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Sætrom, Ola. "Predicting MicroRNA targets." Thesis, Norwegian University of Science and Technology, Department of Computer and Information Science, 2005. http://urn.kb.se/resolve?urn=urn:nbn:no:ntnu:diva-9266.
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MicroRNAs are a large family of short non-encoding RNAs that regulated protein production by binding to mRNAs. A single miRNA can regulate an mRNA by itself, or several miRNAs can cooperate in regulating the mRNAs. This is all dependent on the degree of complementarity between the miRNA and the target mRNA. Here, we present the program TargetBoost that, using a classifier generated by a combination of hardware accelerated genetic programming and boosting, allows for screening several large dataset against several miRNAs, and computes a likelihood of that genes in the dataset is regulated by the set of miRNAs used in the screening. We also present results from comparison of several different scoring functions for measuring cooperative effects. We found that the classifier used in TargetBoost is best for finding target sites that regulate mRNAs by themselves. A demo of TargetBoost can be found on http://www.interagon.com/demo.
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Migliore, Chiara Maria. "RNA-sequencing based identification of microRNA-204 targets." Doctoral thesis, Università degli studi di Trieste, 2011. http://hdl.handle.net/10077/4595.
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2009/2010With the completion of the sequencing and annotation of hundreds of genomes, and the accumulation of data on the mammalian transcriptome, greater emphasis has been placed on elucidating the function of non-coding DNA and RNA sequences. It is well known that the non-coding portion of the genome can transcribe functional RNAs. Several categories of non-coding RNAs (ncRNAs) have been defined, such as transport RNAs (tRNAs) ribosomal RNAs (rRNAs), small nuclear RNAs (snRNAs) and small nucleolar RNAs (snoRNAs). A larger group of ncRNAs comprises the so-called microRNAs (miRNAs) and long non-coding RNAs serving key regulatory roles. It has been shown that miRNAs directly target a large number of genes, thus affecting significantly major pathways. In my project, I focused on miR-204, a microRNA that is highly conserved from zebrafish to human and located in the sixth intron of the human TRPM3 gene. I sought to identify mir-204 targets by using the Medaka fish (Oryzias latipes), where mir-204 is expressed at very low levels in the nervous system, as a model for perturbation of the mir-204 network. Transient transgenic Medaka fish were produced to knock down and over-express mir-204. Next-generation sequencing was used to sequence the Medaka transcriptome, dissect the putative targets of miR-204, and thus gain further insight about its function. Potential target genes of mir-204 were selected by choosing genes, which presented lower expression in the wild-type (wt) fish than in the knock down, a lower expression in the over-expression than in the wt and, finally, a higher expression in the knock down than in the over-expression. At the same time, I collected a list of putative miR-204 mouse and human targets using the prediction softwares miRanda, PicTar and TargetScan, obtained the Medaka orthologues and verified that the selected genes in Medaka had a statistically significant enrichment in miR-204 targets as compared to the complete set of genes obtained from the RNA-Sequencing approach. The combined RNA-Sequencing and bioinformatics analysis revealed 147 predicted targets of mir-204, which showed a significant enrichment for the axon guidance pathway. In order to confirm this data, real time quantitative PCR has been performed on total RNA from wt and morphant fish. Results showed a higher expression in the knock down fish for 15 out of 25 putative targets (Neo1, Trim71, Ddx3y, Prkar1a, MyoX, Sema3B, Sema3F, Ptprg, Slit2, Epha4, Epha7, Amot, Lpp, Odz4, Jarid2). I further validated these genes by both Q-PCR and luciferase assays. To this aim, I cloned five putative target sequences into the 3’UTR of a luciferase reporter vector (pGL3-TK-luc Promega) to use them in luciferase assays: co-transfection with miR-204 reduced the luciferase activity of Sema3F, belonging to the class of receptors involved upstream of the axon guidance pathway. These results indicate that mir-204 directly targets key genes involved in the axon guidance pathway such as Sema3F in the nervous system. Further validation of the disruption of axon guidance in the transgenic fish has been undertaken in vivo by our collaborators: the experiment demonstrated a clear role of this microRNA in axon path finding during retinal development.
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Wang, Qi. "Using Imputed Microrna Regulation Based on Weighted Ranked Expression and Putative Microrna Targets and Analysis of Variance to Select Micrornas for Predicting Prostate Cancer Recurrence." Thesis, North Dakota State University, 2014. https://hdl.handle.net/10365/27341.
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Imputed microRNA regulation based on weighted ranked expression and putative microRNA targets (IMRE) is a method to predict microRNA regulation from genome-wide gene expression. A false discovery rate (FDR) for each microRNA is calculated using the expression of the microRNA putative targets to analyze the regulation between different conditions. FDR is calculated to identify the differences of gene expression. The dataset used in this research is the microarray gene expression of 596 patients with prostate cancer. This dataset includes three different phenotypes: PSA (Prostate-Specific Antigen recurrence), Systemic (Systemic Disease Progression) and NED (No Evidence of Disease). We used the IMRE and ANOVA methods to analyze the dataset and identified several microRNA candidates that can be used to predict PSA recurrence and systemic disease progression in prostate cancer patients.
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Davis, M. P. "Generation of a murine ES cell system deficient in microRNA processing for the identification of microRNA targets." Thesis, University of Cambridge, 2009. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.598389.
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I have developed a system in mouse embryonic stem (ES) cells to simply and rapidly derive gene lists enriched for miRNA targets. I have disrupted miRNA processing by the targeted insertion of a gene trap cassette into the second allele of Dgcr8 in cell lines that already bear a gene trap within their first allele. This led to a broad reduction of miRNA processing in these cells. As a consequence of the disruption of this locus I was able to identify a number of miRNAs that appear to be processed in DGCR8 independent manner. I proceeded to transfect these cells with Es-cell-expressed miRNA mimics. I used microarrays to identify transcripts that are down regulated as a consequence of the miRNA reintroduction. By comparing transcripts that had been up regulated upon the depletion of Dgcr8 to this set I was able to create miRNA target lists for mmu-miR-25 and mmu-miR-291a-3p. These lists should be enriched for functionally relevant, co-expressed targets, moderated for miRNA mimic over expression and to a large extent devoid of interference from target saturation and combinatorial regulation. The system should also not be susceptible to problems associated with functional redundancy. In total I identified 25 target candidates for miR-291a-3p and 40 candidates for miR-25. Amongst these genes are a number of oncogenes and tumour suppressor genes in addition to genes involved in cell cycle regulation and extra-cellular signal transduction. In conclusion it appears that miRNAs play a fundamental role in the regulation of the ES cell transcriptome and as such are deserving of considerable future research. It is my belief that the method presented in this thesis could contribute significantly to this effort by providing substantial and experimentally derived miRNA candidate target lists upon which to base future hypotheses.
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Torkey, Hanaa A. "Machine Learning Approaches for Identifying microRNA Targets and Conserved Protein Complexes." Diss., Virginia Tech, 2017. http://hdl.handle.net/10919/77536.
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Much research has been directed toward understanding the roles of essential components in the cell, such as proteins, microRNAs, and genes. This dissertation focuses on two interesting problems in bioinformatics research: microRNA-target prediction and the identification of conserved protein complexes across species. We define the two problems and develop novel approaches for solving them. MicroRNAs are short non-coding RNAs that mediate gene expression. The goal is to predict microRNA targets. Existing methods rely on sequence features to predict targets. These features are neither sufficient nor necessary to identify functional target sites and ignore the cellular conditions in which microRNA and mRNA interact. We developed MicroTarget to predict microRNA-mRNA interactions using heterogeneous data sources. MicroTarget uses expression data to learn candidate target set for each microRNA. Then, sequence data is used to provide evidence of direct interactions and ranking the predicted targets. The predicted targets overlap with many of the experimentally validated ones. The results indicate that using expression data helps in predicting microRNA targets accurately.
Protein complexes conserved across species specify processes that are core to cell machinery. Methods that have been devised to identify conserved complexes are severely limited by noise in PPI data. Behind PPIs, there are domains interacting physically to perform the necessary functions. Therefore, employing domains and domain interactions gives a better view of the protein interactions and functions. We developed novel strategy for local network alignment, DONA. DONA maps proteins into their domains and uses DDIs to improve the network alignment. We developed novel strategy for constructing an alignment graph and then uses this graph to discover the conserved sub-networks. DONA shows better performance in terms of the overlap with known protein complexes with higher precision and recall rates than existing methods. The result shows better semantic similarity computed with respect to both the biological process and the molecular function of the aligned sub-networks.Ph. D.
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Woodcock, M. Ryan. "Network Analysis and Comparative Phylogenomics of MicroRNAs and their Respective Messenger RNA Targets Using Twelve Drosophila species." VCU Scholars Compass, 2010. http://scholarscompass.vcu.edu/etd/155.
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MicroRNAs represent a special class of small (~21–25 nucleotides) non-coding RNA molecules which exert powerful post-transcriptional control over gene expression in eukaryotes. Indeed microRNAs likely represent the most abundant class of regulators in animal gene regulatory networks. This study describes the recovery and network analyses of a suite of homologous microRNA targets recovered through two different prediction methods for whole gene regions across twelve Drosophila species. Phylogenetic criteria under an accepted tree topology were used as a reference frame to 1) make inference into microRNA-target predictions, 2) study mathematical properties of microRNA-gene regulatory networks, 3) and conduct novel phylogenetic analyses using character data derived from weighted edges of the microRNA-target networks. This study investigates the evidences of natural selection and phylogenetic signatures inherent within the microRNA regulatory networks and quantifies time and mutation necessary to rewire a microRNA regulatory network. Selective factors that appear to operate upon seed aptamers include cooperativity (redundancy) of interactions and transcript length. Topological analyses of microRNA regulatory networks recovered significant enrichment for a motif possessing a redundant link in all twelve species sampled. This would suggest that optimization of the whole interactome topology itself has been historically subject to natural selection where resilience to attack have offered selective advantage. It seems that only a modest number of microRNA–mRNA interactions exhibit conservation over Drosophila cladogenesis. The decrease in conserved microRNA-target interactions with increasing phylogenetic distance exhibited a cure typical of a saturation phenomena. Scale free properties of a network intersection of microRNA target predictions methods were found to transect taxonomic hierarchy.
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Budd, William. "Development and Implementation of a Tissue Specific MicroRNA Prediction Tool for Identifying Targets of the Tumor Suppressor microRNA 17-3p." VCU Scholars Compass, 2010. http://scholarscompass.vcu.edu/etd/2116.
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A unique computational approach was undertaken to identify targets of miR-17-3p that impart an oncogenic potential to the cells of the prostate. Utilizing this approach, we identified insulin growth factor receptor 1 (IGF1R) as a potential target of miR-17-3p. IGF1R imparts an oncogenic approach to the cells by helping cells escape apoptosis, become hypertrophic and increase the production of extracellular proteases that allow cells to detach from neighbors. The regulation of insulin growth factor receptor 1 by human microRNA-17-3p was evaluated using a western blot analysis of prostate cancer cell lines. Protein levels were compared in a cell line that expressed a non-targeting control RNA and a cell line that expressed microRNA-17-3p. The cell line that expressed the non-targeting control had significantly higher levels of IGF1R protein than the cell line expressing more of the active microRNA. Based on this experiment, it appears that microRNA-17-3p might regulate the insulin growth factor receptor 1.
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Joo, Lauren Jin Suk. "RET-regulated microRNAs as Recurrence Biomarkers and Therapeutic Targets in Medullary Thyroid Carcinoma." Thesis, The University of Sydney, 2018. http://hdl.handle.net/2123/19945.
