Análise transcriptômica de miRNAs em pacientes sob dupla terapia de antiagregação

A terapia antiagregante é comumente indicada na prevenção e tratamento de doenças cardiovasculares. A dupla antiagregação com clopidrogrel e ácido acetilsalicílico (AAS) tem sido frequentemente adotada em pacientes com Doença Arterial Coronariana (DAC), mas apresenta ineficácia em uma parcela significativa da população com genótipo de respondedores. Essa falha terapêutica nos leva a questionar se outros mecanismos moleculares podem estar influenciando na resposta a esses fármacos. Recentes estudos sugerem que pequenas sequências de RNA não codificantes denominadas microRNAs (miRNAs) podem estar fortemente relacionadas com resposta ao tratamento fármaco-terapêutico, controlando as proteínas envolvidas na farmacocinética e farmacodinâmica. Entretanto, os principais miRNAs que atuam na dinâmica da resposta medicamentosa ainda não foram bem definidos. O objetivo deste estudo foi avaliar o perfil de miRNAs no sangue total periférico, procurando melhor esclarecer os mecanismos envolvidos na resposta aos antiagregantes plaquetários AAS e clopidogrel. Para isso, selecionou-se pacientes com DAC, os quais apresentavam diferentes respostas à dupla terapia de antiagregação determinadas pelo teste de agregação plaquetária. Baseados nos fenótipos, os perfis de expressão de miRNAs foram comparados entre os valores da taxa de agregação categorizados em tercis (T) de resposta. O grupo T1 foi constituído de pacientes respondedores, o T2 de respondedores intermediários e o T3 de não respondedores. Os perfis de miRNAs foram obtidos após sequenciamento de última geração e os dados obtidos foram analisados pelo pacote Deseq2. Os resultados mostraram 18 miRNAs diferentemente expressos entre os dois tercis extremos. Dentre esses miRNAs, 10 deles apresentaram importantes alvos relacionados com vias de ativação e agregação plaquetária quando analisados pelo software Ingenuity®. Dos 10 miRNAs, 4 deles, os quais apresentaram-se menos expressos no sequenciamento, demonstraram os mesmos perfis de expressão quando analisados pela reação em cadeia pela polimerase quantitativa (qPCR): hsa-miR-423-3p, hsa-miR-744-5p, hsa-miR- 30a-5p e hsa-let-7g-5p. A partir das análises de predição de alvos, pôde-se observar que os quatro miRNAs, quando menos expressos simultaneamente, predizem ativação da agregação plaquetária. Além disso, os miRNAs hsa-miR- 423-5p, hsa-miR-744-5p e hsa-let-7g-5p mostraram correlação com o perfil lipídico dos pacientes que, por sua vez, apresentou influência nos valores de agregação compreendidos no T3 de resposta a ambos os medicamentos. Sendo assim, conclui-se que maiores taxas de agregação plaquetária podem estar indiretamente relacionadas com os padrões de expressão de hsa-miR- 423-3p, hsa-miR-744-5p e hsa-let-7g-5p. Sugere-se que a avaliação do perfil de expressão destes 3 miRNAs no sangue periférico de pacientes com DAC possa predizer resposta terapêutica inadequada ao AAS e ao clopidogrel

