KSHV) encodes a cluster of twelve micro (mi)RNA precursors, which are abundantly expressed during both latent and lytic infection. Previous studies reported that KSHV is able to inhibit apoptosis; we thus tested the involvement of viral miRNAs in this process. We found that both HEK293 epithelial cells and DG-75 cells stably expressing KSHV miRNAs were protected from apoptosis. Potential cellular targets that were significantly down-regulated upon KSHV miRNAs expression were identified by microarray profiling. Among them, we validated by luciferase reporter assays, quantitative PCR and western blotting Caspase 3 (CASP3), a critical factor for the control of apoptosis. Using site-directed mutagenesis, we found that three KSHV miRNAs, miR-K12-1, 3 and 4-3p, were responsible for the targeting of CASP3. Specific inhibition of these miRNAs in KSHV infected cells resulted in increased expression levels of endogenous CASP3 and enhanced apoptosis. Altogether, our results suggest that KSHV miRNAs directly participate to the previously reported inhibition of apoptosis by the virus, and are thus likely to play a role in KSHV-induced oncogenesis

L’espérance de vie des patients atteints de mélanome à haut risque ne peut être prédite d’une façon

fiable en se basant sur les analyses d’histopathologies de la lésion primitive et est souvent ajustée

durant la progression de la maladie. Notre étude vise à élargir nos observations initiales au niveau

des métastases cutanées et d’évaluer la valeur pronostique de tyrosinase related protein 1 (TYRP1)

dans les métastases ganglionnaires des patients atteints de mélanome de stades III et IV. TYRP1 est

une enzyme mélanosomale qui partage des similitudes structurelles avec la tyrosinase, l'enzyme clé

de la mélanogenèse.

L’expression de l'ARNm de TYRP1 a été quantifiée dans 104 métastases ganglionnaires par PCR

en temps réel et normalisée par rapport à l’expression de l’ARNm de S100B (marqueur reconnu du

mélanome) pour corriger l’expression de TYRP1 suivant la charge tumorale de l’échantillon. Le

rapport TYRP1/S100B a été calculé et la médiane a été utilisée en tant que valeur seuil. Ensuite

nous avons étudié la relation entre les valeurs de TYRP1/S100B, le suivi clinique et les

caractéristiques histopathologiques de la tumeur primitive.

Un rapport élevé de l’ARNm TYRP1/S100B corrélait significativement avec une survie sans

récidive et une survie globale plus courtes, avec une épaisseur de Breslow plus élevée et avec la

présence d'une ulcération au niveau de la tumeur primitive. En outre, une expression élevée de

TYRP1/S100B était de meilleure valeur pronostique pour la survie globale que l'épaisseur de

Breslow et l'ulcération des primitifs. De plus, cette expression est bien conservée au cours de la

progression de la maladie par rapport aux groupes de TYRP1 bas/élevé.

Nous avons constaté qu’une expression élevée de TYRP1/S100B dans les métastases de patients

atteints de mélanome est associée à un résultat clinique défavorable et une survie courte. Menée sur

des patients atteints d'un mélanome à haut risque de récidive, cette première étude a suggéré que

l'ARNm de TYRP1 dans les métastases pourrait servir de biomarqueur pour affiner le pronostic

initial des patients surtout ceux ayant des lésions primitives de localisation inconnues ou non

évaluables et peut permettre une gestion différente des deux groupes de patients. Son expression

conservée au cours de la progression de la maladie est en faveur de son utilisation comme cible

thérapeutique.

En second lieu, en évaluant l’expression de la protéine TYRP1 par immunohistochimie dans les

métastases cutanées et ganglionnaires, nous avons observé qu’elle n'était pas détectée dans la moitié

7

des tissus exprimant bel et bien l'ARNm correspondant et qu’elle, contrairement à l'ARNm, n’était

pas associée à la survie.

Des données récentes ont indiqué que le 3'-UTR de l’ARNm de TYRP1 contient trois sites de

liaison putatifs de miR-155 dont deux présentant un polymorphisme d'un seul nucléotide (SNPs:

rs683 et rs910) qui favorisent la dégradation en cas d’hybridation miARN-ARNm parfaite de

l’ARNm ou non en cas d’hybridation imparfaite. Nous avons cherché à examiner si miR-155 peut

affecter l’expression de l’ARNm et de la protéine TYRP1 en fonction de ces SNPs. Tout d'abord,

nous avons transfecté deux lignées de mélanome ayant chacune l’une ou l’autre de l’allèle (au

niveau rs683 et rs910) avec différentes concentrations de pré-miR-155 et nous avons évalué

l’expression du miR-155 et l’ARNm TYRP1 par PCR en temps réel ainsi que l’expression de la

protéine TYRP1 par western blot. Nous avons constaté qu’une surexpression de miR-155 a induit

une dégradation importante des ARNm TYRP1 et a perturbé sa traduction en protéine dans la lignée

avec le génotype “hybridation parfaite”. Ensuite, nous avons examiné l'expression des ARNm et

protéines de TYRP1, le niveau de miR-155 et les SNPs rs683 et rs910 dans 192 échantillons de

métastases cutanées et ganglionnaires de mélanome. Nous avons trouvé que le groupe d'échantillons

avec le génotype “hybridation parfaite” était significativement associé à un niveau de protéine de

TYRP1 plus bas alors qu'aucune différence de niveau d’expression n'a été trouvée pour l’ARNm de

TYRP1 ou miR-155 entre les deux groupes de génotype, confirmant que les SNPs au niveau de 3’-

UTR de TYRP1 peuvent spécifiquement affecter l'expression de la protéine TYRP1. En outre, nous

avons montré que l’ARNm de TYRP1 est inversement corrélé avec l’expression miR-155, mais pas

avec la protéine TYRP1 dans le groupe " hybridation parfaite", alors qu'il corrèle positivement avec

la protéine mais pas avec miR-155 dans le groupe "hybridation imparfaite" où la protéine corrélait

inversement à la survie. Cela montre que les SNPs dans le 3'-UTR de l'ARNm TYRP1 affectent la

régulation de l’ARNm par miR-155 et la traduction en protéine. Ces SNPs rendent la régulation de

l’ARNm et la protéine de TYRP1 indépendante de miR-155 et confèrent une valeur pronostique à

la protéine TYRP1
Doctorat en Sciences biomédicales et pharmaceutiques
info:eu-repo/semantics/nonPublished

博士
國立交通大學
統計學研究所
100
Recent studies have revealed a small non-coding RNA, microRNA (miRNA) down-regulates its mRNA targets, which is regarded as an important role in various biological processes. In recent years, there have been many studies concentrated on the discovery of new miRNAs and identification of their mRNA targets. Although researchers have identified many miRNAs, few miRNA targets have been identified by actual experimental methods. To expedite the identification of miRNA targets for experimental verification, in the literature approaches based on the sequence or microarray expression analysis have been established to discover the potential miRNA targets. We focus on the human miRNA target prediction and propose a generalized relative R squared method (RRSM) to find many high-confidence targets, which is shown to be superior to some existing methods. Although many high-confidence targets from RRSM have been confirmed from previous studies, the thresholds of RRSM are set to be fixed constants, which do not depend on the characteristic of a gene. To find a more feasible method for real data applications, we propose a variable threshold selection method based on the distribution of the relative R squared statistic, which is shown significantly improvement of the prediction results of RRSM only based on a fixed threshold criterion. In addition, we show that the interactions may be only functional on some specific-tissues which depend on the characteristic of a miRNA. There have been no systematic methods established in the literatures to investigate the relationship between miRNA target interactions and tissue specificity through microarray data. In this study, we propose an algorithm to investigate tissue-specificity of miRNAs based on experimental miRNA target interactions. The tissue-specificity result by our method is in accordance with the literatures.

博士
國立交通大學
生物資訊及系統生物研究所
99
MicroRNAs (miRNAs) are small non-coding RNA molecules (~ 22-nt) that can bind to one or more target sites on gene transcript to negatively regulate protein expression and thus control numerous cellular mechanisms. Recent work supports miRNAs downregulate gene expression during various crucial cell processes such as apoptosis, differentiation and development. Previous research has suggested that miRNAs regulate 30% or more of the human protein-coding genes. As the important roles of miRNAs, there were multiple databases storing the miRNA-target interactions (MTIs) identified using different tools. The aim of this work is to systematically analyze the miRNA-target interactions and assess the function of miRNA by developing new methods and resources. In this study, we analyzed 1,524 experimentally verified miRNA-target interactions with strong evidence support in human and elucidate which the more accurate microRNA target prediction database is. Through analyzing the verified MTIs, we could get the overview of relative contribution of sequence and structure features in miRNA targeting. Those analyses are important for identifying putative miRNA targets and are very useful for biologist to choose the proper tool for miRNA research. There are still other factors such as RNA binding protein (RISC), the concentration of miRNA and mRNA, playing an important role in the identification of miRNA-target interaction. We need take into account these factors in future works in order to develop the more reliable miRNA target prediction resources.