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Medullary thyroid carcinoma (MTC) is an aggressive malignancy which accounts for 3 – 5 % of all thyroid cancers. MTC originates from calcitonin-producing parafollicular C-cells of the thyroid gland. Genetically, gain-of-function mutations of the RET tyrosine kinase is known to be the key driver of MTC tumourigenesis. RET has been targeted by tyrosine kinase inhibitors (TKIs), however the efficacy has been modest. MicroRNAs are small non-coding RNAs and are known to suppress gene expression. Deregulation of miRNAs in cancer is often associated with the progression of malignancy, and targeting miRNA expression can modify cancer behaviour. We aimed to identify miRNAs whose expressions are altered by RET in MTC, investigate their cellular and molecular effects on tumourigenesis and on modulation of TKI (cabozantinib) responses, and ultimately establish a novel miRNA therapeutic target for MTC. Small RNA-Sequencing was performed in MTC cells before and after RET inhibition to identify RET-regulated miRNAs. Expressions of potential miRNAs were validated in a large cohort of clinical MTC tissue samples. We identified a specific miRNA, miR-153-3p which was under-expressed in MTC. In vitro gain-of-function studies demonstrated the tumour suppressive roles of miR-153-3p in MTC. Restoration of miR-153-3p reduced cell proliferation, migration and invasion, while enhancing apoptosis. miR-153-3p repressed the expression of ribosomal protein S6 kinase B1 (RPS6KB1) of mTOR signalling and further reduced downstream phosphorylation of Bcl-2 associated death promoter (BAD). Finally, miR-153-3p alone and/or in combination with cabozantinib was tested in mouse xenograft models. miR-153-3p delivery alone significantly impeded the xenograft growth. Combined treatment resulted in greater growth inhibition. Furthermore, miR-153-3p appeared to enhance cell responses to cabozantinib in the xenografts. We report for the first time how RET contributes to tumour behaviour through regulation of specific miRNAs in MTC. We have identified miR-153-3p, which offers potential as a therapeutic target and biomarker for recurrence and disease progression. This study highlights potential for improved therapeutic efficacy with a novel combined treatment of miRNA plus TKIs, especially for patients with advanced, metastatic MTC.
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Rose, Jarod. "An Investigation and Visualization of MicroRNA Targets and Gene Expressions and Their Use in Classifying Cancer Samples." University of Akron / OhioLINK, 2011. http://rave.ohiolink.edu/etdc/view?acc_num=akron1302303717.
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Youssef, Ninwa. "Analysis of conserved microRNA targets in the nematode Caenorhabditis elegans and the fruit fly Drosophila melanogaster." Thesis, Södertörns högskola, Institutionen för naturvetenskap, miljö och teknik, 2013. http://urn.kb.se/resolve?urn=urn:nbn:se:sh:diva-19211.
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MicroRNA (miRNA) is small regulatory non-coding single stranded RNA molecule that can repress protein expression either at transcriptional or translational level. Since their discoveries in nematodes in the early 1990´s extensive research have shown that this mechanism is conserved across species. Because the miRNA is so small, about 22 nucleotides (nt) long and only requires a minimum of 6nt to interact imperfect with its intended target 3´UTR, therefore a single miRNA could potentially have hundreds of potential targets, which have been suggested by computational prediction. The goal of the project is to experimentally verify three predicted Caenorhabditis elegans mir-2 miRNA targets in cell culture, with as candidate targets fos-1, mek-1 and sel-5. In addition C. elegans mir-2 and its mechanism is conserved in Drosophila Melanogaster, miR-2. We want to elucidate if not only mir-2 miRNA is cross species conserved but also it targets. To test this hypothesis we selected the following predicted mir-2 target candidate genes: C. Elegans iff-1 and Drosophila Melanogaster protein ortholog eIF-5A. Validation of miRNA and its functionality was done by transfecting cells with a luciferase-3´UTR reporter only or luciferase-3´UTR and a miRNA-expression plasmid. After the reporter gene was induced, cells were harvested and the luciferase activity measured and the results normalized and compared. Unfortunately our data were inconclusive and future experiments are needed to give a clear picture.
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Martinez-Nunez, Rocio Teresa. "Role of microRNA-155 in dendritic cells and macrophages : MiR-155 directly targets PU.1 and IL13Rα1." Thesis, University of Southampton, 2010. https://eprints.soton.ac.uk/196569/.
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In search of genes differentially expressed between M1 (pro‐Th1 or pro‐inflammatory) and M2 (pro‐Th2 or pro‐tolerogenic) macrophages, BIC (microRNA 155 hosting gene) was found up regulated under inflammatory conditions. MicroRNAs are non coding RNAs of ~22nt in length that inhibit gene expression upon pairing to the 3’UTR (UnTranslated Region) of target mRNAs. In silico analysis predicted two pro‐Th2 targets for miR‐155: PU.1 and IL13R1. PU.1 is a transcription factor essential in myelopoiesis and dendritic cells (DCs) that favours a Th2 profiling; moreover, PU.1 had been shown to regulate the transcription of DC‐SIGN (Dendritic Cell‐ Specific ICAM‐3 Grabbing Non‐integrin 1) which is a pathogen receptor expressed in DCs controlled by Th2 stimuli. IL13R1 is the chain receptor for the Th2 cytokine IL‐13, which promotes M2 differentiation. Pro Th1 stimuli cause maturation of DCs, cells that orchestrate the immune response between Th1 and Th2 profiles; moreover, Th1 stimuli cause classical (M1) macrophages activation versus an alternative (M2) one. My hypothesis was that miR‐155 contributes to the pro‐Th1 profile by down regulating pro‐Th2 factors. MiR‐155 was found up regulated during DC maturation and both PU.1 and IL13R1 were demonstrated as direct targets of miR‐155. Employing a developed monocytic cell line which harbors a miR‐155 transgene under the control of a Tet‐On system (THP1‐155 cells), both PU.1 and IL13R1 were shown to be down regulated following miR‐155 induction in these cells. Moreover, THP1‐155 cells showed that DC‐SIGN transcription is regulated by miR‐155 levels through PU.1 targeting, and that IL‐13 signalling cascade through STAT6 transcription factor was down regulated when miR‐155 was over expressed. Using Anti miR‐155 transfections in DCs, miR‐155 was shown to modulate not only DC‐SIGN expression, but also DC pathogen binding ability. Using the same technique in macrophages, miR‐155 was shown to modulate IL13R1 and STAT6 activation, and to regulate the expression of IL‐13/STAT6 dependent genes. Therefore, miR‐155 contributes to the pro‐Th1 profile by down regulating pro‐Th2 factors, acting as a pro‐Th1/anti‐Th2 modulator in myeloid cells under inflammatory conditions
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Samols, Mark Atienza. "Identification and Functional Analysis of Micro-RNAs Encoded by Kaposi’s Sarcoma-Associated Herpesvirus." Case Western Reserve University School of Graduate Studies / OhioLINK, 2007. http://rave.ohiolink.edu/etdc/view?acc_num=case1181143062.
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CONTU, RICCARDO. "MIRNA-1 IN THE HEART: ITS TARGETS AND ROLE IN CARDIAC HYPERTROPHY AND DIABETIC CARDIOMYOPATHY." Doctoral thesis, Università degli Studi di Milano, 2012. http://hdl.handle.net/2434/168776.
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MicroRNAs (or miRNAs) are small endogenous single-stranded RNA molecules that inhibit the expression of specific mRNA targets through base pairing to their 3' untranslated region (UTR). As the modulatory function of a miRNA is ultimately defined by the genes it targets, identification of these genes is crucial. MicroRNA-1 (miRNA-1) was the first miRNA to be studied in cardiac biology; it is expressed specifically in striated muscle and its sequence is highly conserved across species ranging from fruit flies to humans. MiRNA-1 has been implicated in cardiac hypertrophy, heart development, cardiac stem cell differentiation, and arrhythmias through targeting of several regulatory proteins. In an effort to demystify the biological implications of miR-1 downregulation occurring during hypertrophic responses, bioinformatic softwares were explored for potential miR-1-targeted genes associated with this cardiac phenomenon. Insulin growth factor (IGF)-1 and its receptor, IGF-1R, were identified as potential targets; these are pivotal members of the PI3K/Akt signaling pathway known to play a critical role in many aspects of cardiac development and function, as well as skeletal muscle cell differentiation and proliferation. Moreover, the IGF-1 axis has been implicated in pathological conditions, such as cardiac hypertrophy and diabetic cardiomyopathy, a unique form of cardiac disease. Main findings of this study show that in conditions in which miR-1 is decreased, such as cardiac hypertrophy, IGF-1 is increased. This regulation appears reciprocal since IGF-1 stimulation leads to downregulation of miR-1 levels; thus, by translationally repressing its upstream modulator IGF-1, miR-1 intervenes in regulation of its own expression. This observation is clinically relevant since, in cardiac biopsies from patients affected by acromegaly, miR-1 levels inversely correlate with echocardiographic parameters of cardiac mass. Futhermore, miR-1 and the cotranscribed miR-133a were analyzed in different mouse models of type 1 and type 2 diabetes, where cardiac IGF-1 and Akt signaling cascade have been found dysregulated. Despite previuos findings, both muscle-specific miRNAs were not, or only minimally, affected by the establishment of diabetic cardiomyopathy. In conclusion, the findings show miR-1 as modulator of IGF-1 and IGF-1 receptor expression, confirming this miRNA as an essential regulator in development of cardiac hypertrophy, albeit with some controversy related to its role in the establishement of diabetic cardiomyopathy.
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Savary, Grégoire. "Rôles et mécanismes d’action des microARN dans la fibrogenèse : applications thérapeutiques et diagnostiques dans les fibroses pulmonaires et rénales." Thesis, Université Côte d'Azur (ComUE), 2016. http://www.theses.fr/2016AZUR4151.
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Les maladies fibroprolifératives se caractérisent par l’accumulation de constituants de la matrice extracellulaire en réponse à une agression chronique et répétée conduisant à la destruction de l’architecture tissulaire. Les myofibroblaste, sous l’influence du TGFβ, représentent les cellules effectrices majoritaires dans ce processus. Les miARN, régulateurs négatifs de l’expression génique, interviennent dans de nombreux mécanismes physio-pathologiques dont la fibrose tissulaire mais leur mécanismes d’action et la synergie potentielle entre miARN co-régulés au sein d’un même cluster restent mal compris. Nos travaux ont consisté à caractériser l’implication du cluster miR-199/214 généré à partir du LncARN DNM3os, dans la fibrose pulmonaire. Nous avons montré que les miARN de ce cluster participent à l’activation et à la différenciation des fibroblastes en myofibroblastes via la régulation des voies canoniques et non canoniques du TGF-β. Ce cluster agit également en tant qu’inhibiteur de la réparation épithéliale. In vivo, l’inhibition de l’un de ces « fibromiRs », a permis, dans un modèle murin, de réduire significativement les lésions de fibrose. Par ailleurs, de par leur présence et leur stabilité dans les fluides biologiques, les miARN représentent également une nouvelle classe de biomarqueurs diagnostiques ou pronostiques non invasifs. Nous avons ainsi montré que les niveaux sériques de miR-21-5p étaient augmentés chez les patients présentant une fibrose rénale sévère. Ces travaux soulignent l’importance des miARN dans la pathogénèse des maladies fibroprolifératives et montrent qu’ils représentent de nouvelles cibles thérapeutiques ou des biomarqueurs non invasifsFibrotic diseases are characterized by the accumulation of extracellular matrix components in response to chronic aggression leading to the destruction of tissue architecture. Myofibroblasts, controlled by TGFβ, are central effectors in this process. MiRNAs, negative regulator of gene expression, are involved in many patho-physiological mechanisms, including tissue fibrosis but their mechanisms of action and the potential synergistic activity between co-regulated miRNAs within the same cluster remain poorly understood. Our work consisted in characterizing the involvement of the miR-199/214 cluster, generated from LncRNA DNM3os, in pulmonary fibrosis. We have shown that these miRNAs are involved in the activation and the differentiation of fibroblasts to myofibroblasts through the regulation of canonical and non-canonical TGF-β pathways. This cluster also acts as an inhibitor of epithelial repair. In vivo, the inhibition of one of this “FibromiR”, significantly decrease fibrosis, in a murine lung fibrosis model. In addition, because of their presence and their stability in biological fluids, miRNAs also represent a new class of non-invasive diagnostic or prognostic biomarker. We showed that serum levels of miR-21-5p were increased in patients with severe kidney fibrosis. These studies highlight the importance of miRNAs in pathogenesis of fibrotic disorders and show that they represent new therapeutic targets or non-invasive biomarkers
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Alvarez-Saavedra, Matias Alberto. "MicroRNA-132-Dependent Post-Transcriptional Regulation of Clock Entrainment Physiology Via Modulation of Chromatin Remodeling and Translational Control Gene Targets." Thesis, University of Ottawa (Canada), 2010. http://hdl.handle.net/10393/28722.
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Mammalian circadian rhythms of behaviour are synchronized to external time by daily resetting of the master pacemaker, the suprachiasmatic nucleus (SCN), in response to light, in a process known as light-induced clock entrainment. microRNA-132 (miR-132) is induced by light within the SCN via a MAPK-CREB-dependent mechanism and has the capacity to attenuate the entraining effects of light. However, the identity of the genes that miR-132 regulates and their roles in the light-entrainment process have not yet been characterized. This thesis describes that 2 gene clusters involved in chromatin remodeling (i.e. Mecp2, Ep300, Jarid1a) and translational control (i.e. Btg2, Paip2a) are under the regulation of miR-132 in the SCN and coordinated regulation of these genes underlies miR132-dependent modulation of mPeriod1 (mPer1) and mPeriod2 (mPer2) and the light-entrainment process. I find that the Period genes are bound and transcriptionally modulated by MeCP2. In addition, Paip2a acts as a repressor of Period translation. This work further proposes that miR-132 is enriched for chromatin and translation-associated target genes-and, thus, miR-132 is an important orchestrator of chromatin remodeling and protein translation within the SCN clock, thereby fine-tuning clock entrainment.