IDENTIFICAÇÃO DE SURPRESAS MONETÁRIAS E SEUS IMPACTOS SOBRE A ESTRUTURA A TERMO DA TAXA DE JUROS

A reação dos mercados às alterações na taxa básica de juros é relevante para toda a economia. O entendimento da relação entre a política monetária e as taxas de juros é de extrema importância, uma vez que surpresas monetárias, ou seja, os erros de previsão do mercado a respeito das alterações da meta da Taxa Selic, podem afetar as taxas de juros de diferentes maturidades ou vencimentos, impactando diretamente a Administração Financeira. O objetivo deste estudo foi analisar a variação da Estrutura a Termo da Taxa de Juros (ETTJ) quando verificadas surpresas monetárias na ocasião da decisão do Comitê de Política Monetária (Copom) a respeito da meta da Taxa Selic. Para isso, foi desenvolvido um estudo descritivo quantitativo, que considerou as 88 reuniões ordinárias do Copom realizadas no período de janeiro de 2004 a dezembro de 2013. As surpresas monetárias foram identificadas através de duas formas distintas. Na primeira forma foram consideradas as taxas do contrato de DI1 referente ao último negócio realizado no pregão da data da reunião do Copom, e a taxa do primeiro negócio realizado no pregão seguinte. Desta maneira foram identificadas 11 surpresas monetárias. Na segunda forma foram consideradas as taxas médias verificadas nos mesmos contratos e ocasiões citados anteriormente, sendo assim identificadas 10 surpresas monetárias. Já para a análise da relação entre as variações da ETTJ e as surpresas monetárias foram considerados os vencimentos de 2, 3, 6, 9, 12, 15, 18 e 24 meses. Como resultado foi possível observar que as surpresas monetárias e as variações na ETTJ são diretamente proporcionais, movendo-se na mesma direção, para as duas formas distintas de surpresas monetárias identificadas neste estudo. Além disso, foi empregada nas análises a questão da unanimidade na decisão do Copom, com o objetivo testar o seu conteúdo informacional, e observou-se como resultado uma menor variação da ETTJ em ocasiões em que a decisão do Copom foi unânime. Em resumo, entende-se que os resultados encontrados no presente estudo estão em linha aos apresentados por outros autores, sendo possível comprovar a correlação existente entre as variações da ETTJ e as surpresas monetárias, bem como verificar que a magnitude das variações diminui ao longo da ETTJ, fato este que pode ser relacionado à transparência da política monetária nacional e à experiência na vigência do sistema de metas para a inflação.

Expression and cellular localization of microRNA-29b and RAX, an activator of the RNA-dependent protein kinase (PKR), in the retina of streptozotocin-induced diabetic rats

Purpose: The apoptosis of retinal neurons plays a critical role in the pathogenesis of diabetic retinopathy (DR), but the molecular mechanisms underlying this phenomenon remain unclear. The purpose of this study was to investigate the cellular localization and the expression of microRNA-29b (miR-29b) and its potential target PKR associated protein X (RAX), an activator of the pro-apoptotic RNA-dependent protein kinase (PKR) signaling pathway, in the retina of normal and diabetic rats. Methods: Retinas were obtained from normal and diabetic rats within 35 days after streptozotocin (STZ) injection. In silico analysis indicated that RAX is a potential target of miR-29b. The cellular localization of miR-29b and RAX was assessed by in situ hybridization and immunofluorescence, respectively. The expression levels of miR-29b and RAX mRNA were evaluated by quantitative reverse transcription PCR (qRT-PCR), and the expression of RAX protein was evaluated by western blot. A luciferase reporter assay and inhibition of endogenous RAX were performed to confirm whether RAX is a direct target of miR-29b as predicted by the in silico analysis. Results: We found that miR-29b and RAX are localized in the retinal ganglion cells (RGCs) and the cells of the inner nuclear layer (INL) of the retinas from normal and diabetic rats. Thus, the expression of miR-29b and RAX, as assessed in the retina by quantitative RT-PCR, reflects their expression in the RGCs and the cells of the INL. We also revealed that RAX protein is upregulated (more than twofold) at 3, 6, 16, and 22 days and downregulated (70%) at 35 days, whereas miR-29b is upregulated (more than threefold) at 28 and 35 days after STZ injection. We did not confirm the computational prediction that RAX is a direct target of miR-29b. Conclusions: Our results suggest that RAX expression may be indirectly regulated by miR-29b, and the upregulation of this miRNA at the early stage of STZ-induced diabetes may have a protective effect against the apoptosis of RGCs and cells of the INL by the pro-apoptotic RNA-dependent protein kinase (PKR) signaling pathway.

