Impact of Host Genes and Strand Selection on miRNA and miRNA* Expression

Dysregulation of miRNAs expression plays a critical role in the pathogenesis of genetic, multifactorial disorders and in human cancers. We exploited sequence, genomic and expression information to investigate two main aspects of post-transcriptional regulation in miRNA biogenesis, namely strand selection regulation and expression relationships between intragenic miRNAs and host genes. We considered miRNAs expression profiles, measured in five sizeable microarray datasets, including samples from different normal cell types and tissues, as well as different tumours and disease states. First, the study of expression profiles of “sister” miRNA pairs (miRNA/miRNA*, 5′ and 3′ strands of the same hairpin precursor) showed that the strand selection is highly regulated since it shows tissue-/cell-/condition-specific modulation. We used information about the direction and the strength of the strand selection bias to perform an unsupervised cluster analysis for the sample classification evidencing that is able to distinguish among different tissues, and sometimes between normal and malignant cells. Then, considering a minimum expression threshold, in few miRNA pairs only one mature miRNA is always present in all considered cell types, whereas the majority of pairs were concurrently expressed in some cell types and alternatively in others. In a significant fraction of concurrently expressed pairs, the major and the minor forms found at comparable levels may contribute to post-transcriptional gene silencing, possibly in a coordinate way. In the second part of the study, the behaved tendency to co-expression of intragenic miRNAs and their “host” mRNA genes was confuted by expression profiles examination, suggesting that the expression profile of a given host gene can hardly be a good estimator of co-transcribed miRNA(s) for post-transcriptional regulatory networks inference. Our results point out the regulatory importance of post-transcriptional phases of miRNAs biogenesis, reinforcing the role of such layer of miRNA biogenesis in miRNA-based regulation of cell activities

Computational Prediction of Intronic microRNA Targets using Host Gene Expression Reveals Novel Regulatory Mechanisms

Approximately half of known human miRNAs are located in the introns of protein coding genes. Some of these intronic miRNAs are only expressed when their host gene is and, as such, their steady state expression levels are highly correlated with those of the host gene's mRNA. Recently host gene expression levels have been used to predict the targets of intronic miRNAs by identifying other mRNAs that they have consistent negative correlation with. This is a potentially powerful approach because it allows a large number of expression profiling studies to be used but needs refinement because mRNAs can be targeted by multiple miRNAs and not all intronic miRNAs are co-expressed with their host genes

Identification and Characterization of MicroRNAs in Porcine Gametes and Pre-Implantation Embryos

MicroRNAs (miRNAs) are short ribonucleic acids that ultimately affect the production of proteins. Although miRNAs are involved in nearly every biological process examined to date, little is known of the identity or function of miRNA in porcine reproductive tissues or their potential involvement in reproductive processes in pigs or other species. The objective of this dissertation research was to determine the presence of miRNAs in porcine gametes and both in vivo- and in vitro- produced pre-implantation embryos and to identify differences in miRNA expression between normal and aberrant samples. Using a heterologous RT-PCR approach, we demonstrated the presence of a total of 92 miRNAs in porcine oocytes, spermatozoa, and/ or embryos at the 4-cell, 8-cell, 16-cell, and blastocyst stages, with hundreds more predicted by miRNA microarray. Subsequent qRT-PCR analysis showed differential expression of five miRNAs, let-7a, -7d, -7e, miR-15b, and -22, between normal sperm and morphologically abnormal sperm or sperm samples exhibiting low motility. Messenger RNA targets of the differentially expressed miRNAs encode proteins important for spermatogenesis, sperm structure, and/ or sperm cell metabolism. Differential expression was also shown among embryos at various stages in development, demonstrating a temporal expression pattern of specific miRNAs in pre-implantation embryo growth. More interestingly, miR-24 was differentially expressed between in vivo- and in vitro- produced embryos at the 8-cell and blastocyst stages, supporting the need to characterize aberrant miRNA expression associated with the abnormal embryonic development correlated with assisted reproductive technologies. All of the miRNAs examined demonstrated high sequence similarity to the corresponding human miRNA sequences, indicative of high conservation among species. Understanding miRNA expression in reproductive processes is critical to comprehending the mechanistic roles miRNAs play in the regulation of all physiological processes

MicroRNAs e o processo de inflammaging na esquizofrenia : de mecanismos moleculares a perspectivas na medicina translacional