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Branscheid, Anja. "Phosphate homeostasis and posttranscriptional gene regulation during arbuscular mycorrhizal symbiosis in Medicago truncatula." Phd thesis, Universität Potsdam, 2012. http://opus.kobv.de/ubp/volltexte/2012/6210/.
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Since available phosphate (Pi) resources in soil are limited, symbiotic interactions between plant roots and arbuscular mycorrhizal (AM) fungi are a widespread strategy to improve plant phosphate nutrition. The repression of AM symbiosis by a high plant Pi-status indicates a link between Pi homeostasis signalling and AM symbiosis development. This assumption is supported by the systemic induction of several microRNA399 (miR399) primary transcripts in shoots and a simultaneous accumulation of mature miR399 in roots of mycorrhizal plants. However, the physiological role of this miR399 expression pattern is still elusive and offers the question whether other miRNAs are also involved in AM symbiosis.
Therefore, a deep sequencing approach was applied to investigate miRNA-mediated posttranscriptional gene regulation in M. truncatula mycorrhizal roots. Degradome analysis revealed that 185 transcripts were cleaved by miRNAs, of which the majority encoded transcription factors and disease resistance genes, suggesting a tight control of transcriptional reprogramming and a downregulation of defence responses by several miRNAs in mycorrhizal roots. Interestingly, 45 of the miRNA-cleaved transcripts showed a significant differentially regulated between mycorrhizal and non-mycorrhizal roots.
In addition, key components of the Pi homeostasis signalling pathway were analyzed concerning their expression during AM symbiosis development. MtPhr1 overexpression and time course expression data suggested a strong interrelation between the components of the PHR1-miR399-PHO2 signalling pathway and AM symbiosis, predominantly during later stages of symbiosis. In situ hybridizations confirmed accumulation of mature miR399 in the phloem and in arbuscule-containing cortex cells of mycorrhizal roots. Moreover, a novel target of the miR399 family, named as MtPt8, was identified by the above mentioned degradome analysis. MtPt8 encodes a Pi-transporter exclusively transcribed in mycorrhizal roots and its promoter activity was restricted to arbuscule-containing cells. At a low Pi-status, MtPt8 transcript abundance inversely correlated with a mature miR399 expression pattern. Increased MtPt8 transcript levels were accompanied by elevated symbiotic Pi-uptake efficiency, indicating its impact on balancing plant and fungal Pi-acquisition.
In conclusion, this study provides evidence for a direct link of the regulatory mechanisms of plant Pi-homeostasis and AM symbiosis at a cell-specific level. The results of this study, especially the interaction of miR399 and MtPt8 provide a fundamental step for future studies of plant-microbe-interactions with regard to agricultural and ecological aspects.Phosphat ist ein essentieller Bestandteil der pflanzlichen Ernährung und ein Mangel führt zu schwerwiegenden Folgen für Wachstum, Entwicklung und Reproduktion der Pflanze. Eine der wichtigsten Strategien, um einen Mangel an löslichem Phosphat im Boden auszugleichen, ist die arbuskuläre Mykorrhiza, einer Wurzelsymbiose zwischen Pflanzen und im Boden lebenden Mykorrhizapilzen. Die Symbiose dient dem gegenseitigen Nährstoffaustausch, der über bäumchenartige Strukturen in Wurzelzellen, den Arbuskeln, realisiert wird. Über ein weit reichendes Netzwerk im Boden verbessert der Pilz die Phosphatversorgung der Pflanzen, wohingegen die Pflanze photosynthetisch erzeugte Zucker zur Verfügung stellt. Ein erhöhter Phosphatgehalt in der Pflanze führt zur Unterdrückung der Symbiose. Da weitestgehend unbekannt ist, wie genau Pflanzen diese Einschränkung der Symbiose regulieren, kann die Erforschung dieses Zusammenhangs einen wichtigen Beitrag für Agrarwirtschaft und Umweltschutz leisten. Im Rahmen dieser Arbeit konnte durch die Entdeckung eines neuen, bisher unbekannten Zielgens aufgezeigt werden, dass die für den Ausgleich des pflanzlichen Phosphathaushalts wichtige Mikro-RNA (miR) 399 auch in der Regulation der arbuskulären Mykorrhizasymbiose von besonderer Bedeutung ist. MiRNAs regulieren die Aktivität von Zielgenen indem sie die jeweiligen Transkripte durch Bindung für den Abbau markieren. In kolonisierten Wurzeln, insbesondere in arbuskelhaltigen Wurzelzellen, konnte eine erhöhte Anhäufung der miR399 beobachtet werden. Durch das Verfahren der Hochdurchsatz-Sequenzierung des Wurzeldegradoms, bei dem alle abgebauten Transkripte analysiert werden, konnte das neue Zielgen der miR399 Familie, MtPT8, identifiziert werden. Dieses codiert für einen Phosphat-Transporter, der diesen Studien zufolge ausschließlich in mykorrhizierten Wurzeln vorkommt und dessen Transkription auf arbuskelhaltige Zellen beschränkt ist. Mit der Identifizierung dieses neuen Zielgens konnte erstmals der Beweis für die direkte Verbindung der pflanzlichen Phosphathomöostase durch miR399 und der arbuskulären Mykorrhizasymbiose gezeigt werden. Die Untersuchung der physiologischen Funktion dieses mykorrhizaspezifischen Phosphat-Transporters bietet die Möglichkeit, die Zusammenhänge der phosphatabhängigen Regulation der Symbiose aufzuklären und weit reichende Einblicke in die Regulationsmechanismen während der Pflanze-Pilz-Interaktion zu erhalten.
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El, Hajj Petra. "New prognosis markers and new targets for therapy in high risk melanoma: evaluation of TYRP1 as a melanoma prognostic marker and its regulation by miRNA(s)." Doctoral thesis, Universite Libre de Bruxelles, 2015. http://hdl.handle.net/2013/ULB-DIPOT:oai:dipot.ulb.ac.be:2013/209064.
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L’espérance de vie des patients atteints de mélanome à haut risque ne peut être prédite d’une façonfiable 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
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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
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Webster, Rebecca. "Complementary investigations of the molecular biology of cancer : assessment of the role of Grb7 in the proliferation and migration of breast cancer cells; and prediction and validation of microRNA targets involved in cancer." University of Western Australia. School of Medicine and Pharmacology, 2008. http://theses.library.uwa.edu.au/adt-WU2008.0179.
Full textAbstract:
[Truncated abstract] For this thesis, the molecular biology of cancer was approached from two directions. Firstly, an investigation was conducted on the role of growth factor receptor-bound protein 7 (Grb7) in breast cancer. Grb7 is an adapter molecule that binds to a variety of proteins, including the growth factor receptor and proto-oncogene, ErbB2, and mediates signalling to downstream pathways. It has been linked to cell migration and an invasive phenotype, and is of interest as a therapeutic target. To investigate the role of Grb7 in breast cancer, preliminary experiments were performed that, firstly, determined the expression of wild-type Grb7 and a splice variant, Grb7V, in a range of cell lines, and secondly, aided the development of a protocol for treating cells with short interfering RNA (siRNA) against Grb7 and the ErbB ligand, heregulin (HRG), in a cell system appropriate for measuring the functional outcomes. Using this protocol in conjunction with CellTitre (CT) proliferation assays, it was demonstrated that Grb7 does not play a role in the proliferation of either unstimulated or HRG-stimulated SK-BR-3 breast cancer cells. Furthermore, using the protocol in conjunction with Boyden chamber migration assays, it was shown that inhibition of Grb7 expression has a slight stimulatory effect on HRG-stimulated SK-BR-3 cell migration. Thus, Grb7 was found to play only a minor role in the migration of SK-BR-3 cells, suggesting that it is not an ideal anti-cancer target for breast cancers modelled by this cell system. Concurrently, a second investigation was conducted, which similarly sought insight into the molecular biology of cancer, but adopted a more strategic approach. ... These results provide evidence for a biologically significant role for the miR-7-mediated regulation of EGFR expression. A microarray experiment was also performed to identify genes that were down-regulated following treatment with miR-7 compared to NS precursor. Of 248 down-regulated genes, including EGFR, 37 promising new miR-7 target candidates were identified. Functional clustering of down-regulated genes and promising target candidates suggested that miR-7 may have functionally-related targets involved in processes including cell motility and brain-associated functions. This investigation thus yielded a program capable of accurately predicting a miRNA target not predicted by any other target prediction program, verified a previously unknown miRNA:target interaction with functional consequences in cancer cells and provided the first steps towards investigating miR-7-mediated regulation in greater depth. Furthermore, EGFR was, to our knowledge, the first example of a verified miRNA target with target sites that are not conserved across mammals, an observation with important implications for computational target prediction and the evolution of miRNA regulatory systems. In addition, the demonstrated growth inhibitory and cytotoxic effects of miR-7 on lung cancer cells raise the possibility of a miR-7-based therapeutic for the treatment of EGFR-overexpressing tumours.
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Gao, Cen. "Research in target specificity based on microRNA-target interaction data." Case Western Reserve University School of Graduate Studies / OhioLINK, 2010. http://rave.ohiolink.edu/etdc/view?acc_num=case1275685130.
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Abdelhadi, Ep Souki Ouala. "MicroRNA target prediction based upon metastable RNA conformations." Thesis, King's College London (University of London), 2017. https://kclpure.kcl.ac.uk/portal/en/theses/microrna-target-prediction-based-upon-metastable-rna-conformations(9d426d58-d529-4fe7-a98a-1fa2ecdc7a2e).html.
Full textAbstract:
MicroRNAs (miRNAs) play an important role in biomarker research. Identifying their targets and inferring their functions have been of a great importance to developing our understanding of many biological processes and fundamental novel anti-cancer and viral therapies. Since the discovery and validation of true miRNA-messengerRNA (mRNA) bindings is a laborious and expensive process, computational tools for the prediction of miRNA targets are essential in this research area. Advanced tools of miRNA target prediction incorporate knowledge about secondary structures of mRNA sequences, usually the 3'UTR into the evaluation and assessment of putative miRNAmRNA bindings. The default secondary RNA structure in most target prediction tools of this type is the minimum free energy conformation or a representative of the ensemble of all possible RNA structures. A key indicator of putative miRNA-mRNA bindings is the energy required to open base pairs that are present in the potential binding site within the conformation. However, mRNAs as well as miRNAs are present in a single cell in multiple copies, where the number of copies may range from several tens up to several hundreds of copies, each of them transcribed from DNA at different points of time and therefore, potentially, being present in different folding stages, most likely in metastable conformations. In this thesis we have addressed the problem of miRNA bindings to metastable RNA secondary structures in the context of Single Nucleotide Polymorphisms (SNPs). To this end, we first searched the recent literature for disease-related triples [mRNA/3'UTR; SNP; miRNA] that have been analysed by methods including PCR and/or luciferase reporter assays. We next compared results of two major computational approaches to miRNA target ranking prediction: conservation feature using TargetScan tool and target site accessibility feature using PITA and STarMir tools. We showed that site accessibility may be a better ranking criterion. We then studied the problem of miRNA bindings to metastable secondary structures in the context of SNPs and mRNA concentration levels i.e. whether features of miRNA bindings to metastable conformations could provide additional information supporting the differences in expression levels of the two sequences defined by a SNP. We showed that among the different parameters we introduced and analyzed, we found that three of them, related to the average depth and average opening energy of metastable conformations, may provide supporting information for a stronger separation between miRNA bindings to the two alleles defined by a given SNP. These findings were a trigger to devise a novel target prediction tool that incorporates metastable secondary structures with low energy levels into predictions. We present, RNAStrucTar, a miRNA target prediction tool that analyses putative mRNA binding sites within 3'UTR secondary structures representing metastable conformations. The rst stage consists of generating conformations that can be classified as deep local minima. The second stage incorporates duplex structure prediction through sequence alignment and energy computation. Target site accessibility related to different sets of metastable conformations is also taken into account. An overall interaction score computed from multiple binding sites is returned. The approach is discussed in the context of SNPs where our manually curated [mRNA;SNP;miRNA] dataset is utilised. RNAStrucTar predictions are in favour of the allele with the stronger miRNA binding stated in the underlying literature in 22 instances, while the resulting scores are indifferent in ten cases. For the two other cases (HTR3E and FGF20), the score is in favour of the weaker allele. In this respect, RNAStrucTar results are better than PITA and STarMir, with a positive prediction for RAD51 and MSLN (STarMir favours the weaker allele).