IMP2 provides an alternative to LIN28B for LET-7 target gene protection and glioma stem cell maintenance

Glioblastoma multiforme (GBM) is the most frequent and lethal primary brain tumor in adults. Accumulating evidence suggests that tumors comprise a hierarchical organization that is, at least partially, not genetically driven. Cells that reside at the apex of this hierarchy are commonly referred to as cancer stem cells (CSCs) and are believed to largely contribute to recurrence and therapeutic failure. Although the complexity of epigenetic regulation of the genome precludes prediction as to which epigenetic changes dominate CSC specification in different cancer types, the ability of microRNAs (miRNAs) to fine-tune expression of entire gene networks places them among prime candidates for establishing CSC properties. In this study we characterized the miRNA expression profile of primary GBM grown either under conditions that enrich for GSCs or their differentiated non-tumorigenic progeny (DGCs). Although, we identified a subset of miRNAs that was strongly differentially expressed between GSCs and DGCs, we observed that in GSCs both let-7 and, paradoxically, their target genes are highly expressed, suggesting protection against let-7 action. Using PAR-CLIP we show that insulin-like growth factor-2 mRNA-binding protein 2 (IMP2) provides a mechanism for let-7 target gene protection that represents an alternative to LIN28A/B, which abrogates let-7 biogenesis in normal embryonic and certain malignant stem cells. By direct binding to miRNA recognition elements, IMP2 protects its targets from let-7 mediated decay. Importantly, depletion of IMP2 in GSCs strongly impairs their self- renewal properties and tumorigenicity in vivo, a phenotype that can be rescued by expression of LIN28B, suggesting that IMP2 mainly contributes to GSC maintenance by protecting let-7 target genes from silencing. Using mouse models, we show that depletion of IMP2 in neural stem cells (NSCs) induces let-7 target gene down-regulation, impairs their clonogenic capacity, and affects differentiation. Taken together, our observations describe a novel regulatory function of IMP2 in the let-7 axis whereby it supports GSC and NSC specification. Résumé (Français) Le glioblastome (GBM) est la tumeur primaire maligne du cerveau la plus fréquente. De nombreuses études ont démontré l'existence d'une organisation hiérarchique des cellules cancéreuses liée à des mécanismes épigénétiques. Les cellules qui se trouvent au sommet de cette hiérarchie sont appelées cellules souches cancéreuses (CSC), et contribuent à l'échec thérapeutique. Bien que la complexité des régulateurs épigénétiques permette difficilement de prédire quel mécanisme contribue le plus aux propriétés des CSC, la capacité des microRNAs (miRNAs) de réguler des réseaux entiers de gènes, les placent comme des candidats de premiers choix. Ici, nous avons caractérisé le profil d'expression des miRNAs dans des tumeurs primaires de GBM cultivées dans des conditions qui enrichissent soit pour les CSC, soit pour leur contrepartie de cellules cancéreuses différences (CCD). De manière surprenante et paradoxale la famille de miRNA let-7 et leurs gènes cibles étaient hautement exprimés dans les CSC, suggérant un mécanisme de protection contre l'action des let-7. Avec l'aide de la technologie PAR-CLIP, nous démontrons que la protéine IMP2, protège les mRNAs de l'action des let-7 et représente une alternative à Lin28A/B, qui d'ordinaire réprime fortement la maturation des let-7 dans les cellules souches embryonnaires et divers cancers. En se liant à la région ciblée par les let-7, IMP2 protège ses transcrits de l'action de cette classe de microRNA qui est tumoro-supressive. La déplétion d'IMP2 dans des CSC de GBM réduit fortement leur clonogénicité in vitro et leur tumorigénicité in vivo. Ceci peut être reversé en introduisant Lin28B dans des CSC de GBM, suggérant qu'IMP2 exerce ses fonctions pro-tumorigéniques en modulant l'axe let-7. Avec l'aide de modèles murins, nous observons que la déplétion de IMP2 dans les cellules souches neurales (CSN) induit une baisse de leur clonogénicité et des cibles des miRNAs let-7, suggérant une conservation de ce mécanisme entre les CSC de GBM et les CSN. En résumé, nos observations définissent une nouvelle fonction de IMP2 dans l'axe let-7 par lequel il contribue au maintien des propriétés des CSC et des CSN.