A esquizofrenia (SZ) é uma condição psiquiátrica de etiologia pouco elucidada, caracterizada por um grupo incapacitante de desordens neurais que acomete até 1% da população humana e possui extensa heterogeneidade genética. Indivíduos com o transtorno têm uma expectativa de vida média cerca de 15 anos menor, taxa de mortalidade duas vezes maior que a da população geral e, consequentemente, têm sido associados a um processo contínuo de envelhecimento acelerado. O acúmulo de evidências sugere que pequenas moléculas de RNA não codificantes de fita simples, a família dos microRNAs (miRNAs), de aproximadamente 19-25 nucleotídeos, são reguladores importantes de várias vias de sinalização que desempenham um papel crucial na fisiopatologia da SZ. Porém, os mecanismos regulatórios dos miRNAs em diferentes processos moleculares na SZ, como o envelhecimento precoce presente nesses indivíduos, ainda são um quebra-cabeça a ser resolvido. O avanço da idade associado à inflamação sistêmica de baixo grau é denominado inflammaging, processo caracterizado pelo aumento crônico dos níveis de citocinas e fatores pró-inflamatórios relacionados à progressão da senescência. Portanto, níveis alterados de miRNAs podem estar correlacionados com o processo de inflammaging, principalmente por meio de modificações transcricionais de seus genes-alvo. Guiados por essa hipótese, objetivamos buscar alvos validados para um conjunto de treze miRNAs previamente descritos em SZ, bem como as vias metabólicas que podem ser influenciadas por esses miRNAs em SZ. A busca de alvos foi realizada por meio das plataformas miRTarBase (http://miRTarBase.mbc.nctu.edu.tw/) e miRDB (http://mirdb.org). A análise de alvos dos miRNAs alterados na SZ demonstram um claro envolvimento com sistema imunológico, diferenciação celular, ciclo celular, processo inflamatório, estresse oxidativo e disfunção mitocondrial. Podemos concluir que as alterações encontradas nos perfis de expressão dos miRNAs podem ser utilizadas como ferramentas para elucidar possíveis alvos farmacológicos para vias de sinalização alteradas durante o processo de inflammaging na SZ. Além disso, o estudo da expressão de miRNAs em diferentes fases do transtorno tem potencial na busca de marcadores clínicos para auxiliar o diagnóstico.Schizophrenia (SZ) is a psychiatric condition of poorly understood etiology, characterized by a disabling group of neural disorders that affect up to 1% of the human population and have extensive genetic heterogeneity. Individuals with the disorder have an average life expectancy about 15 years lower, a mortality rate twice that of the general population and, consequently, have been associated with a continuous process of accelerated aging. Accumulation of evidence suggests that small, single-stranded non-coding RNA molecules, the family of microRNAs (miRNAs), with approximately 19-25 nucleotides, are important regulators of several signaling pathways that play a crucial role in the pathophysiology of SZ. Although, the regulatory mechanisms of miRNAs in different molecular processes in SZ, such as the premature aging present in these individuals, are still a puzzle to be solved. Advancing age associated with low-grade systemic inflammation is called inflammaging, a process characterized by the chronic increase in levels of cytokines and pro-inflammatory factors related to the progression of senescence. Therefore, altered levels of miRNAs may be correlated with the inflammaging process, mainly through transcriptional modifications of their target genes. Guided by this hypothesis, we aimed to seek validated targets for a set of thirteen miRNAs previously described in SZ, as well as the metabolic pathways that can be influenced by these miRNAs in SZ. The search for targets was performed using the miRTarBase (http://miRTarBase.mbc.nctu.edu.tw/) and miRDB (http://mirdb.org) platforms. The analysis of altered miRNAs targets in SZ demonstrates a clear involvement with the immune system, cell differentiation, cell cycle, inflammatory process, oxidative stress and mitochondrial dysfunction. We can conclude that the alterations found in the expression profiles of miRNAs can be used as tools to elucidate possible pharmacological targets for altered signaling pathways during the process of inflammaging in SZ. Furthermore, the study of the expression of miRNAs at different stages of the disorder has potential in the search for clinical markers to aid diagnosis

Functional analysis of microRNA-130b in bovine oocyte maturation and preimplantation embryo development