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RUBOLINO, CARMELA. "NEW FINDINGS INTO TARGET-DIRECTED MICRORNA DEGRADATION MECHANISM." Doctoral thesis, Università degli Studi di Milano, 2022. https://hdl.handle.net/2434/945228.
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MiRNAs are a class of small non-coding RNAs that function in post-transcriptional gene silencing by interacting with target RNAs. A novel mechanism in control of miRNA levels, called Target-Directed miRNA degradation (TDMD), has been described, which involves specific transcripts able to interact with miRNAs and induce miRNA degradation. We showed that an endogenous RNA transcript, Serpine1, uses TDMD to control levels and activity of miR:30b/c in murine fibroblasts. It is unknown how many other TDMD transcripts exist; therefore, we developed TDMDfinder, a computational pipeline and free webtool, that identifies “high confidence” TDMD interactions in the Human and Mouse transcriptomes by combining sequence alignment and feature selection approaches. Our predictions suggested that TDMD is widespread, with potentially every miRNA controlled by endogenous targets. We experimentally tested 37 TDMDfinder predictions, of which 17 showed TDMD effects as measured by RT-qPCR and small RNA sequencing, linking the miR-17, miR-19, miR-30, miR-221, miR-26 and miR-23 families to novel endogenous TDMD triggers. Computational analyses performed using the multiomic TCGA platform substantiated the possible involvement of many TDMD transcripts in human cancer and highlighted 36 highly significant pan-cancer interactions, suggesting TDMD as a new potential oncogenic mechanism. Focusing on the SERPINE1:miR30b/c pair as a model, we applied molecular and genetic approaches to manipulate TDMD and investigate the effects afforded by miRNA degradation in breast cancer. Our results suggested that TDMD is used by breast cancer cells to keep low miRNA activity and provide a selective advantage in various cancer phenotypes. In order to identify all TDMD-regulated miRNAs in breast cancer, we inhibited the TDMD mechanism by knocking down a critical player of the pathway, the ZSWIM8 culling-RING ubiquitin ligase substrate adapter. We repressed ZSWIM8 by CRISPR interference in a set of 8 different breast cancer (BC) cell lines, representing the entire spectrum of BC subtypes. Our analyses showed that loss of ZSWIM8 caused significantly increased accumulation of 31 miRNAs, without any evidence of transcriptional regulation, thus expanding the role of endogenous TDMD in sculpting miRNA levels. Finally, to identify the TDMD triggers in BC, we designed a strategy based on the combined use of TDMDfinder predictions and experimental CRISPR-based tools. We performed a proof-of-concept validation of the approach by identifying the endogenous TDMD trigger (NREP) for miR-29b-3p in SUM159PT and BT549, proving that this can be an effective strategy to quickly identify TDMD substrates and triggers.
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Piovezani, Amanda Rusiska. "SIMTar: uma ferramenta para predição de SNPs interferindo em sítios alvos de microRNAs." Universidade de São Paulo, 2013. http://www.teses.usp.br/teses/disponiveis/95/95131/tde-09052014-155546/.
Full textAbstract:
Polimorfismos de um nucleotídeo (SNPs) podem alterar, além de códons de leitura da tradução de genes, posições genômicas importantes do processo de regulação gênica, como sítios de iniciação de transcrição, de splicing e, mais especificamente, sítios alvos de microRNAs (miRNAs). Em parti- cular, a identificação de SNPs alterando sítios alvos de miRNAs é um problema em aberto, embora venha ganhando um importante destaque nos últimos anos decorrente dos avanços descobertos sobre a capacidade dos miRNAs como elementos reguladores do genoma, associados inclusive a muitas doenças como o câncer e vários transtornos psiquiátricos. Os recursos computacionais atualmente disponíveis para esta finalidade (alguns bancos de dados e uma ferramenta) estão restritos à análise de SNPs na região 3UTR (UnTranslated Regions) de RNAs mensageiros, onde miRNAs geralmente se ligam para reprimir sua tradução. No entanto, essa é uma simplificação do problema, dado que já se conhece a regulação por miRNAs ativando ou reprimindo a transcrição gênica quando ligados à sua região promotora, aumentando a efetividade da regulação negativa da tradução quando ligados à região codificante do gene ou ainda miRNAs se ligando a RNAs não codificantes. Esses recursos se limitam também à identificação de SNPs na região seed dos sítios de miRNAs, e portanto só identificam criação ou ruptura de sítios. Porém, SNPs localizados fora dessa região não só podem colaborar na criação e ruptura de sítios como também interferir na estabilidade de ligação de miRNAs e, por- tanto, na efetividade da regulação. Além disso, considerando toda a extensão do sítio, não somente a seed , é possível ocorrer mais de um SNP e, sendo assim, a combinação desses SNPs pode ter uma influência ainda maior na ligação com o miRNA. Os recursos atuais também não informam quais alelos dos SNPs, muito menos quais combinações deles, estão causando qual efeito. Por fim, tais recursos estão restritos a Homo sapiens e Mus musculus. Assim, este trabalho apresenta a ferramenta computacional SIMTar (SNPs Interfering in MicroRNA Targets), desenvolvida para identificar SNPs que alteram sítios alvos de miRNAs e que preenche as lacunas mencionadas. Além disso, é descrita uma aplicação de SIMTar na análise de 114 SNPs associados à esquizofrenia, na qual todos foram preditos interferindo em sítios alvos de miRNAs.Single nucleotide polymorphisms (SNPs) can be involved in alteration of not only open reading frame but also important genomic positions of gene regulation process such as transcription initiation sites, splicing sites and microRNA target sites. In particular, the identification of SNPs interfering on microRNA target sites is still an open problem, despite its increasing prominence in recent years due to the discoveries about the microRNA abilities as regulatory elements in the genome and association with severals diseases such as cancer and psychiatric disorders. The computational resources currently available for this purpose (four databases and one tool) are restricted to the analysis of SNPs in the 3UTR (UnTranslated Regions) of mRNAs, where the microRNAs typically bind in order to repress their translation. However, this is a simplification of the problem, since it is already known the gene transcription activation by microRNAs bound to its promoter region, increasing of the effectiveness of negative regulation of translation when microRNAs are bound to the coding region of the gene or binding of microRNA into non-coding RNAs. These resources are also limited to the identification of SNPs in the seed region of miRNAs, and therefore they can only identify sites creation or disruption. However, SNPs located outside this region can not only create and disrupt target sites but also interfere on the stability of miRNAs binding and therefore on the regulation effectiveness. Moreover, considering the target site length, more than one SNP can occur inside of a site and thus, the combination of these SNPs can have an even greater influence on the microRNA binding. Also, current resources do not display which alleles of SNPs or what combinations of them are causing which effect. Finally, these features are restricted to the Homo sapiens and Mus musculus species. This work presents the computational tool SIMTar (SNPs Interfering on MicroRNA Targets), developed to identify SNPs that alter miRNA target sites and fills the mentioned gaps. Finally, it is described an application of SIMTar on the analysis of 114 SNPs associated with schizophrenia, all of them being predicted interfering with miRNA target sites.
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Bokobza, Cindy. "Neurodevelopmental impact of perinatal inflammation : targets for neuroprotection?" Thesis, Université de Paris (2019-....), 2019. http://www.theses.fr/2019UNIP7082.
Full textAbstract:
Le consensus général concernant les troubles neuro-développementaux, notamment les troubles du spectre autistique (TSA), est qu’ils proviennent de défauts de développement précoces dans la formation du cerveau. Ceci entraînant une modification des circuits neuronaux responsables du comportement pathologique. La prématurité est souvent liée à la survenue d'une inflammation et les nouveau-nés prématurés courent un risque dix fois plus élevé de développer des symptômes analogues aux TSA que les nourrissons nés à terme. De plus, des études cliniques ont démontré des processus neuro-inflammatoires dans différentes régions du cerveau chez les enfants autistes nés à terme : dans le cortex frontal, l'hippocampe et le cervelet. Le principal relais de la réponse environnementale dans le cerveau, y compris les réactions inflammatoires, sont les cellules microgliales, macrophages résidents du cerveau qui surveillent en permanence leur environnement. De plus, au cours du développement, la microglie joue un rôle crucial lors de l’élagage synaptique et de la myélinisation menant à la formation d'un cerveau mature. Dans un contexte inflammatoire, la microglie est activée et participe à la libération locale de cytokines pro-inflammatoires. Notre hypothèse est donc qu’une exposition à l'inflammation périnatale aurait un impact sur les anomalies du développement neurologique conduisant à un TSA. En utilisant un modèle murin d’inflammation périnatale induite par une injection d’IL-1? entre le jour postnatal (P)1-5, ce projet démontre (i) qu’il existe une inflammation région-spécifique et du lien entre l’inflammation périnatale et l’apparition de phénotypes de type autistiques à différents stades du développement ; et (ii) nous avons réussi à identifier deux nouvelles cibles potentielles pour limiter les anomalies du développement neurologique : les microARNs et le récepteur 5HT-7. Ce projet innovant a pour objectif d'identifier des marqueurs de diagnostic potentiels facilitant la détection précoce des TSA chez les prématurés sur la base d'indicateurs inflammatoires et de nouvelles cibles thérapeutiques pour la neuroprotectionA general consensus regarding neurodevelopmental disorders including Autism Spectrum Disorder (ASD) is that they originate from early development defects in brain formation, leading to altered neuronal circuitry responsible for the pathological behavior. Preterm birth is often linked to the occurrence of inflammation and preterm infants have a ten times higher risk of developing ADS-like symptoms than infants born at term. Moreover, some clinical studies reported ongoing neuroinflammation processes in different brain regions in autistic infants including frontal cortex, hippocampus and cerebellum. The major relay of the environmental response in the brain, including inflammatory responses, is microglia cells, the brain resident macrophages that continuously survey their local environment. Moreover, during development microglia play a critical role during the synaptic pruning and myelination to contribute to the formation of a mature brain. In an inflammatory context, MG are activated and participated to the local release of pro-inflammatory cytokines. Our hypothesis is, therefore, that an exposition to perinatal inflammation impacts on neurodevelopmental defects leading to ASD. Using a mouse model of perinatal inflammation induced by IL-1? injection between post-natal day (P)1-5, this project demonstrates that i) there is region specific inflammation and an impact of perinatal inflammation on the onset of ASD-like phenotypes at different developmental stages in mice and (ii) we successfully identify two new potential targets to limit neurodevelopmental defects: microRNAs and the 5HT-7 receptors. This innovative project has as objective to identify potential diagnosis markers to facilitate an early detection of ASD in premature infants based on inflammatory indicators and new therapeutic targets for neuroprotection
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Hanina, Sophie Alexandra. "Identifying direct targets of mouse embryonic stem cell-specific microRNAs." Thesis, University of Cambridge, 2010. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.608582.
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Agarwal, Vikram. "MicroRNAs : principles of target recognition and developmental roles." Thesis, Massachusetts Institute of Technology, 2015. http://hdl.handle.net/1721.1/101295.
Full textAbstract:
Thesis: Ph. D., Massachusetts Institute of Technology, Computational and Systems Biology Program, 2015.This electronic version was submitted by the student author. The certified thesis is available in the Institute Archives and Special Collections.
Cataloged from student-submitted PDF version of thesis.
Includes bibliographical references.
MicroRNAs (miRNAs) are ~21-24 nt non-coding RNAs that mediate the degradation and translational repression of target mRNAs. The genomes of vertebrate organisms encode hundreds of miRNAs, each of which may regulate hundreds of mRNA targets. Thus, miRNAs are crucial post-transcriptional regulators engaged in vast regulatory networks. To date, the characteristics of these networks remain mysterious due to the difficulty of identifying miRNA targets through either experimental or computational means. To understand the physiological roles of miRNAs in animal species, it is of fundamental importance to elucidate the structure of the targeting networks in which they participate. The recognition of a miRNA target is guided largely by perfect Watson-Crick base pairing interactions between nucleotides 2-7 from the 5' end of the miRNA (i.e., the "seed" region) and complementary motifs embedded in the 3' UTRs of the target mRNAs. The prevalence of these motifs throughout the transcriptome poses a challenge to our understanding of how specificity emerges: since the presence of a motif is not sufficient to mediate target repression, what contextual features discriminate effective target sites from ineffective ones? Further complicating this is the proposition that "noncanonical" sites lacking perfect seed pairing might mediate repression, which would expand the potential number of functional target sites by orders of magnitude. In the second chapter of this work, we define the features that predict effective miRNA target sites, incorporating their relative influence into a quantitative model which can outperform existing computational models and experimental approaches in target identification. Though the molecular roles of miRNAs in gene regulation have long been appreciated, the functions of most miRNAs in living organisms has remained elusive. In the third chapter of this work, we discuss the consequences of genetic ablation of miR-196, a deeply conserved miRNA that is predicted to simultaneously repress many HOX genes, in the mouse. We propose a role for miR-196 in the spatial patterning of the vertebrate axial skeleton. Isolating the cell populations that express the miRNA during early mammalian development, we attempt to characterize the direct in vivo targets of miR-196 and dissect the molecular underpinnings of the phenotypes observed.
by Vikram Agarwal.