MicroRNA-499 expression distinctively correlates to target genes sox6 and rod1 profiles to resolve the skeletal muscle phenotype in nile tilapia

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Analisi di espressione di microRNA in Tumore a Cellule Giganti: identificazione dei target con ruolo di potenziali biomarcatori

Il Tumore a Cellule Giganti dell’osso (TCG) è una rara neoplasia che rappresenta il 5% dei tumori di natura ossea; sebbene venga considerato un tumore a decorso benigno può manifestare caratteri di aggressività locale dando origine a recidive locali nel 10-25% dei casi, e nel 2-4% dei casi metastatizza a livello polmonare. In questo studio è stata valutata l’espressione dei miRNA mediante miRNA microarray in 10 pazienti affetti da TCG, 5 con metastasi e 5 liberi da malattia; sono stati riscontrati miRNA differenzialmente espressi tra i 2 gruppi di pazienti e la successiva validazione mediante Real Time PCR ha confermato una differenza significativa per il miR-136 (p=0.04). Mediante analisi bioinformatica con il software TargetScan abbiamo identificato RANK e NF1B come target del miR-136 e ne abbiamo studiato l’espressione mediante Real Time PCR su una più ampia casistica di pazienti affetti da TCG, metastatico e non, evidenziando una maggior espressione di NF1B nel gruppo di pazienti metastatici, mentre RANK non ha dimostrato una differenza significativa. L’analisi di Western Blot ha rilevato una maggiore espressione di entrambe le proteine nei pazienti metastatici rispetto ai non metastatici. Successivamente è stato condotto uno studio di immunoistochimica su TMA di 163 campioni di pazienti affetti da TCG a diverso decorso clinico che ha dimostrato una maggiore e significativa espressione di entrambe i target nei pazienti con metastasi rispetto ai non metastatici; le analisi di popolazione mediante Kaplan-Meier hanno confermato la correlazione tra over-espressione di RANK, NF1B e ricaduta con metastasi (p=0.001 e p<0.0005 rispettivamente). Lo studio di immunoistochimica è stato ampliato alle proteine maggiormente coinvolte nell’osteolisi che risultano avere un significato prognostico; tuttavia mediante analisi di ROC, la co-over-espressione di RANK, RANKL e NF1B rappresenta il migliore modello per predire la comparsa di metastasi (AUC=0.782, p<0.0005).

Statistical prediction of single-stranded regions in RNA secondary structure and application to predicting effective antisense target sites and beyond

Single-stranded regions in RNA secondary structure are important for RNA–RNA and RNA–protein interactions. We present a probability profile approach for the prediction of these regions based on a statistical algorithm for sampling RNA secondary structures. For the prediction of phylogenetically-determined single-stranded regions in secondary structures of representative RNA sequences, the probability profile offers substantial improvement over the minimum free energy structure. In designing antisense oligonucleotides, a practical problem is how to select a secondary structure for the target mRNA from the optimal structure(s) and many suboptimal structures with similar free energies. By summarizing the information from a statistical sample of probable secondary structures in a single plot, the probability profile not only presents a solution to this dilemma, but also reveals ‘well-determined’ single-stranded regions through the assignment of probabilities as measures of confidence in predictions. In antisense application to the rabbit β-globin mRNA, a significant correlation between hybridization potential predicted by the probability profile and the degree of inhibition of in vitro translation suggests that the probability profile approach is valuable for the identification of effective antisense target sites. Coupling computational design with DNA–RNA array technique provides a rational, efficient framework for antisense oligonucleotide screening. This framework has the potential for high-throughput applications to functional genomics and drug target validation.