The possibility of selectively alter the expression pattern of a particular gene has been sought by scientists and clinicians for a long time. Nowadays, RNA interference (RNAi)-based technology has become a novel tool for silencing gene expression in cells. In addition, this strategy encloses an enormous therapeutic potential that could change the course of the currently applied treatments in several life threatening pathologies and it is expected that this technology can be translated onto clinical applications in a near future. MicroRNA (miRNA) has become a commonly employed tool for gene silencing, since it prevents protein synthesis by inducing the messenger RNA (mRNA) degradation, with a high specificity degree. Consequently, in the last years, the miRNAs have emerged as biopharmaceuticals to regulate several pathways involved in the insurgence and progression of the Alzheimer’s disease (AD), since they might have key regulatory roles in many neuronal functions, such as differentiation, synaptic plasticity and memory formation, and typically they are down-regulated in disease conditions. In the literature there are some studies describing a causal relationship between miR-29 expression and AD, since a loss of miR-29 cluster can contribute to increased beta-amyloid precursor protein-converting enzyme 1 (BACE1) and Amyloid-β (Aβ) levels in sporadic AD patients. Thus, this evidence supports the possibility to use miR-29 as a potential therapeutic target for AD therapy. In general, miRNA-based therapy relies on the use of synthetic microRNAs. However, these synthesized formulations typically present contaminants that can lead to non-targeted gene silencing, which still restricts the pre-clinical or clinical application of these RNAs. Thus, considering this therapeutic purpose and the global distribution of novel biopharmaceuticals it is necessary to develop efficient processes for their preparation. The development of new strategies for microRNA production with high purity degree and biologically active is extremely required. One of the strategies might be the use of the recombinant production of biomolecules using prokaryotic hosts. Hence, the present work intends to develop and establish an integrative biotechnological platform to biosynthesize and purify a recombinant miRNA precursor (pre-miR-29b) to act in the selective silencing of endogenous pathways directly related with AD, in particular BACE1 and Aβ. In addition, the success of these therapies also depends upon the ability to selectively and efficiently deliver the pre-miR-29b in the cytoplasmic compartment of neuronal cells, the location where their function is exerted; therefore the development of miRNA delivery systems was also envisioned. The expression system Rhodovulum sulfidophilum (R. sulfidophilum) DSM 1374 allowed, for the first time, the production of human pre-miR-29b with a straightforward recuperation of pre-miR-29b in a single step, maintaining its biological active form. The application of this recombinant bacterial microorganism is innovative and is supported by the unusual capacity of secreting the nucleic acids to the extracellular space and the absence of host ribonucleases in the culture medium. Therefore, it is expected that the secreted miRNA will be devoid of main bacterial associated impurities. Regarding the growth conditions, and conversely to what was previously described for this bacterium, our results showed to be possible to develop an original approach for the aerobic growth of the R. sulfidophilum, which results in a cell growth improvement followed by an enhanced production of human pre-miR-29b. The extracellular pre-miR-29b concentration was approximately 182 μg/L, after 40 hours of bacterial growth and the total intracellular pre-miR-29b was of about 358 μg/L, at 32 hours of cell growth. To further develop a potential therapeutic application, the major interest is not only to produce high quantities of RNA but also to obtain and preserve its biological active form, fulfilling the requirements of regulatory agencies. Hence, to assure that this prerequisite is met it was used a novel and effective purification strategy, based on affinity chromatography, to purify the pre-miR-29b. Therefore, in order to achieve the selectivity towards the target pre-miRNA and the maximum resolution between the pre-miR-29b and other host biomolecules (transfer RNAs and proteins) it was used an affinity support that exploits the same biological interactions that are established within the cell, by using immobilized amino acids (L-lysine and L-arginine), as specific ligands. The recognition of the pre-miR-29b achieved with these supports, allowed its selective recovery from a complex mixture with high efficiency and high purity. In parallel, the binding of pre-miRNA to these different amino acids was studied by Surface Plasmon Resonance. This information brings important insights concerning the characterization of the pre-miRNA binding onto chromatographic supports. Moreover, it was possible to determine some particular conditions enabling the improvement of the binding specificity of the amino acid ligands used to purify miRNA, preserving the RNA integrity. Taking into account that the structure of the chromatographic supports has been continuously developed to afford rapid and efficient separations, namely for the purification of nucleic acids, it was also tested a monolithic support to purify the pre-miR-29b. The association of the high capacity of these supports with the specificity conferred by the agmatine ligand (a derivative of L-arginine) represented a novelty and an advantage to obtain highly pure pre-miR-29b (90%) with a high recovery yield (95%). The establishment of an effective application of miRNAs is usually constrained by different phenomena, namely their easy degradation when in contact with the body fluids. To overcome this limitation, delivery systems, such as polymeric systems (polyplexes), were developed and characterized in order to encapsulate and protect the pre-miR-29b biopharmaceuticals from degradation, allowing their sustained and targeted release. The formulations prepared with chitosan and polyethylenimine demonstrated high loading capacity, small sizes and exhibited a strong positive charge on their surface. In addition, considering the application field of this work, the delivery systems should also have the ability to penetrate the Blood-Brain Barrier (BBB), causing an increase of the pre-miRNAs concentration in the brain and, consequently the improvement of the therapeutic effect. Actually, BBB is an intrinsic barrier limiting miRNA therapeutic effect on the central nervous system. Thus, to improve the delivery of pre-miRNA therapeutics in the brain, the polyplexes were functionalized with specific ligands, namely lactoferrin and stearic acid which are recognized by cell surface receptors of BBB. Finally, it was evaluated the biological activity of the recombinant pre-miR-29b by measuring the efficiency on human BACE1 knockdown, using in vitro neuronal cell lines. The effect of recombinant pre-miR-29b administration was verified by both assessing the mRNA and protein human BACE1 levels, by using RT-qPCR, Western blot and Imunocytochemistry. Results suggest that recombinant pre-miR-29b can represent a novel biopharmaceutical product for the therapeutic modulation of human BACE1 levels, because high levels of inhibition were achieved, namely 80% of reduction for BACE1 protein expression and 45% for Aβ42 levels. Globally, the implementation of these cutting-edge technologies can have a great impact on the biopharmaceutical industry, providing the basis for the implementation of novel miRNA-based therapeutics, not only for neurological disorders but also for future therapeutic targets that can be of potential interest.
A recente descoberta da tecnologia do RNA de interferência tornou-se numa nova ferramenta que permite regular seletivamente o padrão de expressão de um ou mais genes, o que pode ser explorado no âmbito de aplicação terapêutica. Deste modo, os resultados promissores desta nova abordagem têm vindo a reforçar a investigação relacionada com o RNA, avaliando e compreendendo os mecanismos celulares em que está envolvido, com o objetivo de o usar como uma nova classe de produtos bioterapêuticos, de fácil translação e implementação para o âmbito clínico. Na verdade, o mecanismo celular responsável pelo silenciamento da expressão génica apresenta um enorme potencial terapêutico que poderá alterar os tratamentos atualmente disponíveis para diversas patologias, como por exemplo as doenças neurológicas. Os microRNAs (miRNAs) constituem uma classe de RNAs de baixo peso molecular e, nos últimos anos, têm sido cada vez mais reconhecidos como moléculas endógenas reguladoras em numerosos processos biológicos. Devido à sua especificidade e eficiência, os miRNAs tornaram-se uma das ferramentas mais utilizadas no silenciamento de genes pelo mecanismo de RNA de interferência, uma vez que podem bloquear a síntese de proteínas através da indução da degradação do RNA mensageiro. Assim, os miRNAs podem ser considerados agentes terapêuticos promissores. Durante a última década, a hipótese de que os miRNAs podem ser usados como biofármacos para regular e controlar várias vias envolvidas no desenvolvimento e progressão da doença de Alzheimer (DA) ganhou consistência, uma vez que estes apresentam um papel crucial em muitas funções neuronais, tais como diferenciação, plasticidade sináptica, formação da memória, e normalmente estão sub-expressos em doentes com esta patologia. A doença de Alzheimer é a forma mais comum de demência e caracteriza-se por uma perda neuronal e sináptica generalizada, que causa um declínio progressivo e irreversível em diversas funções cognitivas. Embora, as causas ainda não sejam completamente compreendidas, sabe-se que esta doença neurodegenerativa está associada ao aparecimento de dois tipos de agregados proteicos, nomeadamente placas extracelulares beta-amiloides (Aβ) e complexos neurofibrilares intraneuronais. A formação das placas de Aβ resulta da clivagem sequencial da proteína percursora amiloide (APP) pela BACE1 e, posteriormente, pelo complexo gama-secretase dando origem às espécies Aβ tóxicas. Alguns estudos têm descrito uma relação causal entre a expressão do miR-29 e a DA, uma vez que a diminuição dos níveis de expressão da família miR-29 tem sido associada ao aumento da expressão da BACE1 e, consequentemente