MicroRNAs (miRNAs) are well known to regulate the proteins involved in various biological processes including development. The expression pattern of miRNAs is believed to vary between immature and in vitro matured bovine oocytes. Among these, miR-130b was reported to upregulated in immatured compared to matured oocytes. However, its functional role in cell viability, proliferation or transcription during bovine oocyte maturation and preimplantation embryo development is not known. Therefore, this experiment was aimed to investigate the functional role of miR-130b in oocyte maturation and oocyte surrounding cells and its involvement in preimplantation embryo development. For this, the spatiotemporal expression pattern of miR-130 family was performed throughout the bovine preimplantation stage embryos. Accordingly, miR-130b was found to be highly expressed in cumulus and granulosa cells, immature oocyte, morula and blastocyst stage embryos. Once the expression pattern of miR-130b was evaluated, its target genes were in silico analyzed and experimentally validated. Accordingly, MSK1, SMAD5, MEOX2, DOC1R and EIF2C4 were found to be the real targets of miR-130b. To investigate the involvement of miR-130b during oocyte maturation, immatured oocytes were microinjected with pre-miR-130b (precursor) or sequence specific antisense (inhibitor) of miR-130b, while scramble miRNA injected and uninjected oocytes were used as controls. The maturational status of the oocytes and the level of miR-130b target genes expression were assessed 22 hours post microinjection. The result showed that the first polar body extrusion was 86.3, 73, 85 and 84.6% in oocytes injected with pre-miR-130b, anti-miR-130b, scramble and uninjected controls, respectively. Similarly, mitotic staining showed that majority of oocytes injected with anti-miR-130b remains arrested at the telephase I stage (22%) and significantly reduced to reach Metaphase II compared to other oocyte groups. In addition, the mitochondrial activity was higher in pre-mir-130b and lower in anti-miR-130b injected oocytes compared to scramble and uninjected oocytes. This was associated with the reduction of miR-130b and increase of its target genes SMAD5 and MSK1 expression. Furthermore, oocyte surrounding cells are required for oocyte maturation, the involvement of miR-130b in cumulus and granulosa cell proliferation, lactate production and cholesterol level was assessed after transfection of pre-miR-130b or anti-miR-130b in both cell types. The inhibition of miR-130b resulted in reduction of cell proliferation and lactate production. However, knockdown of miR-130b did not change the cholesterol level in the granulosa or cumulus cells. Apart from oocyte maturation and oocyte companion cell function, the role of miR-130b was investigated during preimplantation embryo development by microinjecting zygotes with pre-miR-130b or anti-miR-130b. The result has shown that the first cleavage rate was unaffected by knockdown or ectopic expression of miR-130b, but the rate of morula and blastocyst were significantly reduced in anti-miR-130b injected zygotes. Therefore this study provides the significant evidence that miR-130b may be required during bovine oocyte in vitro maturation and granulosa cell proliferation, morula and blastocyst formation, further functional in depth studies are necessary to understand whether miR-130b is involved in bovine oocyte in vivo maturation or embryo implantation.Funktionelle Analyse der microRNA miR-130b während der bovinen Oozytenmaturation und der preimplantativen Embryonalentwicklung MicroRNAs (miRNAs) sind dafür bekannt, dass sie eine regulatorische Rolle in verschiedenen biologischen Prozessen, wie in der Embryonalentwicklung spielen. Es wir angenommen, dass das Expressionsmuster von miRNAs zwischen immaturen und in vitro maturierten bovinen Oozyten variiert, wobei gezeigt wurde, da miR-130b in inmaturierten Oozyten hoch reguliert ist. Allerdings ist seine funktionelle Rolle in der Zellvitalität, Proliferation und Transkription während der bovinen Oozytenmaturation und der Präimplantiationsembryoentwicklung noch nicht bekannt. Daher war das Ziel dieser Studie die Bedeutung von miR-130b in der Maturation von Oozyten-, Granulosa- und Kumuluszellen und in der präimplantativen Embryoentwicklung zu untersuchen. Dafür wurde das Expressionsmuster der miR-130-Familie im bovinen embryonalen Präimplantationsstadium erstellt. MiR-130b war in Kumulus- und Granulosazellen, in immaturen Oozyten sowie im Morula- und Blastozystenstadium höher exprimiert. Die miR-130b Zielgen Identifizierung erfolgte mittels der In silico Analyse und der experimentellen Validierung durch den Luciferase-Assay. Dementsprechend konnten MSK1, SMAD5, MEOX2, DOC1R und EIF2C4 als Zielgene von miR-130b ermittelt werden. Um den Einfluss der miR-130b während der Maturation der Oozyten zu untersuchen, wurden in immaturierten Oozyten pre-miR-130b und sequenz-spezifische miR-130b Antisense (Inhibitor) mikroinjiziert, während mit scrambled miRNA injizierte und nicht injizierte Oozyten als Kontrolle dienten. 24 Stunden nach der Mikroinjektion wurde der Reifungsstatus der Oozyten mittels der ersten Polkörper-Extrusion, festgestellt. Sie betrug 86,3, 73, 85, und 84,6% bei Oozyten mit injizierter pre-miR-130b, anti-miR-130b, scramble RNA und nicht injizierte Kontrolle. Die Mehrheit der anti-miR-130b injizierten Oozyten blieb in der Telophase 1 (22%) stehen. Darüber hinaus konnte eine höhere mitrochendriale Aktivität in pre-miR-130b und eine niedrigere in anti-miR-130b injizierten Oozyten im Vergleich zu scramble RNA und nicht injizierte Oozyten gefunden werden. Dies konnte mit der Zunahme der Proteinexpression der miR-130b Zielgene SMAD5 und MSK1 assoziiert werden. Oozyten Companionzellen sind für die Oozytenmaturation erforderlich. Der Einfluss der miR-130b konnte bei der Zellproliferation, Laktatproduktion und beim Cholesterinspiegel durch die Transfektion der miR-130b Precursor RNA oder anti-miR-130b RNA in Kumulus- und Granulosazellen beobachtet werden. Mit der Reduktion der miR-130b folgte einen Reduzierung in der Zellproliferation und der Laktatproduktion, allerdings keine Änderungen im Cholesterinspiegel in Granulosa- oder Kumuluszellen. Neben der Maturation der Oozyten und der Oozyten Companionzellfunktion wurde die Rolle der miR-130b während der präimplantations Embryoentwicklung nach einer Mikroinjektion der miR-130b Precursor oder – Inhibitor in Zygoten untersucht. Das Ergebnis zeigte, dass die erste Teilungsrate unbeeinflusst vom Knockdown oder der Überexpression der miR-130b war, jedoch war die Morula/Blastozysten rate der anti-miR-130b injizierten Eizellen signifikant reduziert. Diese Studie liefert Hinweise dafür, dass die miR-130b während der bovinen Oozytenmaturation, der Granulosazellproliferation und der Morula- und Blastozystenformation funktionell beteiligt ist