Ph. D.
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Ebert, Margaret S. (Margaret Sarah). "Molecular titration by MicroRNAs and target mimic inhibitors." Thesis, Massachusetts Institute of Technology, 2010. http://hdl.handle.net/1721.1/61886.
Full textAbstract:
Thesis (Ph. D.)--Massachusetts Institute of Technology, Dept. of Biology, 2010.Vita. Cataloged from PDF version of thesis.
Includes bibliographical references.
MicroRNAs (miRNAs) are short, highly conserved non-coding RNA molecules that repress gene expression in a sequence-dependent manner. Each miRNA is predicted to target hundreds of genes, and a majority of protein-coding genes are computationally predicted to be miRNA targets. To test miRNA functions experimentally, we introduced the miRNA "sponge" method, which uses miRNA target mimics to sequester mature miRNAs and thereby create continuous miRNA loss of function in cell lines and transgenic organisms. Sponge RNAs contain complementary binding sites to a miRNA of interest, and are produced from transgenes within cells. As with most miRNA target genes, a sponge's binding sites are specific to the miRNA seed region, which allows them to block a whole family of related miRNAs. This transgenic approach has proven to be a powerful tool to generate miRNA phenotypes in a variety of experimental systems. Bulk measurements on populations of cells have indicated that, although pervasive, repression due to miRNAs is on average quite modest. To assay repression in single cells, we performed quantitative fluorescence microscopy and flow cytometry to monitor a target gene's protein expression in the presence and absence of regulation by miRNA. We found that repression among individual cells varies dramatically. miRNAs establish a threshold level of target mRNA below which protein production is highly repressed and above which expression responds ultra sensitively to target mRNA input until reaching high enough mRNA levels to almost escape repression. We constructed a mathematical model describing molecular titration of target mRNAs by miRNAs. The model predicted, and experiments confirmed, that the ultrasensitive regime could be shifted to higher target mRNA levels by increasing the miRNA concentration or the number of miRNA binding sites in the 3' untranslated region (UTR) of the target mRNA. Thus even a single species of miRNA can act both as a switch to effectively silence gene expression and as a fine-tuner of gene expression. This fits the emerging paradigm in which miRNAs help to confer robustness to biological processes by reinforcing transcriptional programs, attenuating leaky transcripts, and perhaps buffering random fluctuations in transcript copy number.
by Margaret S. Ebert.
Ph.D.
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Liu, Wai-man Raymond, and 廖偉文. "Identification of microRNA-184 target genes in squamous cell carcinomaof tongue." Thesis, The University of Hong Kong (Pokfulam, Hong Kong), 2010. http://hub.hku.hk/bib/B45208207.
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Viaut, Camille. "Investigating microRNA-target interactions during skeletal muscle development in chicken embryos." Thesis, University of East Anglia, 2017. https://ueaeprints.uea.ac.uk/63691/.
Full textAbstract:
MicroRNAs (miRNAs), short non-coding RNAs, which act post-transcriptionally to regulate gene expression, are of widespread significance and have been implicated in many biological processes during development and disease, including muscle disease. In addition to the myomiRs, which are miRNAs highly enriched in striated muscles, recent advances in sequencing technology and bioinformatics led to the identification of a large number of miRNAs in vertebrates and other species. However, for many of these miRNAs specific roles, in particular during myogenesis, have not yet been determined. Here, I investigated the potential functions of miR-128, confirmed an interaction with one of its candidate targets, Eya4, and looked at the impact of its knock-down on skeletal myogenesis in the chicken embryo. The expression pattern of miR-128, as well as 22 other somitic miRNAs, were characterised by LNA in situ hybridisation (LNA ISH). Eya4 was identified as a candidate ‘muscle’ target of miR-128 by computational analysis. Its expression pattern was characterised; miR-128 and Gga-Eya4 displayed similar profiles in developing somites. Using the miRanda algorithm potential miRNA binding sites were identified in the 3’ untranslated region (UTR) of other transcription factors, which along with Eya4 are members of the PAX-SIX-EYA-DACH (PSED) network (Six1/4, Eya1/2/3, and Dach1). These miRNA/target interactions were examined in vitro and in vivo. Gga-Eya4 was confirmed as a target of miR-128 as well as miR-206 by luciferase reporter assays. MiR-128/Gga-Eya4 interaction was validated by RNA ISH and RT-qPCR after antagomiR (AM)-128 injection in chicken embryos. Knock-down of miR-128 resulted in a significant de-repression of Gga-Eya4 expression; an increase in Gga-Six4 and Gga-Pax3 expression was also observed, whereas Gga-MyoD1 expression was decreased. With this project, using a combination of cell-based experiments and animal studies, I showed that miR-128 could play an important role in the regulation of skeletal myogenesis in the chicken embryo by targeting Gga-Eya4, a member of the PSED network.
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Zahra, Latib. "Identification of novel microRNAs as potential biomarkers for the early diagnosis of ovarian cancer using an in-silico approach." University of the Western Cape, 2019. http://hdl.handle.net/11394/7415.
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Philosophiae Doctor - PhDOvarian 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.
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Park, Jong Kook. "Target Identification, Therapeutic Application and Maturation Mechanism of microRNAs." The Ohio State University, 2012. http://rave.ohiolink.edu/etdc/view?acc_num=osu1331096696.
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Schulz, Nikola. "microRNA profiling and target identification in a mouse model for allergic asthma." Diss., lmu, 2012. http://nbn-resolving.de/urn:nbn:de:bvb:19-143012.
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Sualp, Merter. "Machine Learning Methods For Using Network Based Information In Microrna Target Prediction." Phd thesis, METU, 2013. http://etd.lib.metu.edu.tr/upload/12615645/index.pdf.
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Computational microRNA (miRNA) target identification in animal genomes is a challenging problem due to the imperfect pairing of the miRNA with the target site. Techniques based on sequence alone are prone to produce many false positive interactions. Therefore, integrative techniques have been developed to utilize additional genomic, structural features, and evolu- tionary conservation information for reducing the high false positive rate. We propose that the context of a putative miRNA target in a protein-protein interaction (PPI) network can be used as an additional filter in a computational miRNA target pr ediction algorithm. We compute several graph theoretic measures on human PPI network as indicators of network context. We assess the performance of individual and combined contextual measures in increasing the precision of a popular miRNA target prediction tool, TargetScan, using low throughput and high throughput datasets of experimentally verified human miRNA targets. We used clas- sification algorithms for that assessment. Since there exists only miRNA targets as training samples, this problem becomes a One Class Classification (OCC) problem. We devised a novel OCC method, DiVo, based on simple distance metrics and voting. Comparative analysis with the state of the art methods show that, DiVo attains better classification performance. Our eventual results indicate that topological properties of target gene products in PPI networks are valuable sources of information for filtering out false positive miRNA target genes. We show that, for targets of a number of miRNAs, netwo rk context correlates better with being a target compared to a sequence based score provided by the prediction tool.
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Samenuk, Thomas. "Incorporation of Organ-Specific MicroRNA Target Sequences to Improve Gene Therapy Specificity:." Thesis, Boston College, 2021. http://hdl.handle.net/2345/bc-ir:109174.
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Thesis advisor: Vassilios BezzeridesThe aim of this study was to utilize a massively parallel reporter assay (MPRA) to identify organ-specific microRNA (miRNA) target sequences to refine the timing and expression of transgene expression for gene therapy. We previously had developed a cardiac gene therapy for Catecholaminergic Polymorphic Ventricular Tachycardia (CPVT) using a systemically delivered adeno-associated virus (AAV9) vector. We hypothesized that incorporation of organ specific miRNA target sites into our vector construct could improve our therapy’s tissue specificity due to the ability of miRNAs to silence transgene expression. Initially, we attempted to incorporate mir-124 target sequences into our vector to detarget the brain. Although these initial attempts were unsuccessful, the study allowed us to develop a protocol to test the effectiveness of miRNA target sequences. Thereafter, we developed a method to screen thousands of putative miRNA target sequences simultaneously. In this study, target sequences of miRNAs specific to the heart, brain and liver were incorporated into a plasmid library. This plasmid library was subsequently made into AAV and injected into mice from a CPVT transgenic line. Total DNA and RNA was later extracted from the target organs, converted into genomic DNA (gDNA) and complementary DNA (cDNA) libraries respectively, and sent for amplicon sequencing. We analyzed the results using Comparative Microbiome Analysis 2.0 software (CoMA) and a custom python script to count the occurrence of each specified barcode per sample. In doing so, we showed that the miRNA suppression mechanism is not only effective but also organ specific. Furthermore, we developed a second script to create a combinatorial library from a set list of miRNA target sequences enabling us to efficiently test thousands of target sequence combinations at once. In doing so, we will be able to identify effective miRNA target sequence combinations to further improve gene therapy specificity
Thesis (BS) — Boston College, 2021
Submitted to: Boston College. College of Arts and Sciences
Discipline: Departmental Honors
Discipline: Biology
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Winata, Patrick. "The Development of Artificial microRNAs (amiRs) to Target Multiple Oncogenes in Malignant Pleural Mesothelioma (MPM)." Thesis, The University of Sydney, 2018. https://hdl.handle.net/2123/21883.
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Malignant pleural mesothelioma (MPM) is an aggressive tumour induced by asbestos exposure. It is associated with a poor prognosis and there are limited treatment options available. Standard first line treatment includes a combination of cisplatin and pemetrexed, which is mainly palliative with a modest 40% response rate. MicroRNAs (miRNAs) are small, non-coding RNAs that are able to post-transcriptionally regulate the expression of multiple genes via binding to their 3 prime untranslated regions (3’UTR). Our previous studies identified four genes (PLK1, NDC80, CDK1 and BCL2) with upregulated expression to be involved in MPM cell survival, proliferation and differentiation. Targeting each oncogene using siRNA led to suppression of MPM colony formation and cell growth in vitro. In mesothelioma, multiple molecular pathways are dysregulated to contribute to the malignant phenotype. Consequently, therapeutic options should focus on attenuating numerous oncogenic targets. In contrast to siRNA silencing of single targets, miRNAs are able to repress multiple genes via imperfect base-pairing with 3’UTR of mRNAs. MiRNA replacement has elicited anti-tumour effects in MPM and has recently been administered to MPM patients in the phase 1 clinical trial MesomiR-1. Adding to this strategy, miRNAs with pre-selected oncogenic targets of interest would serve as an ideal molecular tool for MPM therapy. In this project, we designed two artificial miRNAs (amiRs) to target all four oncogenes of interest simultaneously by inputting their 3’UTR sequence into the miR-Synth software algorithm. Hypothesis: Newly developed amiRs with pre-selected gene targets able to attenuate mesothelioma cells growth. Aims: The multi-targeting and anti-tumour potential of the designed amiRs was investigated by (1) validating their binding efficacy to each of four pre-selected target oncogenes, (2) quantitatively determining the repression of target genes by measuring mRNA and protein expression and (3) observing the functional effect of amiR transfection in MPM cells. Results: Dual luciferase assays following co-transfection of amiRs and 3’UTR reporter constructs demonstrated direct binding of each amiR with selected regions. Additionally, amiR transfection expectedly led to gene downregulation and reduction of cell proliferation and migration. These results collectively highlight the potential of amiR transfection as an alternative therapy for MPM.
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Chow, Hiu Tung. "Arabidopsis miR163 and its target are involved in defense against Pseudomonas syringae." HKBU Institutional Repository, 2016. https://repository.hkbu.edu.hk/etd_oa/312.