MicroRNA modulation of aldosterone production in the adrenal gland

Hypertension is the major risk factor for coronary disease worldwide. Primary hypertension is idiopathic in origin but is thought to arise from multiple risk factors including genetic, lifestyle and environmental influences. Secondary hypertension has a more definite aetiology; its major single cause is primary aldosteronism (PA), the greatest proportion of which is caused by aldosteroneproducing adenoma (APA), where aldosterone is synthesized at high levels by an adenoma of the adrenal gland. There is strong evidence to show that high aldosterone levels cause adverse effects on cardiovascular, cerebrovascular, renal and other systems. Extensive studies have been conducted to analyse the role that regulation of CYP11B2, the gene encoding the aldosterone synthase enzyme plays in determining aldosterone production and the development of hypertension. One significant regulatory factor that has only recently emerged is microRNA (miRNA). miRNAs are small non-coding RNAs, synthesized by a series of enzymatic processes, that negatively regulate gene expression at the posttranscriptional level. Detection and manipulation of miRNA is now known to be a viable method in the treatment, prevention and prognosis of certain diseases. The aim of the present study was to identify miRNAs likely to have a role in the regulation of corticosteroid biosynthesis. To achieve this, the miRNA profile of APA and normal human adrenal tissue was compared, as was the H295R adrenocortical cell line model of adrenocortical function, under both basal conditions and following stimulation of aldosterone production. Key differentially-expressed miRNAs were then identified and bioinformatic tools used to identify likely mRNA targets and pathways for these miRNAs, several of which were investigated and validated using in vitro methods. The background to this study is set out in Chapter 1 of this thesis, followed by a description of the major technical methods employed in Chapter 2. Chapter 3 presents the first of the study results, analysing differences in miRNA profile between APA and normal human adrenal tissue. Microarray was implemented to detect the expression of miRNAs in these two tissue types and several miRNAs were found to vary significantly and consistently between them. Furthermore, members of several miRNA clusters exhibited similar changes in expression pattern between the two tissues e.g. members of cluster miR-29b-1 (miR-29a-3p and miR-29b-3p) and of cluster miR-29b-2 (miR-29b-3p and miR-29c- 3p) are downregulated in APA, while members of cluster let-7a-1 (let-7a-5p and let-7d-5p), cluster let-7a-3 (let-7a-5p and let-7b-5p) and cluster miR-134 (miR- 134 and miR-382) are upregulated. Further bioinformatic analysis explored the possible biological function of these miRNAs using Ingenuity® Systems Pathway Analysis software. This led to the identification of validated mRNAs already known to be targeted by these miRNAs, as well as the prediction of other mRNAs that are likely targets and which are involved in processes relevant to APA pathology including cholesterol synthesis (HMGCR) and corticosteroidogenesis (CYP11B2). It was therefore hypothesised that increases in miR-125a-5p or miR- 335-5p would reduce HMGCR and CYP11B2 expression. Chapter 4 describes the characterisation of H295R cells of different strains and sources (H295R Strain 1, 2, 3 and HAC 15). Expression of CYP11B2 was assessed following application of 3 different stimulants: Angio II, dbcAMP and KCl. The most responsive strain to stimulation was Strain 1 at lower passage numbers. Furthermore, H295R proliferation increased following Angio II stimulation. In Chapter 5, the hypothesis that increases in miR-125a-5p or miR-335-5p reduces HMGCR and CYP11B2 expression was tested using realtime quantitative RT-PCR and transfection of miRNA mimics and inhibitors into the H295R cell line model of adrenocortical function. In this way, miR-125a-5p and miR-335-5p were shown to downregulate CYP11B2 and HMGCR expression, thereby validating certain of the bioinformatic predictions generated in Chapter 3. The study of miRNA profile in the H295R cell lines was conducted in Chapter 6, analysing how it changes under conditions that increase aldosterone secretion, including stimulation Angiotensin II, potassium chloride or dibutyryl cAMP (as a substitute for adrenocorticotropic hormone). miRNA profiling identified 7 miRNAs that are consistently downregulated by all three stimuli relative to basal cells: miR-106a-5p, miR-154-3p, miR-17-5p, miR-196b-5p, miR-19a-3p, miR-20b- 5p and miR-766-3p. These miRNAs include those derived from cluster miR-106a- 5p/miR-20b-5p and cluster miR-17-5p/miR-19a-3p, each producing a single polycistronic transcript. IPA bioinformatic analysis was again applied to identify experimentally validated and predicted mRNA targets of these miRNAs and the key biological pathways likely to be affected. This predicted several interactions between miRNAs derived from cluster miR-17-5p/miR-19a-3p and important mRNAs involved in cholesterol biosynthesis: LDLR and ABCA1. These predictions were investigated by in vitro experiment. miR-17-5p/miR-106a-p and miR-20b-5p were found to be consistently downregulated by stimulation of aldosterone biosynthesis. Moreover, miR-766-3p was upregulation throughout. Furthermore, I was able to validate the downregulation of LDLR by miR-17 transfection, as predicted by IPA. In summary, this study identified key miRNAs that are differentially-expressed in vivo in cases of APA or in vitro following stimulation of aldosterone biosynthesis. The many possible biological actions these miRNAs could have were filtered by bioinformatic analysis and selected interactions validated in vitro. While direct actions of these miRNAs on steroidogenic enzymes were identified, cholesterol handling also emerged as an important target and may represent a useful point of intervention in future therapies designed to modulate aldosterone biosynthesis and reduce its harmful effects.