da formação dos péptidos Aβ em doentes com DA. Desta forma, a utilização do miR-29 pode proporcionar uma estratégia terapêutica eficaz na prevenção, controlo da progressão e tratamento desta patologia. As terapêuticas baseadas no uso de miRNAs recorrem, na sua maioria, à utilização de miRNAs sintéticos. Embora a síntese de miRNAs possa ser muito eficiente, verifica-se normalmente a presença de contaminantes nas amostras de RNA sintetizado o pode levar à inespecificidade do silenciamento génico. Além disso, por norma, no processo de preparação do RNA sintético é necessário recorrer à utilização de solventes tóxicos, solventes orgânicos e condições desnaturantes, que podem comprometer a qualidade e integridade do produto alvo. Por todos estes motivos, e considerando o objetivo de aplicação terapêutica destes novos produtos biofarmacêuticos, torna-se evidente a necessidade de desenvolver novos processos eficientes ou melhorar as metodologias atualmente empregues para a sua preparação. Assim, um dos desafios mais importantes no desenvolvimento destas estratégias terapêuticas surge com a necessidade de produzir miRNA com elevado grau de pureza e atividade biológica, de modo a satisfazer os requisitos necessários à sua aplicação. Deste modo, uma das estratégias que pode ser aplicada consiste na produção recombinante de biomoléculas utilizando hospedeiros procarióticos. Desta forma, o objetivo principal do presente trabalho consiste no desenvolvimento e implementação de uma plataforma biotecnológica para a produção e purificação de precursores de miRNAs, em particular o pre-miR-29b (percursor do miR-29), cuja aplicação visa o silenciamento seletivo de vias endógenas diretamente relacionados com DA, nomeadamente a BACE1 e os péptidos Aβ. Paralelamente, serão desenvolvidas e caracterizadas nanopartículas poliméricas para a entrega do pre-miR-29b no citoplasma de células neuronais de forma seletiva e eficaz, de modo a assegurar o sucesso destas administrações. O sistema de expressão recombinante utilizado permitirá, pela primeira vez, a produção e o isolamento do pre-miR-29b humano na bactéria Rhodovulum sulfidophilum (R. sulfidophilum) DSM 1374, mantendo a sua atividade biológica. A utilização deste hospedeiro bacteriano é inovadora e vantajosa, devido à sua capacidade invulgar de secretar os ácidos nucleicos para o meio de cultura, bem como devido à ausência de ribonucleases no mesmo, permitindo a obtenção do miRNA de interesse com baixo conteúdo de impurezas bacterianas. A fim de otimizar a produção e acumulação de miRNAs no espaço extracelular, as condições de crescimento foram estudadas, nomeadamente no que diz respeito ao efeito da temperatura e concentração de cloreto de sódio. Os ensaios realizados demonstraram ser possível desenvolver um protocolo para o crescimento aeróbio da bactéria R. sulfidophilum, na ausência de luz, a 30ºC, o que resulta num melhoramento do crescimento das células, seguido de um aumento da produção do pre-miR-29b humano. Neste trabalho foi possível atingir uma concentração de pre-miR-29b no meio extracelular de aproximadamente 182 μg/L, após 40 horas de crescimento bacteriano e uma concentração de 358 μg/L de pre-miR-29b intracelular, após 32 horas de fermentação. Para que seja possível a aplicação terapêutica do pre-miR-29b é necessário ter em conta as exigências estabelecidas pelas agências reguladoras internacionais, que requerem a produção de miRNA com elevada qualidade e atividade biológica. Para assegurar esta condição, foram desenvolvidas novas estratégias de purificação para o pre-miR-29b, baseadas em cromatografia de afinidade. A fim de alcançar a máxima seletividade e especificidade na separação do pre-miRNA de outras biomoléculas do hospedeiro (outras espécies de RNA e proteínas), foram desenvolvidos suportes de afinidade baseados nas interações biológicas que são estabelecidas a nível celular, usando como ligandos de afinidade, aminoácidos básicos como a L-lisina e a L-arginina. Na estratégia de purificação com o aminoácido de L-lisina foi demonstrada pela primeira vez a purificação de pre-miRNA utilizando um gradiente decrescente de concentração de sulfato de amónio em três passos, o que permitiu explorar maioritariamente interações hidrofóbicas. Contudo, a necessidade de aplicar elevadas concentrações de sal pode ser visto como uma desvantagem devido aos custos e ao impacto ambiental associado ao processo, principalmente no que diz respeito à aplicação ao nível industrial. De modo a ultrapassar estas limitações foi usada L-arginina como aminoácido imobilizado. Este estudo demonstrou a possibilidade de purificar o pre-miR-29b utilizando três estratégias diferentes de eluição, nomeadamente concentrações decrescentes de sulfato de amónio e duas condições de eluição moderadas, tais como usando um gradiente crescente de cloreto de sódio e a adição de um agente de competição (arginina) ao tampão de eluição. A versatilidade da matriz de arginina na purificação do pre-miR-29b sugeriu que o mecanismo de interação envolveria uma multiplicidade de interações não-covalentes, que globalmente resultam no bioreconhecimento do RNA de interesse. O reconhecimento bioespecífico e seletivo do pre-miR-29b por estes suportes cromatográficos permitiu a sua purificação e recuperação de forma eficiente, com elevados rendimentos, grau de pureza e integridade, a partir de uma mistura complexa. Além disso, a utilização da cromatografia de afinidade com arginina resultou na eliminação das impurezas associadas à produção recombinante, nomeadamente proteínas e endotoxinas, respeitando os critérios estabelecidos pelas agências reguladoras (por exemplo “Food and Drug Administration” – FDA). Considerando que esta estratégia cromatográfica requer condições suaves de eluição, torna-se um método de purificação mais económico do que a lisina-agarose. Em paralelo, a ligação do pre-miR-29b aos aminoácidos em estudo foi avaliada por biosensor. Este estudo também permitiu compreender as interações envolvidas entre o pre-miR-29b e as matrizes de lisina- e arginina-agarose, assim como determinar as melhores condições que favorecem o bioreconhecimento e a especificidade de ligação dos aminoácidos ao pre-miR-29b, preservando a sua estabilidade e integridade. Os resultados obtidos neste estudo mostraram a existência de várias interações entre o pre-miR-29b e as matrizes de afinidade com os aminoácidos imobilizados, tais como interações hidrofóbicas, eletrostáticas, catião-π, pontes de hidrogénio e forças de “van der Waals”. Tendo em conta que a estrutura dos suportes cromatográficos está em contínuo desenvolvimento de modo a proporcionar separações rápidas e eficientes, nomeadamente para a purificação de ácidos nucleicos, foi também testado um suporte monolítico na purificação do pre-miR-29b. Esta estratégia que associa a alta capacidade destes suportes com a especificidade e seletividade conferida pelo ligando de agmatina (um derivado da L-arginina), permitiu a recuperação do pre-miR-29b com alta eficiência (95%) e com elevado grau de pureza (90%) para posterior aplicação nos ensaios in vitro. Além disso, este suporte monolítico revelou elevada capacidade de ligação para o RNA, permitindo uma rápida e eficiente separação do pre-miR-29b, independentemente da taxa de fluxo aplicada. No geral, foi possível desenvolver métodos de purificação simples, robustos, versáteis e de elevada reprodutibilidade, que permitiram minimizar o manuseamento das amostras e evitar o uso de condições desnaturantes e solventes orgânicos, contribuindo para o sucesso das aplicações terapêuticas do RNA. No entanto, o sucesso das terapêuticas baseadas em miRNAs depende também da capacidade de entrega do miRNA, de forma seletiva e eficiente, aos órgãos-alvo, com a mínima toxicidade. De facto, a entrega cerebral de fármacos é limitada por diversos fatores intrínsecos, nomeadamente a sua rápida degradação quando em contato com os fluidos corporais e a reduzida permeabilidade ao longo da barreira hematoencefálica (BHE). Para ultrapassar estas limitações, vários sistemas de entrega de fármacos não-virais têm sido desenvolvidos e caracterizados, nomeadamente os sistemas poliméricos (poliplexos) que possuem características intrínsecas ideais para a transfecção, proteção e libertação controlada e direcionada de RNA. No presente trabalho, as formulações foram preparadas com polímeros comerciais, tais como quitosano e polietilenimina e demonstraram elevada capacidade de transporte de RNA, apresentando pequenas dimensões e uma forte carga superficial positiva. Além disso, e considerando o campo de aplicação do presente trabalho, estes sistemas devem também ter a capacidade de penetrar a BHE, levando a um aumento da concentração do pre-miRNA no cérebro e, consequentemente uma melhoria da sua ação terapêutica. Deste modo, a fim de potenciar o efeito terapêutico das abordagens baseadas no RNAi no sistema nervoso central, os poliplexos desenvolvidos foram funcionalizados com ligandos específicos, tais como a lactoferrina e o ácido esteárico, os quais são reconhecidos pelos recetores localizados à superfície da BHE. Este estudo revelou que os sistemas de entrega desenvolvidos conseguem penetrar a BHE e assim entregar o pre-miR-29b no cérebro. Finalmente, avaliou-se a atividade biológica do pre-miR-29b recombinante através da verificação da sua eficiência na regulação dos níveis de expressão dos genes relacionados com a DA, em particular no silenciamento da BACE1 humana, utilizando modelos in vitro (linhas de células neuronais). O efeito da administração do pre-miR-29b recombinante foi verificado tanto ao nível da expressão do RNA mensageiro como ao nível da expressão da proteína BACE1, através de RT-qPCR, Western blot e Imunocitoquímica. Os resultados sugerem que o pre-miR-29b recombinante pode funcionar como biofármaco para a modulação terapêutica dos níveis de BACE1, uma vez que foram atingidos elevados níveis de inibição, ou seja 80% de redução para a expressão da proteína BACE1 e 45% para os níveis dos péptidos beta amiloides, quando comparados com as células não transfectadas e células transfectadas como um RNA não relacionado bem como um miR-29b sintético. Em suma, a implementação destas metodologias terá um grande impacto na indústria farmacêutica e biotecnológica, fornecendo a base para a utilização de novas formas terapêuticas baseadas na utilização de miRNAs, não apenas para aplicação em doenças neurológicas, mas também para futuros alvos terapêuticos que possam ser de interesse.