Post-transcriptional regulation of Estrogen Receptor-α by miR-17-92 interaction and LMTK3 phosphorylation in Breast Cancer

Estrogen receptor-α (ERα) is expressed in two-thirds of BCs and is a well-known prognostic and predictive marker. For this reason it is one of the most studied proteins in BC. To understand how ERα positive BC develops, it is crucial to investigate both how this protein is regulated and which genes are modulated by it. MicroRNAs (miRNAs) control gene expression post-transcriptionally by interacting through sequence complementarity to their target transcripts. Through a microarray approach, we identified the subset of miRNAs modulated by ERα, that include up-regulation of miRNAs derived from the processing of two paralogous primary (pri-) transcripts, pri-miR-17-92 and pri-miR-106a-363. Characterisation of the miR-17-92 locus confirmed that the ERα target protein c-MYC binds its promoter in an estrogen-dependent manner. These findings indicated that miRNAs derived from these pri-miRNAs (miR-18a, miR-19b and miR-20b) target and down-regulate ERα, whilst a subset of pri-miRNA-derived mature miRNAs inhibit protein translation of the ERα transcriptional p160 co-activator, AIB1. Therefore, different subsets of the miRNAs identified act as part of a negative autoregulatory feedback loop. We observed that levels of pri-miR-17-92 increase earlier than the mature miRNAs derived from it, implicating precursor cleavage modulation after transcription. Pri-mir-17-92 is immediately cleaved by Drosha to pre-miR-18a, indicating that its regulation occurs during the formation of the mature molecule from the precursors. Furthermore, we wanted to explore the new kinases that regulate the ERα activity. Thereby, we performed kinome screening (by RNAi technologies) to determine kinases that regulate ERα in MCF-7 BC cells and identified a novel kinase, LMTK3, which acts as positive regulator of ERα's transcriptional activity. This could be a new therapeutic target and/or a novel biomarker for BC, although further studies are required to validate this. Together, these studies identify new transcriptional and translational factors that regulate ERα expression in BC.Open Acces

The Complex Network of miRNA and mRNA Target Interactions in Pancreatic Cancer

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