MicroRNA transcriptome profiles during swine skeletal muscle development

Background: MicroRNA (miR) are a class of small RNAs that regulate gene expression by inhibiting translation of protein encoding transcripts. To evaluate the role of miR in skeletal muscle of swine, global microRNA abundance was measured at specific developmental stages including proliferating satellite cells, three stages of fetal growth, day-old neonate, and the adult. Results: Twelve potential novel miR were detected that did not match previously reported sequences. In addition, a number of miR previously reported to be expressed in mammalian muscle were detected, having a variety of abundance patterns through muscle development. Muscle-specific miR-206 was nearly absent in proliferating satellite cells in culture, but was the highest abundant miR at other time points evaluated. In addition, miR-1 was moderately abundant throughout developmental stages with highest abundance in the adult. In contrast, miR-133 was moderately abundant in adult muscle and either not detectable or lowly abundant throughout fetal and neonate development. Changes in abundance of ubiquitously expressed miR were also observed. MiR-432 abundance was highest at the earliest stage of fetal development tested (60 day-old fetus) and decreased throughout development to the adult. Conversely, miR-24 and miR-27 exhibited greatest abundance in proliferating satellite cells and the adult, while abundance of miR-368, miR-376, and miR-423-5p was greatest in the neonate. Conclusion: These data present a complete set of transcriptome profiles to evaluate miR abundance at specific stages of skeletal muscle growth in swine. Identification of these miR provides an initial group of miR that may play a vital role in muscle development and growth.

MicroRNA-375 plays a dual role in prostate carcinogenesis

Background: Prostate cancer (PCa), a highly incident and heterogeneous malignancy, mostly affects men from developed countries. Increased knowledge of the biological mechanisms underlying PCa onset and progression are critical for improved clinical management. MicroRNAs (miRNAs) deregulation is common in human cancers, and understanding how it impacts in PCa is of major importance. MiRNAs are mostly downregulated in cancer, although some are overexpressed, playing a critical role in tumor initiation and progression. We aimed to identify miRNAs overexpressed in PCa and subsequently determine its impact in tumorigenesis. Results: MicroRNA expression profiling in primary PCa and morphological normal prostate (MNPT) tissues identified 17 miRNAs significantly overexpressed in PCa. Expression of three miRNAs, not previously associated with PCa, was subsequently assessed in large independent sets of primary tumors, in which miR-182 and miR-375 were validated, but not miR-32. Significantly higher expression levels of miR-375 were depicted in patients with higher Gleason score and more advanced pathological stage, as well as with regional lymph nodes metastases. Forced expression of miR-375 in PC-3 cells, which display the lowest miR-375 levels among PCa cell lines, increased apoptosis and reduced invasion ability and cell viability. Intriguingly, in 22Rv1 cells, which displayed the highest miR-375 expression, knockdown experiments also attenuated the malignant phenotype. Gene ontology analysis implicated miR-375 in several key pathways deregulated in PCa, including cell cycle and cell differentiation. Moreover, CCND2 was identified as putative miR-375 target in PCa, confirmed by luciferase assay. Conclusions: A dual role for miR-375 in prostate cancer progression is suggested, highlighting the importance of cellular context on microRNA targeting.