MicroRNAs (miRNAs) are small RNAs that have important roles in post-transcriptional gene regulation in a wide range of species. This regulation is controlled by having miRNAs directly bind to a target messenger RNA (mRNA), causing it to be destabilized and degraded, or translationally repressed. Identifying miRNA targets has been a large area of focus for study; however, a lack of generally high-throughput experiments to validate direct miRNA targeting has been a limiting factor. To overcome these limitations, computational methods have become crucial for understanding and predicting miRNA-gene target interactions.

While a variety of computational tools exist for predicting miRNA targets, many of them are focused on a similar feature set for their prediction. These commonly used features are complementarity to 5'seed of miRNAs and evolutionary conservation. Unfortunately, not all miRNA target sites are conserved or adhere to canonical seed complementarity. Seeking to address these limitations, several studies have included energy features of mRNA:miRNA duplex formation as alternative features. However, different independent evaluations reported conflicting results on the reliability of energy-based predictions. Here, we reassess the usefulness of energy features for mammalian target prediction, aiming to relax or eliminate the need for perfect seed matches and conservation requirement.

We detect significant differences of energy features at experimentally supported human miRNA target sites and at genome-wide interaction sites to Argonaute (AGO) protein family members, which are essential parts of the miRNA machinery complex. This trend is confirmed on data sets that assay the effect of miRNAs on mRNA and protein expression changes, where a statistically significant change in expression is noted when compared to the control. Furthermore, our method also allows for prediction of strictly imperfect sites, as well as non-conserved targets.

Recently, new methods for identifying direct miRNA binding have been developed, which provides us with additional sources of information for miRNA target prediction. While some computational target predictions tools have begun to incorporate this information, they still rely on the presence of a seed match in the AGO-bound windows without accounting for the possibility of variations.

We investigate the usefulness of the site level direct binding evidence in miRNA target identification and propose a model that incorporates multiple different features along with the AGO-interaction data. Our method outperforms both an ad hoc strategy of seed match searches as well as an existing target prediction tool, while still allowing for predictions of sites other than a long perfect seed match. Additionally, we show supporting evidence for a class of non-canonical sites as bound targets. Our model can be extended to predict additional types of imperfect sites, and can also be readily modified to include additional features that may produce additional improvements.

Dissertation

碩士
國立清華大學
資訊系統與應用研究所
99
MicroRNAs are a class of small non-coding RNAs which play important regulatory roles in animals and plants. They cause transcriptional cleavage or translational repression through binding their target mRNAs. MicroRNAs affect a variety of cellular processes such as development, cell proliferation, apoptosis, and stress response. Thus identification of mRNA targets is an essential step to understand microRNA functions. Currently several microRNA target prediction tools have been developed. The majority of these algorithms are based on the sequence alignment or the minimum free energy of the hybridization. However, due to the omission of gene expression information in the screening process, a number of candidate targets could be false positives which are too large to validate. In this work, a filtering strategy which was implemented based on SVM machine learning has been built in order to reduce the false positives. Information of sequence alignment retrieved from existing database and microarray expression data were both used to classify mRNA candidates into non-target or target group by SVM, trying to separate microRNA target genes from non-target genes. Besides, the concept of conservation between species has been included to mitigate the problem of noisy data then decrease false positive predictions.

Dissertação para obtenção do grau de Mestre no Instituto Superior de Ciências da Saúde Egas Moniz
For a diverse range of diseases there are no effective treatments. A refined understanding of the underlying molecular mechanisms of disease and effective targeted therapies are still required for several life-threatening disorders. In the past decade, microRNAs have been discovered as master regulators of thousands of genes at the post-transcriptional level in both normal physiological conditions and in disease. These small non-coding RNAs are highly conserved among animals and their inappropriate expression has been linked to a variety of diseases, such as, cancer, neurodegenerative, autoimmune and cardiovascular diseases. Based on these remarks, miRNA-based therapies are being developed by several pharmaceutical companies with the principle to enhance disease response and elevate cure rates. Therapeutic strategies based on modulation of miRNA expression and function, namely, miRNA blocking and miRNA replacement therapies have been studied in recent years. Once, miRNAs act as key molecules affecting many cellular processes through the regulation of different genes, therapies based on miRNAs are expectable to be particularly effective in heterogeneous diseases that cannot be treated by a single therapeutic agent. However, off-target effects are expected as a result of pleiotropic nature of microRNAs. Despite the fact of many drug discovery programs are ongoing, the most advanced of these programs are yet in phase 2 clinical trials for the treatment of hepatitis C virus infection. An additional effort need to be made to bring these microRNA-based approaches to the clinic.
Para uma grande variedade de doenças ainda não existe um tratamento eficaz. Uma compreensão aprofundada dos mecanismos moleculares de doença e terapias específicas eficazes são ainda necessárias para várias doenças potencialmente fatais. Na última década os microRNAs foram descobertos como reguladores chave de milhares de genes a nível pós-transcricional tanto no estado fisiológico normal como em situação patológica. Estes pequenos RNAs não codificantes são altamente conservados entre os animais e a sua expressão inapropriada tem sido associada a várias doenças, tais como, cancros, doenças neurodegenerativas, autoimunes e cardiovasculares. Com base nestas observações, a terapia com miRNAs está a ser desenvolvida por várias companhias farmacêuticas com o objetivo de aumentar a resposta à doença e elevar as taxas de cura. As estratégias terapêuticas baseadas na modulação da expressão e função dos miRNAs, nomeadamente, o bloqueio e a restituição dos miRNAs foram estudadas nos últimos anos. Uma vez que os miRNAs atuam como moléculas-chave afetando diversos processos celulares através da regulação de diferentes genes, é expetável que as terapias com miRNAs sejam particularmente efetivas em doenças heterogéneas que não podem ser tratadas com um único agente terapêutico. No entanto, os efeitos fora do alvo são esperados como resultado da natureza pleiotrópica dos microRNAs. Apesar do fato de estarem a decorrer vários programas de descoberta de fármacos, o mais avançado desses programas está ainda em ensaio clínico de fase 2 para o tratamento da infeção pelo vírus da hepatite C. Um esforço adicional necessita de ser realizado para trazer essas abordagens terapêuticas baseadas em microRNAs para a prática clínica.