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Small RNAs are important regulators for a variety of biological processes, including leaf development, flowering-time, embryogenesis and defense responses. Most ancient miRNAs are conserved among different plant species and well characterized, while young MIRNA genes are considered to be non-conserved, highly species-specific and less well-studied. miR163 is a non-conserved miRNA and its locus has evolved recently by inverted duplication events of its target gene. Previously, we have shown that miR163 acts as a negative regulator of defense response. However, it remains unclear how miR163 and its targets are being regulated in response to pathogen attacks. Here, we further elucidated the molecular controls and the involvement of miR163 and its targets in plant defense response. Elevated level of miR163 was observed by Pst treatment in Arabidopsis thaliana, and this upregulation was found to be important in controlling the accumulation of its targets (PXMT1 and FAMT), to which they were also inducible by Pst treatment. Transcript and protein level analyses in transgenic plants overexpressing miR163-resistant form of PXMT1 or FAMT provided evidence for miR163 in fine-tuning its targets, suggesting that the stress-inducible miR163 and its targets act in concert in affecting defense genes expression. Epigenetically, histone deacetylation was found to involve in the repression of miR163 targets before and after Pst infection. Our findings revealed additional mechanistic insights to the controls and the evolutionary significance of young miRNA in mediating plant defense pathways against biotic stresses.
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Ariede, Jovita Ramos. "MicroRNoma dos carcinomas de fígado." Botucatu, 2017. http://hdl.handle.net/11449/148986.
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Orientador: Patricia Pintor dos ReisResumo: Introdução: O carcinoma hepatocelular (CHC) está entre as neoplasias de alta complexidade no diagnóstico, determinação do prognóstico e tratamento, sendo o sexto tipo de câncer mais comum no mundo e a segunda causa mais comum de morte por câncer. Uma variedade de genes alterados foram relatados, revelando heterogeneidade genética entre tumores de diferentes pacientes. Portanto, o insucesso terapêutico da terapia convencional pode ser parcialmente atribuído a essa heterogeneidade associada ao comportamento biológico tumoral. Sendo assim, justifica-se a necessidade de identificação de vias moleculares as quais podem conter biomarcadores clinicamente aplicáveis para a melhoria do diagnóstico e tratamento dos pacientes com CHC. Os resultados podem ter futuras aplicações clínicas utilizando miRNAs e os genes-alvo regulados por miRNAs como biomarcadores com valor diagnóstico, prognóstico e terapêutico. Objetivos: Identificar o perfil global de expressão de miRNAs (microRNoma) e os mRNAs-alvo potencialmente regulados por miRNAs em CHC. Pacientes e Métodos: Foram incluídas 18 amostras de tecido, fixadas em formalina e emblocadas em parafina (FFPE) de carcinoma hepatocelular, sendo 18 amostras tumorais e 18 amostras de tecido hepático histologicamente normal, adjacente ao tumor, dos mesmos pacientes. O perfil de expressão de miRNAs das amostras tumorais foi determinado utilizando o ensaio TaqMan Array Human MicroRAN Cards (TLDA) (card A) (Life Technologies). A análise dos dados util... (Resumo completo, clicar acesso eletrônico abaixo)
Doutor
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Tan, Yi. "Functional analysis of microRNA-181a : identification of target proteins and application in HCC therapy." Thesis, Imperial College London, 2014. http://hdl.handle.net/10044/1/24698.
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Hepatocellular carcinoma (HCC), or the cancer of the liver, is of great concern due to its poor patient outcome despite the various treatments available. It is imperative, therefore, that a novel, viable treatment method is developed such that patient survival rates may be improved from current statistics of less than 50%. The role of miRNAs in the regulation of gene expression and cellular development makes it an important player in cancer development process, as it is found that the aberrant expression of miRNAs is a typical feature of cancer cells or even pre-disposed cancer cells. MiR-181a has been shown to be an important miRNA involved in HCC. In this study, we investigated the potential effects of miR-181a in HepG2 cells and the mechanisms in which it works in controlling cell fate. As chemotherapy is widely used in liver cancer treatment, we also study the use of miR-181a along with chemotherapy (i.e. Cisplatin). Using iTRAQ-coupled 2D LC-MS/MS analysis, we report here the study of protein profile of HepG2 cells transfected with miR-181a and its inhibitor respectively. Three main types of cellular proteins including metabolic enzymes, protein binding and stress proteins displayed changes. The changes in the level of proteins (14-3-3σ, Hsp-90β and NPM1) involved in important cancer processes like cell growth were further supported by a Western blot analysis. MiR-181a was subsequently found to significantly increase HepG2 cell viability while inhibiting it displayed the opposite effect. Inhibiting miR-181a also sensitized HepG2 cells to cisplatin treatment and retards cell cycle progression by decreasing the proportion of cells in S and G2/M phases. We next investigated the reasons behind these observations at a molecular level. As miRNAs are known to regulate genes by binding to and targeting mRNAs, we first used bioinformatics to screen out potential cellular targets. Two important genes identified, cyclin-dependent kinase inhibitor 1B (CDKN1β) and transcriptional factor E2F7 (E2F7), which are involved in cell cycle and cell proliferation, were chosen to be further experimentally studied. In vitro validation via surface plasmon resonance (SPR) technique showed a positive binding between miR-181a and the seed regions of the 3'UTRs of the two putative mRNA targets, with dissociation constants being 272.5 ± 0.008 nM and 1.186 ± 0.009 uM for CDKN1β and E2F7 respectively. In vivo luciferase assay studies further validated the miR-181a:mRNA bindings, in both cases displaying significant decrease in luciferase activity when HepG2 cells were co-transfected with the 3'UTR-containing reporter plasmids and miR-181a. A positive binding, however, may not necessarily lead to a lowered expression of protein levels. A Western blot study on the expression levels of the two proteins, however, showed a decrease in the levels of CDKN1β and E2F7. Lastly, to gain an insight into the overall effects miR-181a has in HepG2 cells, a microarray analysis was performed. Cellular pathways important in cancer were studied and results show that miR-181a significantly activated the MAPK/JNK pathway by increasing the expression levels or activity of transcription factor activator protein 1 (AP-1). Inhibiting miR-181a, on the other hand, abolished this observation and significantly decreased expression levels or activity of hypoxia-inducible factors (HIF) and also significantly upregulated the expression levels or activity of SMAD2/3/4 proteins, possibly inducing a cancer-suppressing effect. Overall, miR-181a appears to activate mainly cancer-promoting pathways, and may act as an oncogene in HepG2 cells. Inhibiting it, on the other hand, activates mainly the tumour-suppressing pathways, making it a possible option for therapy. A separate microarray analysis on gene expression showed that one way in which miR-181a could have activated the SMAD, NFκB and MAPK pathways is via the significant increase in gene expression of bone morphogenetic protein receptor type II (BMPR2), a cellular receptor that mediates the signal transduction of these pathways. Our findings provide a new platform of identifying miRNA targets, in the process offering molecular evidence on the mechanism of action of miR-181a, including the beneficial effects of inhibiting miR-181a in HCC therapy.
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Parveen, Alisha [Verfasser], and Norbert [Akademischer Betreuer] Gretz. "Advanced hierarchical learning approach for microRNA and target prediction / Alisha Parveen ; Betreuer: Norbert Gretz." Heidelberg : Universitätsbibliothek Heidelberg, 2020. http://d-nb.info/1205807489/34.
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Fleischmann, Katrin Kristina. "Identification of MLL-A9 related target genes and microRNAs involved in leukemogenesis." Diss., Ludwig-Maximilians-Universität München, 2012. http://nbn-resolving.de/urn:nbn:de:bvb:19-161310.
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Malik, Juliane [Verfasser]. "Evaluierung der diagnostischen Rolle von microRNAs und Validierung ihrer Targets in hepatozellulärem Karzinom / Juliane Malik." Ulm : Universität Ulm, 2021. http://d-nb.info/1238690440/34.
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Rieger, Megan Elizabeth. "Transcription Cofactor LBH is a Direct Target of the Oncogenic WNT Pathway with an Important Role in Breast Cancer." Scholarly Repository, 2010. http://scholarlyrepository.miami.edu/oa_dissertations/659.
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Limb-Bud and Heart (LBH) is a novel key transcriptional regulator of vertebrate development. However, the molecular mechanisms upstream of LBH and its role in adult development are unknown. Here we show that in epithelial development, LBH expression is tightly controlled by Wnt signaling. LBH is transcriptionally induced by the canonical Wnt pathway, as evident by the presence of functional TCF/LEF binding sites in the LBH locus and rapid beta-catenin-dependent upregulation of endogenous LBH by Wnt3a. In contrast, LBH induction by Wnt/beta-catenin signaling is inhibited by Wnt7a, which in limb development signals through a non-canonical pathway involving Lmx1b. Furthermore, we show that Lbh is aberrantly overexpressed in mammary tumors of MMTV-Wnt1 transgenic mice and in aggressive basal-subtype human breast cancers that display Wnt/beta-catenin hyperactivation. Deregulation of LBH in human breast cancer appears to be Wnt/beta-catenin dependent as DKK1 and Wnt7a inhibit LBH expression in breast tumor cells. RNAi mediated knockdown of LBH in basal breast cancer cell lines resulted in loss of CD44high/CD24low tumor cells, luminal differentiation, reduced cell growth, reduced colony forming ability, and increased apoptosis, suggesting a novel pro-survival and stem cell maintenance function of LBH in breast cancer. Reciprocal overexpression studies in the basal breast carcinoma line BT549 resulted in increased tumorigenicity in vitro, suggesting that LBH overexpression is indeed oncogenic. Finally, we further characterized LBH protein expression patterns and post-transcriptional regulation. Collectively, this thesis demonstrates that LBH is a direct Wnt target gene in both development and basal breast cancer that promotes the undifferentiated phenotype and survival of basal breast tumor cells.
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Torossian, Avédis. "Contrôle de l'expression de Bcl-2 dans les lymphomes anaplasiques à grandes cellules par la protéine HuR en réponse au crizotinib : impact sur l'apoptose et l'autophagie." Thesis, Toulouse 3, 2017. http://www.theses.fr/2017TOU30190/document.