碩士
國立陽明大學
醫學生物技術暨檢驗學系暨研究所
98
MicroRNAs (miRNAs) are ~22 nt endogenous RNAs that direct their posttranscriptional repression by binding to the complementary sites in the 3’ untranslated region (UTR) of target mRNAs. As a result, translation of target mRNAs is repressed to some extent. Most miRNA target sites are defined by Watson-Crick pairing with seed region (positions ~2-7 nt of the mature miRNA). The binding sites of target mRNAs can be mainly categorized as the 7mer-m8, the 7mer-1A and the 8mer site. In this study, we did systemic comparison of repression force contributed by different types of seed /target sequences association under a unified model. miR-155 and one of its target sequence were chosen or modified. Different types of monomer (7mer-m8, 7mer-1A, 8mer site); dimer, dimer with one mismatch to the seed region, or sequence perfect match to miR-155 were inserted into the 3’UTR of a reporter gene for monitoring fold repression. It showed perfect-match target >> dimer targets >> monomer targets. For dimeric targets, 2X 8mer > 2X 7mer-m8 > 2X 7mer-1A. Fold repression of 8mer + 7mer-1A lied in between 2X 8mer and 2X 7mer-1A and a mismatch to the seed region in one monomer dramatically decreases repression force of dimer. It indicates that the repression force can be synergistically enhanced by the cooperation of the two RISC-bound monomers. miR-155 and siRNA-155 (siRNA carrying miR-155 sequence) elicited equally high repression forces as they bound to the perfect-match target. Moreover, we found that strong repression of miR-155 and siRNA-155 with perfect-match target was mainly due to translational repression. Cleavage of target mRNA, however, was a minor cause. In the process of miRNA maturation, Drosha and Dicer cut the precursor transcript, thereby producing a 21 23-nt short RNA duplex with 5' phosphates and 2-nt overhangs at 3' ends. Despite the structural symmetry of the miRNA, the nucleotide sequence asymmetry can generate a bias for preferred loading of one of the two duplex-forming strands into the RNA-induced silencing complex (RISC). This strand, namely guide strand, becomes a functional component of the RNA-Induced Silencing Complex (RISC), while the other is eliminated. Thermostability of the 5’ end appears to play a decisive role in the strand selection process. miR-155 is used to elucidate if single point mutation near 3’ end of the miRNA may result in the strand-selection shift, i.e. increasing miR-155* and decreasing miR-155. That fine-tunes the translation of their target genes. Consequently, the butterfly effect cause alteration in transcripome. Based on microarray data, there were 10489 genes increased over 1.3 fold, 344 genes increased over 1.5 fold, 6258 genes decreased over 0.77 fold, and 132 genes decreased over 0.66 fold. RT-PCR analysis of 4 arbitrarily selected genes showed that the change of the mRNA levels were in consistent with the data of microarray. According to our findings, a single point mutation of miRNA may affect wide range of gene expression and result in the change of cell proliferation, differentiation, apoptosis and metabolism characters.

碩士
國立成功大學
電機工程學系
104
MicroRNAs (miRNAs) are functional RNA molecules which play important roles in post-transcriptional regulation. miRNAs regulate their targets by repressing translation or inducing degradation of target mRNAs. Several databases have been constructed to deposit predicted miRNA-target information by using different algorithms, but these databases usually contains lots of false positives. Besides, the validated databases provides only a few miRNA-target information compared to the predicted databases. To reduce incorrect records and increase the number of reliable records, many other databases integrate these predicted miRNA-target information from the databases mentioned above. However, the expression of the same miRNAs in different tissues are different, the realistic regulatory mechanisms could not be figured out in these databases. Moreover, they cannot return the common targets with multiple input miRNAs. To solve these two problems, we construct a database called CSmiRTar (Condition-Specific miRNA Targets). CSmiRTar collects computationally predicted targets of 2588 miRNAs in Human (or 1945 miRNAs in Mouse) from four existing databases (microRNA.org, TargetScan, DIANA-microT and miRDB), and it provides some biological filters which enabling users to search miRNA targets which are expressed only in a specific tissue or related to a specific disease. Moreover, CSmiRTar allows users to search the common targets of multiple miRNAs under a specific biological condition. We believe that CSmiRTar could be helpful for biologists whom want to study the regulatory mechanisms of miRNAs. The CSmiRTar database is available at http://cosbi.ee.ncku.edu.tw /CSmiRTar/.

Dissertação de Mestrado em Biodiversidade e Biotecnologia Vegetal apresentada à Faculdade de Ciências e Tecnologia
A indução de embriogénese somática (ES) é uma técnica de clonagem que permite a obtenção de embriões a partir de outros tecidos além do embrião zigótico. De maneira a induzir competência embriogénica, os explantes devem ser expostos a auxinas ou outros stresses. Em Solanum betaceum, a etapa de indução irá promover a formação de calos que tanto podem ser embriogénicos (CE) ou não-embriogénicos (CNE) e apenas transferindo massas de CE para um meio de cultura sem auxina e com baixas concentrações de sacarose, os embriões e as plantas se desenvolvem.A via de sinalização das auxinas é portanto essencial para a regulação da ES, e genes como TIR, AFB ou GRF’s são fulcrais em processos de desenvolvimento de plantas mediados por auxinas. MicroRNA’s (miRNA’s) são pequenos RNA’s de cadeia única que funcionam como reguladores pós-transcricionais, reprimindo a expressão por clivagem endonucleotídica. Dois destes miRNA’s são o miR393 e o miR396 que desempenham papéis importantes no crescimento, desenvolvimento e maturação de plantas. Ambos são cruciais na via das auxinas, regulando a expressão dos genes TIR e AFB (miR393) e da família dos GRF’s (miR396).Este estudo visou a analisar a expressão dos miR393 e miR396 e dos seus genes alvo durante o processo de ES em tamarilho (Solanum betaceum Cav.), a partir de embriões zigóticos e folhas. Amostras de RNA total e enriquecidos em smallRNAs foram extraídas de calos embriogénico e não embriogénico assim como de explantes recolhidos durante a indução de ES e durante o processo de desenvolvimento dos embriões somáticos. As amostras de RNA foram convertidas em cDNA por transcrição reversa. O RNA total foi usado para a quantificação dos níveis de expressão dos genes alvo enquanto que as amostras de smallRNAs foram usadas para quantificar a expressão dos miRNAs, por análise de PCR quantitativo.Os resultados mostram que as expressões dos miR393, miR396 e dos seus genes alvo, durante os vários estágios de ES e nos diferentes calos, sugerem a sua modulação durante a ES em tamarilho, onde ambos os miRNA’s compartilham uma forte correlação inversa com os genes alvo correspondentes. Mir393 e miR396 parecem regular negativamente os seus genes alvo durante a indução de ES, sendo que durante o desenvolvimento e germinação dos embriões somáticos, os valores de expressão dos genes alvo aumentam (miR393 - TIR1 e AFB2; miR396 - GRF1 e GRF4).Estes resultados fornecem novos conhecimentos sobre a aquisição de competência embriogénica a partir de diferentes explantes de tamarilho, assim como sobre o modo como essa competência é mantida durante as subculturas de calos e expressa durante o desenvolvimento dos embriões.
Somatic embryogenesis (SE) is a cloning technique that allows the development of embryos from tissues other than the zygotic embryo. In order to induce embryogenic competence, the explants must be exposed to auxins or to other different stresses. In the case of Solanum betaceum, this induction stage will promote the formation of callus that can be embryogenic (EC) or non-embryogenic (NEC) by transferring EC to culture media devoid of auxins and with lower sucrose concentrations, embryos and plantlets develop. Thus, auxin pathway is essential in the regulation of SE, involving genes such as TIR, AFB or GRF’s that play a key role in auxin-mediated plant development processes. MicroRNA’s (miRNA’s) are small, single-stranded RNA’s known to function as post-transcriptional regulators of genes, repressing their expression through endonucleotidic cleavage. Two of those miRNA’s are miR393 and miR396 that play important roles in plant growth, development and maturation. Both are crucial in the auxin pathway, by regulating TIR and AFB genes expression (miR393), and the expression of GRF’s genes (miR396). This study aimed to analyse the expression of miR393 and miR396 and their target genes during tamarillo’s (Solanum betaceum Cav.) SE from zygotic embryos and leaf explants. Total RNA and small RNA’s enriched samples were extracted from embryogenic and non-embryogenic callus as well as from explants collected throughout the SE induction and embryo development processes. RNA samples were reverse transcribed into cDNA. Total RNA was used for the quantification of the expression levels of the target genes whereas the small RNA’s were used for the miRNA’s expression quantification by qPCR analysis.Results show that stage and tissue-specific expressions of miR393 and miR396 and their targets suggest their possible modulation on tamarillo SE where both miRNA’s share a strong inverse correlation with the corresponding target genes. MiR393 and miR396 seem to downregulate their target genes expression values during SE induction, while during somatic embryo development and germination, target genes expression values increased (miR393 - TIR1 and AFB2; miR396 - GRF1 and GRF4). These results provide new insights into embryogenic competence acquisition by tamarillo tissues, as well as how that competence is maintained during callus subcultures and expressed during embryo development.
Outro - Project PTDC/BAA-AGR/32265/2017 "Tamarillo breeding: better plants for better products"
Outro - Project “RENATURE - Valorization of the Natural Endogenous Resources of the Centro Region” (CENTRO-01-0145-FEDER-000007), funded by the Comissão de Coordenação da Região Centro (CCDR-C) and subsidized by the European Regional Development Fund (FEDER).

Accumulating evidence suggests that non-coding RNAs (ncRNAs) play key roles in gene regulation and may form the basis of an inter-gene communication system. MicroRNAs are a class of small non-coding RNAs found in both plants and animals that regulate the expression of other genes. Identification and analysis of microRNAs enhances our understanding of the important roles that microRNAs play in this complex regulatory network. The work presented in this thesis constitutes the first large-scale prediction and characterization of both ncRNAs and miRNAs in the model legume Medicago truncatula and Lotus japonicus, and provides a basis for further research on elucidating ncRNA function in legume genomics...

Research Doctorate - Doctor of Philosophy (PhD)
This thesis explores the role of microRNAs (miRNAs) that target the placental renin-angiotensin system (RAS) in placental development and function. The placental RAS contributes to trophoblast proliferation, migration and invasion, as well as vasoconstriction and angiogenesis. As such, the placental RAS is essential for adequate placental development and function. miRNAs targeting RAS mRNAs are present in the developing placenta, and their expression varies across gestation, along with RAS expression. Furthermore, changes in expression of these miRNAs are seen in pregnancy complications. This research investigated: the effects of low oxygen tension, such as the low oxygen environment of the first trimester placenta, on expression of miRNAs targeting the placental RAS ; the effect of miRNAs targeting the placental RAS on the functional ability of trophoblasts to proliferate, and ; the in vitro and in vivo roles of miR-155 in the placenta. I found that in HTR-8/SVneo cells (an immortalised human extravillous trophoblast cell line) cultured in low oxygen tension (~1%), there was suppressed expression of ten miRNAs predicted to target the RAS. Furthermore, this allowed increased expression of two critical RAS components. This would suggest that a low oxygen environment encourages placentation by suppressing miRNA expression to allow RAS activity. These experiments were then repeated in first trimester chorionic villus explants (CVE) obtained from primary tissue, where the expression of only four of the tested miRNAs was suppressed by low oxygen tension. The RAS components that were increased in this low oxygen milieu were also different to those increased in the cell line experiments. These experiments taken together illustrate the differing roles of miRNAs and the RAS in the extravillous trophoblasts and the chorionic villi. Investigation into the effect of miRNAs predicted to target the RAS on trophoblast proliferation showed that treatment with mimics of these miRNAs suppressed, or completely inhibited, trophoblast proliferation. In the case of many miRNAs tested, a dose-dependent response was observed, with higher mimic concentrations leading to lower proliferation of the cells. The effect of these miRNAs on their intended RAS targets was also assessed, with 7 of the 9 miRNAs suppressing mRNA expression of their RAS targets. These experiments demonstrated the functional effect of miRNA dysregulation of placental development through trophoblast proliferation. This has particular implications for a number of pregnancy complications that arise from poor placental development such as fetal growth restriction and preeclampsia. Finally, the role of miR-155 in placentation was observed, as a miR-155^-/- mouse model revealed the consequences of miR-155 deletion. Placentae from miR-155^-/- dams had significantly larger labyrinthine zones (responsible for substrate transfer to the fetus), but no change in placental weight, indicating an increase in the labyrinth zone to placental area ratio that would suggest that the placenta has improved efficiency of substrate transfer. These dams also had larger fetuses, possibly as a consequence of the changes in placental morphology. As mIR-155 is known to target the angiotensin II type 1 receptor (AT₁R), mRNA and protein was measured and was significantly increased in miR-155^-/- placentae. Additionally, in vitro investigation into the functional effects of miR-155, utilising a miR-155 mimic, showed that treatment with this miRNA decreases trophoblast proliferation in a dose-dependent manner. Altogether, this study clarified the importance of appropriate miR-155 regulation during pregnancy. This was particularly important as miR-155 upregulation is seen in preeclampsia (PE), a dangerous pregnancy complication. Therefore, through utilising a cell line, primary tissue explants and a murine model, I have been the first to demonstrate the effects of various miRNAs targeting the placental RAS on RAS expression, both in and out of a low oxygen milieu, trophoblast proliferation, placental morphology and fetal growth.

碩士
國立交通大學
生物資訊及系統生物研究所
102
MicroRNAs (miRNAs) are small non-coding RNAs of ~22 nucleotides that play an important role for most organisms through regulating gene expression in the post-transcriptional level. By base-pairing with 3’ untranslated region (3’-UTR) of mRNA, miRNAs function as degradation of mRNAs and repression of translation to achieve gene silencing. Owing to the importance of miRNA, computational prediction of miRNA-mRNA pairs is entry for us to learn the relationship between miRNAs and its targets. However, existing methods strongly focused on the seed-region complementarity or the cross-species conservation. Even though there has been significant progress on miRNA target prediction algorithm, it still have room for improvement. So in this study, we aim to construct a model to predict the miRNA-mRNA interaction with high accuracy. We apply GA-SVM algorithm which combines SVM (support vector machine) and GA (genetic algorithm) to increase prediction accuracy of miRNA target classification through select optimal feature subset. High-throughput datasets of miRNA-mRNA interaction are utilized for training and testing. Furthermore, data coming from miRTarBase were used for testing to improve the performance. The performance of the model is further evaluated by independent set and compare to other algorithms. In addition, the datasets are carried out for further analysis of miRNA-mRNA interaction and characteristics. In conclusion, we constructed a comprehensive dataset that comes from different methods especially negative data which was generated by the expression profiles from TCGA through selecting high Pearson correlation coefficient of miRNA-target pairs. A GA-SVM model was built for miRNA target prediction. Several information about miRNA-target interaction were taken into account and it lay a foundation for researchers to investigate miRNA and target interaction.

Generally Hepatitis C Virus tropism is limited to hepatocytes. This limited tropism is a result of the receptors HCV requires for cellular entry and other host cellular factors including, uniquely, a liver specific miRNA, miR-122. The relationship between HCV and miR-122 is interesting, as commonly, miRNA are associated with suppression of function, but in the case of HCV, miR-122 actively promotes HCV proliferation. In-depth studies have demonstrated that miR-122 along with the RNA induced silencing complex (RISC) protein Argonaute 2 (Ago2) binds directly to two seed sequences separated by 8-9 nucleotides on HCV 5’UTR. Binding to the 5’UTR results in an increase in viral replication and translation. The method by which miR-122 promotes HCV translation and replication is not fully understood but evidence suggests that part of the function of miR-122 is to stabilize the HCV genome and protect it from exonuclease degradation by Xrn1, but other mechanisms remain to be identified. The reliance of HCV on miR-122 is best exemplified by the fact that removal of miR-122 by a miR-122 antagonist drastically impedes HCV viral titers in Chimpanzees and humans with no indication of escape mutants. The observation that HCV augmentation of the HCV life cycle by miR-122 requires Ago2 suggests that other components downstream in the miRNA suppression pathway may also be part of the mechanism of action. Our studies focused specifically on the processing body (p-body) associated DEAD-box helicase DDX6. DDX6 is essential for p-body assembly, required for robust miRNA suppression activity and elevated in HCV associated hepatocellular carcinomas. As such we hypothesized that DDX6 and p-bodies were directly or in-directly associated with the mechanism of action of miR-122. Knocking down DDX6 with siRNA indicated that DDX6 augments both HCV replication and translation. To examine whether DDX6 augmentation of HCV replication was related to the effects of miR-122 on the HCV life cycle, HCV replication and translation were assessed in the presence or absence of miR-122 when DDX6 was knocked down. Our data indicated that HCV replication and translation were augmented equally by miR-122 whether DDX6 was present or not. Our data also demonstrated that HCV replication and translation that was occurring independent of miR-122 was also still affected by DDX6 knockdown. Taken together our observations strongly suggest that the role DDX6 has on HCV is independent of HCV and miR-122’s relationship. In order to better understand miR-122’s relationship with HCV, we hypothesized that targeting the miR-122 binding region with siRNA would inhibit HCV replication initially, but that over the course of several rounds of treatment with the same siRNA, HCV would mutate to escape the siRNA, producing escape mutants that replicate without a dependency on miR-122. These escape mutants could be evaluated on how they replicate without using miR-122, shedding light on miR-122 and HCV’s relationship. Conversely if no escape mutants arose the siRNA could be further studied as a potential therapeutic for HCV. siRNA designed to target the miR-122 binding region inhibited HCV replication, confirming that the designed siRNAs could access the miR-122 binding region and function as an siRNA. Interestingly, when the siRNAs were used against a replication competent HCV RNA having a single nucleotide mutation in the first miR-122 binding site, instead of abolishing siRNA knockdown, two of the siRNA showed enhanced inhibition activity. The target sequences of these siRNAs spanned both miR-122 binding sites and we speculate that their inhibitory activity was due to competition for miR-122 binding to site 2. This observation indicates that siRNA targeting the miR-122 binding region have dual activity, by siRNA induced cleavage, and as a competitive inhibitor of miR-122 binding. Selection for viral escape mutants of the miR-122-binding site targeting siRNAs revealed viral RNAs having mutations within the miR-122 binding sites, in the surrounding region, and to other areas within the HCV IRES. The mutant viruses will be used to assess the influence of miR-122 binding site mutations on HCV replicative fitness, and to determine if the virus can evolve to replicate independent from augmentation by miR-122.