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Les lymphomes anaplasiques à grandes cellules (LAGC) sont des lymphomes non-hodgkiniens dits de type T ou nul, représentant la majorité des lymphomes T pédiatriques (20 à 30%). Dans plus de 80% des cas, une translocation chromosomique réciproque aboutissant à l'expression anormale de protéines chimères de type X-ALK qui arborent constitutivement et de manière anormale l'activité tyrosine-kinase ALK (Anaplastic Lymphoma Kinase) est le moteur de la tumorigenèse (LAGC dits "ALK+ "). L'une des particularités de ces lymphomes, mise en évidence par mon équipe, est le fait que B-Cell Lymphoma-2 (BCL-2) demeure indétectable dans les cas ALK+ contrairement aux cas ALK-. Ce point est d'autant plus surprenant que BCL-2, oncogène largement établi comme prototype de protéines anti-apoptotiques ainsi que régulateur clé de l'autophagie, est fortement surexprimé dans la majorité des lymphomes. A l'inverse, Human Antigen R (HuR) est surexprimée dans les LAGC (comme dans la plupart des cancers). Il a été démontré que cette protéine de liaison aux ARN participait au maintien du phénotype tumoral, et que sa localisation subcellulaire et ses fonctions dépendaient étroitement de son statut de phosphorylation, lequel est régulé par ALK dans les LAGC ALK+. Au niveau du cytoplasme, HuR permet de stabiliser et d'augmenter la traduction d'ARNm possédant, dans leur région 3'-UTR, des séquences riches en adénine et uridine (AU-rich elements, "ARE"). De manière plus générale, HuR a la capacité de dialoguer avec les microARNs (miARN), soit en empêchant leur action par compétition, soit à l'inverse en coopérant avec ces derniers et en promouvant ainsi leur fonction de régulateur négatif sur certains transcrits cibles communs. Le transcrit Bcl-2, dont l'expression est réprimée dans les LAGC ALK+, fait partie des cibles potentielles de HuR. Au cours de ma thèse, j'ai ainsi cherché à comprendre les mécanismes moléculaires mis en jeu dans la répression de l'expression de Bcl-2, en me focalisant sur le rôle de HuR et de miARN "partenaires" dans ce processus. Mes données semblent indiquer que ce mécanisme implique le recrutement par HuR du miR-34a sur l'ARNm Bcl-2, conduisant à la mise en silence de ce dernier. A l'inverse, quand l'activité tyrosine- kinase de ALK est inhibée, l'interaction entre HuR et le transcrit Bcl-2 diminue, ce qui limite le recrutement de miR-34a et conduit à une restauration de l'expression de cette oncogène majeur dans les cellules lymphomateuses. Dans le contexte des essais cliniques d'inhibiteurs ciblant l'activité tyrosine kinase de ALK tels que le Crizotinib, la question de cette ré-expression de BCL-2 éclaire d'une lumière nouvelle certains échecs thérapeutiques subis par cette molécule pourtant prometteuse. Je me suis donc également consacré, pendant ma thèse, à l'étude des conséquences de cette ré-expression de BCL-2 sur les LAGC ALK+ traités par le Crizotinib. Les résultats que j'ai obtenus in vitro et in vivo montrent que contrecarrer, par interférence à l'ARN, l'élévation du taux de BCL-2 consécutive au traitement par le Crizotinib, permet de potentialiser les effets de la drogue : cela se traduit en particulier par une potentialisation de la mort par apoptose induite par le traitement mais aussi, de manière fascinante, par une conversion de la réponse autophagique initialement cytoprotectrice et pro-tumorale en une autophagie incontrôlée et délétère, qui participe alors à l'effet thérapeutique accru de la drogue. De manière globale, ce travail permet d'envisager de nouvelles combinaisons et alternatives thérapeutiques pour les patients souffrants de LAGC ALK+, et illustre la complexité des régulations croisées entre processus apoptotiques et autophagiquesAnaplastic large cell lymphoma (ALCL) are T/-null non-hodgkin lymphoma representing most of childhood T-cell lymphoma (up to 30%). More than 80% of cases bear reciprocal chromosomic translocation responsible for abnormal expression and constitutive activation of X-ALK type (Anaplastic Lymphoma Kinase) chimeric proteins (ALK+ ALCL). A striking characteristic of this lymphoma is that B-Cell Lymphoma-2 (BCL-2) remains undetectable in ALK+ cases compared to ALK- cases. This is all the more surprising as the BCL-2 oncogene, which is firmly established as a prototypic anti-apoptotic factor as well as a key autophagy regulator, has been shown to be overexpressed in a majority of lymphomas. On the other hand, the RNA-binding protein HuR (Human Antigen R) is overexpressed in ALCL (as in most cancers). It has been demonstrated that this protein was involved in the sustainability of the tumoral phenotype, and that its subcellular localization and functions were closely related to its phosphorylation status, which in turn heavily depends on ALK activity in ALK+ ALCL. In the cytoplasm, HuR has the ability to bind adenine and uridine-rich elements (ARE) located on the 3'-UTR of target mRNAs, and both protect them from degradation and increase their translation. From a general point of view, HuR is able to establish an interplay with microRNAs (miRNAs), either blocking them through competition, or actually cooperating with them and thus promote their function of negative regulators of gene expression on common target transcripts. The BCL-2 transcript, which expression seems to be silenced in ALK-expressing ALCL, has been described as a potential target of HuR. During my PhD work, I dedicated myself to understand the molecular mechanism at work in the silencing of BCL-2 expression with a focus on HuR and collaborating miRNA. The data I obtained point at a cooperation between HuR and miR-34a leading to the silencing of the BCL-2 transcript. However, when the ALK tyrosine kinase activity is inhibited, it appears the interaction between the BCL-2 mRNA diminishes, which limitates the miR-34a 's access to this transcript and ultimately results in a re-expression of the BCL-2 oncogene in these lymphoma cells. In the current context of clinical trials for ALK-targeting inhibitors, such as the Crizotinib, this BCL-2 re-expression observed upon ALK inhibition shed light on potential reasons behind some therapeutic failures that have recently been reported. Indeed, during my PhD work, I also studied the consequences of the BCL-2 re-expression observed in Crizotinib-treated cells. The data I obtained in vitro and in vivo show that, by blocking this re-expression using RNA interference, the Crizotinib anti-tumoral efficiency can be greatly potentiated. This potentiation took the form of an increase of apoptotic cell death induction and, interestingly, also affected the autophagic response triggered by the drug, making it switch from a cytoprotective- type, protumoral autophagic flux to an enhanced, deletary-type and tumor suppressive flux, adding to the therapeutic effect of the drug. This work in general provides insights for new therapeutic combinations that could potentially benefit to ALK+ ALCL patients, and illustrates the complex cross-regulations between apoptotic and autophagic pathway
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Limbu, Sarita. "Identifying Differentially Expressed Human Lung MicroRNAs and Their Molecular Functions." University of Akron / OhioLINK, 2009. http://rave.ohiolink.edu/etdc/view?acc_num=akron1259543051.
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Arif, Km Taufiqul. "Functional association of Micrornas with molecular subtypes of breast cancer." Thesis, Queensland University of Technology, 2021. https://eprints.qut.edu.au/213110/1/Km%20Taufiqul_Arif_Thesis.pdf.
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This research study investigated the association of microRNA related single nucleotide polymorphisms (miRSNPs) with breast cancer susceptibility in Australian Caucasian women. The thesis then progressed with developing an in silico methodology for miRNA-target identification followed by the validation of miRNA-target relationships regarding the distinctive molecular subtypes of human breast cancers.
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"Identification of microRNA targets." NEW YORK UNIVERSITY, 2010. http://pqdtopen.proquest.com/#viewpdf?dispub=3365704.
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Lin, Li_Zen, and 林立人. "Computational prediction of host microRNA targets against virus genome and identification of virus microRNAs." Thesis, 2006. http://ndltd.ncl.edu.tw/handle/73394184058148358652.
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碩士國立交通大學
生物資訊研究所
94
Recent work has indicated that microRNAs (miRNAs) play important roles on development, oncogenesis and apoptosis by binding to mRNAs to regulate the post-transcriptional level of gene expression in mammals, plants and insects. Notably, miRNAs also can be produced from virus, and the expression of virus genes are potentially regulated by host miRNAs after infecting host. In this thesis, two databases, VirMiR and ViTa, are developed to compile the virus miRNAs and the regulatory relationships between host miRNAs and virus genes, respectively. Known virus microRNAs in four species, such as humans, mouse, rat and chicken, were obtained from miRBase and miRNAMap. Virus information was collected from NCBI, ICTVdB, VBRC and VirGen. Experimentally validated host miRNA targets on viruses are identified via a literature survey. Then, miRanda and TargetScan are applied to identify the miRNA targets within virus genomes. The virus miRNAs were detected based on comparative sequence analysis. Finally, virus annotations, virus infected tissues from literatures and tissue-specificity of miRNAs from MIT are integrated, and the possible relationship between miRNAs and viruses are identified. This work also provides comparisons between subtypes in some common viruses, such as influenza viruses, liver viruses, and conserved regions between different strains of the same virus species for users. Both textual and graphical web interfaces are provided to facilitate data retrieval from the two databases. The two databases can be available on the Internet at http://vita.mbc.nctu.edu.tw/ and http://virmir.mbc.nctu.edu.tw/.
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"The Functional Evolution of Human microRNA Families." Doctoral diss., 2016. http://hdl.handle.net/2286/R.I.41220.
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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
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Chou, Yi-Hsuan, and 周宜璇. "Genome-Wide RNAi Screen to Identify Genetic Interactions with microRNA-26a, and Its Potential Targets." Thesis, 2011. http://ndltd.ncl.edu.tw/handle/43335235472204846860.
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碩士國立臺灣大學
生物化學暨分子生物學研究所
99
In post genomic era, a major biological challenge is to observe how genes function as networks to carry out and regulate cellular processes. Genetic interaction analysis is a powerful tool for establishing functional linkages between genes. Hepatocellular carcinoma (HCC) is the third-leading cause of death from cancer and the fifth most common malignancy worldwide. Despite great advances in the diagnosis and treatment of this cancer, relapse and metastasis is largely unavoidable and the 5-year survival rate remains unsatisfactory. MicroRNAs (miRNAs) are a class of highly conserved small RNA molecules that function as critical regulators of gene expression. Mature miRNAs bind to the target mRNAs, resulting in mRNA degradation or translation repression dependent on the sequence complementarily. Importantly, the ability of individual miRNAs to regulate hundreds of mRNAs allows these RNAs to coordinate complex programs of gene expression. Dysregulated miRNA expression has been linked to many human diseases. Previous studies have shown that HCC cells exhibit reduced expression of miR-26a compared to the normal tissue. Activation of nuclear factor κB and interleukin-6 was observed in tumors with reduced miR-26a expression. Moreover, ectopically expressed miR-26a in mouse HCC results in inhibition of cell proliferation and restrains disease progression. These findings indicate that miR-26a may function as a tumor suppressor gene and provide a new approach for HCC treatment. Although the biochemical mechanisms of miRNA have been specified and some candidate miRNA target genes may be predicted by bioinformatics approaches, identification of physiologically relevant targets of individual miRNA remains challenging. In this research, we carried out high-throughput RNA interference screen in human hepatocellular carcinoma cells to identify genes interacting with the query miR-26a gene, which is able to reveal the upstream regulators and downstream effectors. We generated query stable HepG2 cell lines harboring overexpression constructs. We delivered miR26a expression construct into HepG2 cells using lentiviruses followed by TaqMan miRNA assay to confirm the overexpression level. Furthermore, we found that forced expression of miR-26a2 in these stable cell lines render them less capable of growing without anchorage, which is consistent with the proposed tumor suppressor phenotype of this miRNA. Finally, we performed genome-wide genetic interaction screens to identify some interesting genetic interacting partner genes of miR-26a2. These may provide us direct informations on networks, pathways and dynamics of miRNA in hepatocarcinogenesis.
49
Sims, Emily K. "Microrna 21 targets B Cell Lymphoma 2 (Bcl2) Mrna to increase beta cell apoptosis and exosomal Microrna 21 could serve as a biomarker of developing Type 1 Diabetes Mellitus." 2018. https://doi.org/10.7912/C2T366.
Full textAbstract:
Indiana University-Purdue University Indianapolis (IUPUI)The role of beta cell miR-21 in Type 1 Diabetes (T1D) pathophysiology has been controversial. Here, we sought to define the context of beta cell miR-21 upregulation in T1D and the phenotype of beta cell miR-21 overexpression through target identification. Furthermore, we sought to identify whether circulating extracellular vesicle (EV) beta cell-derived miR-21 may reflect inflammatory stress within the islet during T1D development.. Results suggest that beta cell miR-21 is increased in in-vivo models of T1D and cytokine-treated cells/islets. miR-21 overexpression decreased cell count and viability, and increased cleaved caspase-3 levels, suggesting increased cell death. In silico prediction tools identified the anti-apoptotic mRNA B Cell Lymphoma 2 (BCL2) as a conserved miR-21 target. Consistent with this, miR-21 overexpression decreased BCL2 transcript and protein expression, while miR-21 inhibition increased BCL2 protein levels and reduced cleaved caspase-3 levels following cytokine-treatment. miR-21-mediated cell death was abrogated in 828/33 cells, which constitutively overexpress BCL-2. Luciferase assays suggested a direct interaction between miR-21 and the BCL2 3’untranslated region. With miR-21 overexpression, PRP revealed a shift of BCL-2 message toward monosome-associated fractions, indicating inhibition of BCL2 translation. Finally, overexpression in dispersed human islets confirmed a reduction in BCL2 transcripts and increased cleaved caspase 3 production. Analysis of EVs from human beta cells and islets exposed to cytokines revealed a 3-5-fold increase in miR-21. Nanoparticle tracking analysis showed no changes in EV quantity in response to cytokines, implicating specific changes within EV cargo as responsible for the miR-21 increase. Circulating EVs from diabetic non-obese diabetic (NOD) mice displayed progressive increases in miR-21 that preceded diabetes onset. To validate relevance to human T1D, we assayed serum samples collected from 19 pediatric T1D subjects at the time of diagnosis and 16 healthy controls. Consistent with our NOD data, EV miR-21 was increased 5-fold in T1D samples. In conclusion, in contrast to the pro-survival role reported in other systems, our results demonstrate that miR-21 increases beta cell death via BCL2 transcript degradation and inhibition of BCL2 translation. Furthermore, we propose that EV miR-21 may be a promising marker of developing T1D.
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"Identification of molecular targets for Brucein D and metastasis suppressor genes in cancer through microRNA and RNAi screening." 2012. http://library.cuhk.edu.hk/record=b5549124.