Pinus pinaster is a conifer species with high ecologic and economic value which covers a vast area of the Mediterranean region. However, the area occupied by P. pinaster has been decreasing mainly due to environmental causes. Somatic embryogenesis may contribute to circumvent this problem as it allows the large-scale propagation of plants adapted to several stresses. However, this technique is poorly understood in P. pinaster and the study of gene expression regulators like miRNAs may contribute to its improvement. MiRNAs regulate gene expression at post-transcriptional level by mRNA cleavage or translation repression. In this work, a set of four miRNA-mRNA target cleavage interactions were studied. First, mRNAs targets were predicted for a list of miRNAs using bioinformatics tools to analyze available sequencing data, and seven miRNA-mRNA pairs were selected. Second, the expression of four of the mRNAs were analyzed by RT-PCR in two embryo developmental stages, T4B (pre-cotyledonary) and T7 (mature). The four transcripts included ARF 10, 16 ou 17 (regulated by miR160), F-box (regulated by miR482a), LEA (regulated by miRM09664) and PPR (regulated by miRM06658). All transcripts show a higher expression in T4B than in T7 stage. Several parameters were studied to ensure that high-confidence pairs were selected, namely conservation of miRNA and respective target, complementarity between miRNA and respective target, relation between miRNA expression and target expression. Only miRNA160-ARF target fulfilled all the conditions. It was the first time that potential miRNA targets were identified in P. pinaster. This work contributed to uncover the miRNA landscape in conifers, pointing to potential functions in embryo development. However, more experiments are needed to further validate the interaction between the miRNA-mRNA targets studied.

碩士
國立中興大學
分子生物學研究所
99
MiRNA (miRNAs), a group of single-stranded small RNAs, are about 21 nucleotides in length. These miRNAs regulate their target genes by mRNA cleavage or transcriptional repression. MiRNAs play an important role in plant growth and development, including down-regulating transcription factors to operate the transcript levels of downstream genes. Auxin response factors (ARFs) are one of the transcription factor families which are regulated by miRNAs. There have been many miRNAs found in rice, but few of them have been carefully investigated. It has been shown that transgenic rice over-expressing miR160a could suppress the expressions of OsARF18 and OsARF22 genes, and their targeted-cleavage sites has been identified by RLM-5’RACE assay. Further treatment with exogenously added ABA showed more severe inhibition on seedling roots growth in miR160a over-expression transgenic rice than those of the wild-type. In addition, a wider tiller growth angle since the booting stage was observed in miR160a over-expression transgenic rice suggesting a regulatory role of miR160a in rice architecture development. Another rice miR167b has been studied to analyze its effects on plant growth and target gene OsARF25 by creating miR167b over-expression transgenic rice. The expression levels of miR167b and its putative target gene OsARF25 of five transgenic rice lines were analyzed. Results showed that the higher the expression levels of miR167b in the transgenic rice lines, the lower the expression levels of OsARF25 were detected, suggesting that OsARF25 is the target gene of miR167b. A phenotype with dwarf and wider tiller growth angle was observed in two independent transgenic lines that expressed approximately 9 to 11-fold of miR167b than that of the wild-type, while the other three lines that expressed approximately 4 to 7-fold of miR167b revealed the same phenotype as the wild-type. How can this phenotype be attributed by the different expression levels of miR167b and OsARF25 require further investigation. In this study, an approach to establish a quick miRNA target genes screening method using the known relationship between miR160a and OsARF18 was also performed, but was not successful. Further investigations to increase the sensitivity and stability of the assay method are required.

Research Doctorate - Doctor of Philosophy (PhD)
microRNA (miRNA) are small non-coding RNA molecules that function to guide the miRNA-induced silencing complex (miRISC) to regions of complementarity within mRNA transcripts, thereby mediating sequence-specific post-transcriptional gene silencing (PTGS). This form of gene regulation is vital to a variety of biological processes in animals, from fundamental developmental functions including cellular proliferation and differentiation, to more complex and specialised roles such as long-term potentiation and synapse-specific modifications in neurons. While the complementarity of miRNA-mRNA interactions enables bioinformatic algorithms to predict potential target genes, the capacity for miRNA to interact with their targets through elements of only partial complementarity renders high false discovery rates to these predictions. With the discovery of increased cortical miRNA expression in schizophrenia, biological evidence was therefore required to investigate the function of schizophrenia-associated miRNA including miR-181b, miR-107, and members of the miR-15 and miR-17 families. Functional genomic techniques were established with genome-wide expression analysis and miRNA reporter-gene assays to characterise the biology of these miRNA in a variety of cellular contexts. In vitro characterisation of miR-181b revealed target gene regulation through interactions at both conserved and non-conserved MREs, with primary and secondary effects of miRNA modulation affecting many genes enriched within neurodevelopmental pathways vital to synaptic plasticity, including a number of schizophrenia candidate genes. Surprisingly, comparisons of miR-181b and miR-107 function against target prediction frameworks revealed a subset of predicted targets displaying positive correlation with cognate miRNA expression. Context-specific functional pleiotropy was also observed in different cellular environments, and this was further explored through miR-17/20a in T lymphocyte activation, and through miR-16 in cell cycle progression. Ultimately, substantial evidence was obtained to establish biological plausibility for miR-181b dysregulation as a potential aetiological factor in the development of schizophrenia, though the emergent theme of context-specific miRNA function requires future methodological developments and experiments to consider this functional pleiotropy when exploring miRNA function in normal developmental and pathophysiological states.

MicroRNAs (miRNAs) are a class of short (21-25 nt) non-coding endogenous RNAs that mediate the expression of their direct target genes post-transcriptionally. The goal of this thesis is to identify the target genes of miRNAs using computational methods. The most popular computational target prediction methods rely on sequence based determinants to predict targets. However, these determinants are neither sufficient nor necessary to identify functional target sites, and commonly ignore the cellular conditions in which miRNAs interact with their targets \emph{in vivo}. Since miRNAs activity reduces the steady-state abundance of mRNA targets, the main goal of this thesis is to augment large scale gene expression profiles as a supplement to sequence-based computational miRNA target prediction techniques. We develop two computational miRNA target prediction methods: InMiR and BayMiR; in addition, we study the interaction between miRNAs and lncRNAs using long RNA expression data. InMiR is a computational method that predicts the targets of intronic miRNAs based on the expression profiles of their host genes across a large number of datasets. InMiR can also predict which host genes have expression profiles that are good surrogates for those of their intronic miRNAs. Host genes that InMiR predicts are bad surrogates contain significantly more miRNA target sites in their 3 UTRs and are significantly more likely to have predicted Pol II-III promoters in their introns. We also develop BayMiR that scores miRNA-mRNA pairs based on the endogenous footprint of miRNAs on gene expression in a genome-wide scale. BayMiR provides an ``endogenous target repression" index, that identifies the contribution of each miRNA in repressing a target gene in presence of other targeting miRNAs. This thesis also addresses the interactions between miRNAs and lncRNAs. Our analysis on expression abundance of long RNA transcripts (mRNA and lncRNA) shows that the lncRNA target set of some miRNAs have relatively low abundance in the tissues that these miRNAs are highly active. We also found lncRNAs and mRNAs that shared many targeting miRNAs are significantly positively correlated, indicating that these set of highly expressed lncRNAs may act as miRNA sponges to promote mRNA regulation.

博士
國立成功大學
基礎醫學研究所
104
Oral cancer is a deadly disease, ranking the eighth among all cancers in mortality. MicroRNAs (miRNAs) are small non-coding RNAs that mediate gene expression at the post-transcriptional level by degrading or repressing the translation of target mRNAs. Although accumulating evidence suggests that miRNAs function as oncogenes or tumor suppressors in human malignancy, there are still few studies focused on the role of miRNA(s) in oral carcinogenesis. To clarify the miRNAs involved in oral cancer, human miRNA microarray was used to identify the down-regulation of 54 miRNAs in three oral cancer cell lines when compared to normal oral keratinocytes (NOK). miR-99a as the most down-regulated in oral cancer cells (~32 folds). MiR-99a down-regulation was also confirmed both in tested oral cancer cell lines and clinical specimens. To study the role of miR-99a in oral cancer, etopic miR-99a expression inhibited oral cancer cell migration and invasion. Anti-miR-99a, silencing miR-99a functions, had the opposite effect. Myotubularin- related protein 3 (MTMR3) with one evolutionarily conserved seed region in the 3’-untranslated region was a novel miR-99a target. Depleting MTMR3 expression significantly reduced cell proliferation, migration, or invasion. There was an inverse expression of miR-99a and MTMR3 protein in oral cancer lines and clinical specimens. Together, miR-99a repressed oral cancer cell migration and invasion partly through decreasing MTMR3 expression. In addition to miR-99a, we also identified the downregulation of miR-22 in oral cancer cell lines and clinical specimens. Hyaluronan synthases 3 (HAS3) was predicted as a novel target of miR-22 and ectopic miR-22 repressed the levels of both endogenous and ectopic HAS3. The expression of HAS3 mRNA, involved in pro-inflammatory low molecular mass HA (LMM-HA) accumulation, was also highly expressed in oral cancer. To assess the functionality, ectopic HAS3 expression significantly increased oral cancer cell migration, invasion and xenograft tumorigenesis accompanied with the increase of pro-inflammatory TNF-α and MCP-1 expression. Conversely, HAS3 depletion significantly abrogated HAS3-mediated stimulation. The oncogenic action of HAS3 was partly through the activation of EGFR-Src signaling axis. The release of HAS3-derived LMM-HA into extracellular milieu enhanced transendothelial monocyte migration and MCP-1 expression, which could be attenuated by the addition of HAS3 antibody or an inhibitor, 4-Methylumbelliferone (4-MU). Consistent with the stimulatory effects of cytokines and growth factors on HAS3 expression, we also found TNF-α dose-dependently increased HAS3 mRNA expression partly through the activation of NF-κB in oral cancer cells. The increase of HAS3 mRNA expression significantly reduced the overall survival of late-stage oral cancer patients and high TNF-α expression further negated the clinical outcome among these patients. Overexpressed HAS3, a target of miR-22, enhanced crucial biological activities necessary for tumorigenesis and thus offered an advantageous environment for oral cancer progression. Taken together, MTMR3 and HAS3, rather than miR-99a or miR-22, might serve as theranostic targets for oral cancer treatment although more studies are needed to validate this possibility.