Full textAbstract:
微小RNA是内源性小非编码RNA，在肿瘤生成中扮演重要角色。Brucein D(BD)是一种B. javanica果实提取物，已被报道在胰腺癌中具有抗肿瘤作用。在此研究中，我们证明了BD在体内和体外均可抑制肝癌细胞生长。为了研究BD是否通过调节微小RNA来执行其抗肿瘤功能，我们进行了一个肿瘤微小RNA定量PCR阵列谱分析。此阵列包括95个已被报道与肿瘤有关的微小RNA。通过对比BD处理前后微小RNA谱的变化，我们发现微小RNA-95在BD处理后被显著下调了。其后促凋亡的CUGBP2被确定为微小RNA-95的下游靶基因。胰腺癌是一种预后很差的恶性肿瘤，常常在确诊时已发生转移。为了找出在胰腺癌转移过程中发挥决定性作用的基因，我们进行了全基因组范围的RNA干扰筛选。一个包含针对全部人类基因的shRNA文库被导入胰腺癌细胞系capan-2.然后将这些细胞移植到裸鼠的胰腺中来建立一个原位胰腺癌小鼠模型。我们的假设是下调某个基因会促使低转移潜力的capan-2细胞转移到肝脏。通过从肝转移结节中回收shRNA模板，我们找到了几个推定的转移抑制基因。其中之一，SOX9，通过体内实验验证，证明下调SOX9基因的表达可促进胰腺癌转移。
化疗适用于进展期胰腺癌病人。然而他们对一线化疗药吉西他滨的反应并不乐观，这进一步使胰腺癌的预后变差。我们展开了一个全基因组范围的RNA干扰筛选来确定一些在化疗耐药过程中起关键作用的基因。携带上述shRNA文库的capan-2细胞被用于吉西他滨药物处理之下的筛选。通过微阵列分析，一些基因被筛选成为可影响癌细胞对药物敏感性的潜在的靶基因。通过进一步验证，LLGL1基因被确定为在调节癌细胞对化疗敏感性过程中起重要作用的基因。
MicroRNAs (miRNAs) are endogenous small non-coding RNAs that have been shown to play important roles in tumorigenesis. Brucein D (BD), a chemical compound isolated from Brucea javanica fruit, has previously been reported to have anti-cancer effect in pancreatic cancer. In this study, we showed that BD also inhibited the growth of liver cancer cells both in vitro and in vivo. To investigate whether BD exerts its anti-cancer effect through regulation of miRNAs, we performed a cancer miRNA qPCR array profiling. From the profiling, miR-95 was found to be significantly down-regulated after BD treatment. Subsequently, a pro-apoptotic gene CUGBP2 was identified as a direct downstream target of miR-95. These findings suggested BD suppressed liver cancer cell growth through down-regulation of miR-95 and reinforcing CUGBP2.
Pancreatic cancer is an aggressive malignancy with extremely poor prognosis. It is usually diagnosed when metastases are already present. To identify genes that play critical roles in the processes of pancreatic cancer metastasis, a whole genome RNAi screening was performed. An shRNA library targeting all human genes was introduced into a human pancreatic cancer cell line capan-2. The infected cells were then transplanted into the pancreas of nude mice. Because capan-2 is of low metastatic potential, we hypothesized that knocking down of metastasis suppressor genes would facilitate capan-2 cells to spread to the liver. By retrieving shRNA templates from the liver metastatic nodules, several candidate genes were found. One of them, SOX9, has been validated as metastasis suppressor gene in vivo, implying that loss of expression of SOX9 promotes pancreatic cancer metastasis.
Chemotherapy is recommended for patients of pancreatic cancer in advanced stage. However, their response to the first-line chemotherapy drug gemcitabine is not satisfactory. A genome-wide RNAi screening was conducted to identify genes that were critical in chemotherapy resistance. Capan-2 cells containing the above shRNA library were applied for the screening under gemcitabine treatment. Through microarray analysis, a number of genes were screened as potential gemcitabine sensitivity genes. Validation experiments implied that the gene LLGL1 may play an important role in modulating pancreatic cancer cells’ sensitivity to gemcitabine.
Detailed summary in vernacular field only.
Detailed summary in vernacular field only.
Detailed summary in vernacular field only.
Xia, Tian.
Thesis (M.Phil.)--Chinese University of Hong Kong, 2012.
Includes bibliographical references (leaves 125-134).
Abstracts also in Chinese.
Chapter Abstract --- p.I
Chapter Acknowledgements --- p.V
Chapter Abbreviations --- p.VI
Chapter List of Figures --- p.XV
Chapter List of Tables --- p.XVI
Chapter Part I: --- Brucein D-modulated microRNA-95 expression inhibits hepatocellular carcinoma cell growth --- p.1
Chapter 1 --- Introduction --- p.1
Chapter 1.1 --- Hepatocellular carcinoma --- p.1
Chapter 1.1.1 --- Definition and classification --- p.1
Chapter 1.1.2 --- Epidemiology --- p.1
Chapter 1.1.3 --- Etiology --- p.3
Chapter 1.1.4 --- Molecular pathogenesis of HCC --- p.4
Chapter 1.1.4.1 --- Genomic instability --- p.4
Chapter 1.1.4.2 --- Deregulation of key signaling pathways --- p.5
Chapter 1.1.4.3 --- Epigenetic changes of HCC --- p.6
Chapter 1.1.4.4 --- Two models of HCC pathogenesis --- p.7
Chapter 1.1.5 --- Therapeutic methods and prognosis of HCC --- p.8
Chapter 1.2 --- Apoptosis --- p.9
Chapter 1.2.1 --- Types of cell death --- p.9
Chapter 1.2.2 --- Apoptosis --- p.10
Chapter 1.2.3 --- Morphological features of apoptosis --- p.10
Chapter 1.2.4 --- Molecular mechanisms of apoptosis --- p.11
Chapter 1.2.5 --- Apoptosis and cancer --- p.13
Chapter 1.2.5.1 --- Imbalance of pro-apoptotic proteins and anti-apoptotic proteins --- p.13
Chapter 1.2.5.2 --- Impaired caspases activity --- p.14
Chapter 1.2.5.3 --- Deregulated death receptor signaling --- p.15
Chapter 1.2.6 --- Cancer therapy targeting apoptotic defects --- p.15
Chapter 1.3 --- microRNA --- p.16
Chapter 1.3.1 --- Overview --- p.16
Chapter 1.3.2 --- Biogenesis and maturation of microRNA --- p.17
Chapter 1.3.3 --- Gene silencing by microRNA --- p.18
Chapter 1.3.4 --- MicroRNA and cancers --- p.19
Chapter 1.3.5 --- MicroRNA’s involvement in HCC --- p.21
Chapter 1.3.6 --- Involvement of miR-95 in cancer --- p.22
Chapter 1.4 --- Brucein D --- p.22
Chapter 1.5 --- Aims of study --- p.23
Chapter 2 --- Materials and Methods --- p.25
Chapter 2.1 --- Cell Culture --- p.25
Chapter 2.1.1 --- Mammalian Cell Culture --- p.25
Chapter 2.1.2 --- Preparation of cell stock --- p.25
Chapter 2.1.3 --- Cell recovery from liquid nitrogen stock --- p.26
Chapter 2.1.4 --- Preparation of drugs for treatments --- p.26
Chapter 2.1.5 --- Drug treatment --- p.26
Chapter 2.1.6 --- Transfection of siRNA --- p.27
Chapter 2.1.7 --- MTT Assay --- p.28
Chapter 2.1.8 --- Luciferase reporter assays --- p.28
Chapter 2.1.9 --- Annexin V Assay --- p.29
Chapter 2.2 --- In vivo mouse model --- p.29
Chapter 2.3 --- Tunel Assay (Terminal deoxynucleotide transferase dUTP Nick End Labeling Assay) --- p.30
Chapter 2.4 --- RNA manipulation --- p.31
Chapter 2.4.1 --- RNA Isolation --- p.31
Chapter 2.4.2 --- Synthesis of cDNA from miRNA --- p.32
Chapter 2.4.3 --- Synthesis of cDNA from RNA and quantitative PCR --- p.33
Chapter 2.4.4 --- miRNA qPCR array --- p.34
Chapter 2.5 --- DNA manipulation --- p.34
Chapter 2.5.1 --- Agarose gel electrophoresis and purification of DNA --- p.34
Chapter 2.5.2 --- Restriction enzymes digestion --- p.35
Chapter 2.5.3 --- Ligation of DNA fragments --- p.36
Chapter 2.5.4 --- Polymerase chain reaction --- p.36
Chapter 2.5.5 --- Preparation of competent E. coli cells --- p.37
Chapter 2.5.6 --- Transformation of E. coli cells --- p.37
Chapter 2.5.7 --- Small scale plasmid isolation from E. coli (mini-prep) --- p.38
Chapter 3 --- Results --- p.39
Chapter 3.1 --- Brucein D inhibited the growth of HCC cells both in vitro and in vivo --- p.39
Chapter 3.2 --- BD induced apoptosis in HCC cells --- p.43
Chapter 3.3 --- miR-95 is an target of BD to modulate cell growth --- p.46
Chapter 3.4 --- Identification of CUGBP2 as a downstream target of miR-95 --- p.55
Chapter 4 --- Discussion --- p.60
Chapter Part II: --- Genome-wide RNAi screening identifies tumor metastasis suppressor genes and drug sensitivity genes in pancreatic cancer --- p.65
Chapter 1 --- Introduction --- p.65
Chapter 1.1 --- Pancreatic cancer --- p.65
Chapter 1.1.1 --- Overview --- p.65
Chapter 1.1.2 --- Pancreatic ductal adenocarcinoma (PDAC) --- p.67
Chapter 1.1.3 --- Molecular basis of PDAC --- p.67
Chapter 1.1.3.1 --- KRAS --- p.67
Chapter 1.1.3.2 --- TP53 --- p.68
Chapter 1.1.3.3 --- CDKN2A --- p.69
Chapter 1.1.4 --- Gemcitabine treatment in PDAC --- p.69
Chapter 1.2 --- Metastasis --- p.71
Chapter 1.2.1 --- Overview --- p.71
Chapter 1.2.2 --- The stepwise process of metastasis --- p.72
Chapter 1.2.3 --- Metastasis of pancreatic cancer --- p.74
Chapter 1.3 --- SOX9 --- p.75
Chapter 1.4 --- Aims of study --- p.77
Chapter 2 --- Materials and Method --- p.78
Chapter 2.1 --- Cell culture --- p.78
Chapter 2.1.1 --- Mammalian Cell Culture --- p.78
Chapter 2.1.2 --- MTT Assay --- p.78
Chapter 2.1.3 --- Colony formation assay --- p.79
Chapter 2.1.4 --- Wound healing assay --- p.79
Chapter 2.1.5 --- Transwell migration chamber assay --- p.80
Chapter 2.1.6 --- Immunocytochemistry --- p.80
Chapter 2.1.7 --- Transient transfection of siRNA --- p.81
Chapter 2.2 --- Establishment of in-vivo and in-vitro models --- p.82
Chapter 2.2.1 --- shRNA library introduction --- p.82
Chapter 2.2.2 --- Establishment of the orthotopic pancreatic cancer mouse model --- p.82
Chapter 2.2.3 --- Package of lentivirus expressing shRNA --- p.83
Chapter 2.2.4 --- Generation of stable cell line expressing shRNA --- p.84
Chapter 2.3 --- DNA manipulation --- p.84
Chapter 2.3.1 --- Large scale plasmid isolation from E. coli (maxi-prep) --- p.84
Chapter 2.4 --- Analysis of Protein --- p.85
Chapter 2.4.1 --- Preparation of protein cell lysates --- p.85
Chapter 2.4.2 --- Protein concentration determination --- p.86
Chapter 2.4.3 --- SDS-PAGE --- p.86
Chapter 2.4.4 --- Immunoblotting (Western blotting) --- p.87
Chapter 2.5 --- RNA manipulations --- p.88
Chapter 2.5.1 --- RNA Isolation --- p.88
Chapter 2.5.2 --- Synthesis of cDNA from RNA and quantitative PCR --- p.89
Chapter 2.6 --- Analysis of Clinical Samples --- p.90
Chapter 2.6.1 --- Clinical specimens --- p.90
Chapter 2.6.2 --- Immunohistochemistry --- p.90
Chapter 3 --- Results --- p.92
Chapter 3.1 --- Genome-wide RNAi screening identifies genes as metastasis suppressors in an orthotopic pancreatic cancer mouse model --- p.92
Chapter 3.2 --- SOX9 is a metastasis suppressor gene in pancreatic cancer --- p.97
Chapter 3.3 --- Investigation into cellular functions of SOX9 --- p.102
Chapter 3.3.1 --- SOX9’s effect on cell growth --- p.102
Chapter 3.3.2 --- SOX9’s effect on cell migration --- p.105
Chapter 3.4 --- Implication of SOX9 in human pancreatic cancer samples --- p.109
Chapter 3.5 --- Genome-wide RNAi screening for the identification of gemcitabine sensitivity genes --- p.113
Chapter 4 --- Discussion --- p.120
Chapter General conclusions --- p.125