abstract: MicroRNAs (miRNAs) are short non-coding RNAs that play key roles during metazoan development, and are frequently misregulated in human disease. MiRNAs regulate gene output by targeting degenerate elements primarily in the 3´ untranslated regions of mRNAs. MiRNAs are often deeply conserved, but have undergone drastic expansions in higher metazoans, leading to families of miRNAs with highly similar sequences. The evolutionary advantage of maintaining multiple copies of duplicated miRNAs is not well understood, nor has the distinct functions of miRNA family members been systematically studied. Furthermore, the unbiased and high-throughput discovery of targets remains a major challenge, yet is required to understand the biological function of a given miRNA. I hypothesize that duplication events grant miRNA families with enhanced regulatory capabilities, specifically through distinct targeting preferences by family members. This has relevance for our understanding of vertebrate evolution, as well disease detection and personalized medicine. To test this hypothesis, I apply a conjunction of bioinformatic and experimental approaches, and design a novel high-throughput screening platform to identify human miRNA targets. Combined with conventional approaches, this tool allows systematic testing for functional targets of human miRNAs, and the identification of novel target genes on an unprecedented scale. In this dissertation, I explore evolutionary signatures of 62 deeply conserved metazoan miRNA families, as well as the targeting preferences for several human miRNAs. I find that constraints on miRNA processing impact sequence evolution, creating evolutionary hotspots within families that guide distinct target preferences. I apply our novel screening platform to two cancer-relevant miRNAs, and identify hundreds of previously undescribed targets. I also analyze critical features of functional miRNA target sites, finding that each miRNA recognizes surprisingly distinct features of targets. To further explore the functional distinction between family members, I analyze miRNA expression patterns in multiple contexts, including mouse embryogenesis, RNA-seq data from human tissues, and cancer cell lines. Together, my results inform a model that describes the evolution of metazoan miRNAs, and suggests that highly similar miRNA family members possess distinct functions. These findings broaden our understanding of miRNA function in vertebrate evolution and development, and how their misexpression contributes to human disease.
Dissertation/Thesis
Doctoral Dissertation Molecular and Cellular Biology 2016

CUNHA, Pedro Miguel Pacheco de Jesus da - On the development of a novel targeted miRNA-based therapy towards glioblastoma. Coimbra : [s.n.], 2015. Dissertação de Mestrado em Biologia Celular e Molecular.
Glioblastoma (GBM) is a fast growing, highly vascularized and chemoresistant cancer, being the most common and malignant type of brain tumor. Despite recent advances in the identification of genomic/molecular alterations that fuel the tumor and the aggressive standard post-surgery treatment regimen of radiotherapy and concomitant or adjuvant chemotherapy with temozolomide (TMZ), the average patient survival post-diagnosis remains very low (14.6- months). In this work, we aim at finding new therapeutic targets for GBM treatment and eradication. Abnormal microRNA (miRNA) expression has been clearly associated with cancer development and progression, including GBM pathogenesis and resistance to therapy. Therefore, we hypothesized that a gene therapy based on the modulation of miRNAs aberrantly expressed in GBM cells might promote cancer cell killing and tumor eradication, either per se or in combination with chemotherapy. We have found that the transient increase in the levels of miR-128 and miR-302a, downregulated in two human GBM cell lines (U87 and DBTRG), including isolated CD133+ cells, promoted anti-proliferative effects, which were strengthened upon cell treatement with TMZ, sunitinib and axitinib. On the other hand, we have demonstrated that miR-302a arrested cell cycle in S phase, which was further potentiated by chemotherapeutic drug combination. Next, we conducted in vivo studies using an orthotopic GBM mouse model to test the anti-tumoral potential of the combined therapy involving miR- 21 modulation and sunitinib treatment, which previous studies in our laboratory had demonstrated to constitute a promising approach for GBM treatment. We demonstrated that stable nucleic acid lipid particles targeted to GBM cells, through covalent coupling of chlorotoxin (CTX-SNALPs) were able to specifically deliver nucleic acids to intracranial tumors in mice upon intravenous injection, without causing toxicity. Moreover, silencing of miR-21 (overexpressed in GBM) via CTX-SNALPs containing anti-miR-21 oligonucleotides rendered GBM cells highly susceptible to the anti-angiogenic drug sunitinib. Altogether, our results clearly show that miRNA modulation working alongside chemotherapy constitutes a promising strategy towards GBM eradication. Importantly, the CTX-SNALP formulations show to be an appropriate vehicle for a clinical application towards GBM.
O glioblastoma (GBM) é o tipo de cancro cerebral mais comum e mais maligno, caracterizandose por elevada taxa de proliferação, abundante vascularização e alta resistência à quimioterapia. Apesar dos recentes avanços científicos na identificação das alterações genómicas e moleculares responsáveis pela progressão deste tumor e do agressivo tratamento pós-cirúrgico, que tipicamente alia à radioterapia a administração do fármaco temozolomide (TMZ), a verdade é que a sobrevivência dos pacientes após diagnóstico desta patologia permanece muito baixa (em média 14,6 meses). O objetivo do presente trabalho reside em encontrar novos alvos para o tratamento de GBM, com vista à sua erradicação. A expressão anómala de microRNAs (miRNAs) tem sido claramente associada ao desenvolvimento e progressão de tumores, incluindo GBM, e à aquisição de resistência aos tratamentos convencionais. Desta forma, o trabalho realizado no âmbito da presente tese emergiu da hipótese de que uma terapia génica, baseada na manipulação dos níveis de expressão de miRNAs em GBM, per se ou coadjuvada com quimioterapia, seria capaz de conduzir à morte das células cancerígenas e, assim, à erradicação do tumor. Consistentemente, demonstrou-se neste trabalho que um aumento transitório dos níveis de miR-128 e miR-302a, que se encontravam sub-expressos em duas linhas celulares humanas de GBM (U87 e DBTRG), e inclusivamente nas células CD133+ isoladas a partir dessas linhas, promovia efeitos antiproliferativos que se tornavam mais acentuados após tratamento das células com TMZ, sunitinib e axitinib. Por outro lado, verificou-se que miR-302a induzia a paragem do ciclo celular na fase S, efeito que era potenciado pela combinação com fármacos antitumorais. Posteriormente, foram conduzidas experiências in vivo, com recurso a um modelo animal ortotópico de GBM, de forma a testar o potencial antitumoral de uma estratégia combinada, envolvendo regulação dos níveis de expressão de miR-21 e administração de sunitinib, que estudos prévios in vitro, levados a cabo no nosso laboratório, haviam demonstrado constituir uma abordagem promissora para o tratamento de GBM. Partículas lipídicas estáveis de ácidos nucleicos, endereçadas a células de GBM através da conjugação de clorotoxina (CTX-SNALPs), mostraram a capacidade de entregar os ácidos nucleicos a tumores intracranianos em murganhos, após administração intravenosa, sem causarem toxicidade. Para além disso, o silenciamento de miR-21 (sobreexpresso em GBM) através de CTX-SNALPs contendo oligonucleótidos anti-miR-21 tornou as células de GBM mais susceptíveis ao fármaco antiangiogénico sunitinib. No seu conjunto, os nossos resultados mostraram claramente que a manipulação dos níveis de miRNAs em paralelo com quimioterapia constitui uma estratégia promissora visando a erradicação de GBM. Adicionalmente, CTX-SNALPs revelaram-se formulações adequadas ao transporte de fármacos, com potencial aplicação clínica para o tratamento de GBM.