Prediction of drug targets in human pathogens

The identification of new and druggable targets in bacteria is a critical endeavour in pharmaceutical research of novel antibiotics to fight infectious agents. The rapid emergence of resistant bacteria makes today's antibiotics more and more ineffective, consequently increasing the need for new pharmacological targets and novel classes of antibacterial drugs. A new model that combines the singular value decomposition technique with biological filters comprised of a set of protein properties associated with bacterial drug targets and similarity to protein-coding essential genes of E. coli has been developed to predict potential drug targets in the Enterobacteriaceae family [1]. This model identified 99 potential target proteins amongst the studied bacterial family, exhibiting eight different functions that suggest that the disruption of the activities of these proteins is critical for cells. Out of these candidates, one was selected for target confirmation. To find target modulators, receptor-based pharmacophore hypotheses were built and used in the screening of a virtual library of compounds. Postscreening filters were based on physicochemical and topological similarity to known Gram-negative antibiotics and applied to the retrieved compounds. Screening hits passing all filters were docked into the proteins catalytic groove and 15 of the most promising compounds were purchased from their chemical vendors to be experimentally tested in vitro. To the best of our knowledge, this is the first attempt to rationalize the search of compounds to probe the relevance of this candidate as a new pharmacological target.

MicroRNA-375 plays a dual role in prostate carcinogenesis

Background: Prostate cancer (PCa), a highly incident and heterogeneous malignancy, mostly affects men from developed countries. Increased knowledge of the biological mechanisms underlying PCa onset and progression are critical for improved clinical management. MicroRNAs (miRNAs) deregulation is common in human cancers, and understanding how it impacts in PCa is of major importance. MiRNAs are mostly downregulated in cancer, although some are overexpressed, playing a critical role in tumor initiation and progression. We aimed to identify miRNAs overexpressed in PCa and subsequently determine its impact in tumorigenesis. Results: MicroRNA expression profiling in primary PCa and morphological normal prostate (MNPT) tissues identified 17 miRNAs significantly overexpressed in PCa. Expression of three miRNAs, not previously associated with PCa, was subsequently assessed in large independent sets of primary tumors, in which miR-182 and miR-375 were validated, but not miR-32. Significantly higher expression levels of miR-375 were depicted in patients with higher Gleason score and more advanced pathological stage, aswellaswithregionallymph nodesmetastases. Forced expression of miR-375 in PC-3 cells, which display the lowest miR-375 levels among PCa cell lines, increased apoptosis and reduced invasion ability and cell viability. Intriguingly, in 22Rv1 cells, which displayed the highest miR-375 expression, knockdown experiments also attenuated the malignant phenotype. Gene ontology analysis implicated miR-375 in several key pathways deregulated in PCa, including cell cycle and cell differentiation. Moreover, CCND2 was identified as putative miR-375 target in PCa, confirmed by luciferase assay. Conclusions: A dual role for miR-375 in prostate cancer progression is suggested, highlighting the importance of cellular context on microRNA targeting.

Conserved microRNA editing in mammalian evolution, development and disease.

BACKGROUND: Mammalian microRNAs (miRNAs) are sometimes subject to adenosine-to-inosine RNA editing, which can lead to dramatic changes in miRNA target specificity or expression levels. However, although a few miRNAs are known to be edited at identical positions in human and mouse, the evolution of miRNA editing has not been investigated in detail. In this study, we identify conserved miRNA editing events in a range of mammalian and non-mammalian species. RESULTS: We demonstrate deep conservation of several site-specific miRNA editing events, including two that date back to the common ancestor of mammals and bony fishes some 450 million years ago. We also find evidence of a recent expansion of an edited miRNA family in placental mammals and show that editing of these miRNAs is associated with changes in target mRNA expression during primate development and aging. While global patterns of miRNA editing tend to be conserved across species, we observe substantial variation in editing frequencies depending on tissue, age and disease state: editing is more frequent in neural tissues compared to heart, kidney and testis; in older compared to younger individuals; and in samples from healthy tissues compared to tumors, which together suggests that miRNA editing might be associated with a reduced rate of cell proliferation. CONCLUSIONS: Our results show that site-specific miRNA editing is an evolutionarily conserved mechanism, which increases the functional diversity of mammalian miRNA transcriptomes. Furthermore, we find that although miRNA editing is rare compared to editing of long RNAs, miRNAs are greatly overrepresented among conserved editing targets.

Let-7a Is a Direct EWS-FLI-1 Target Implicated in Ewing's Sarcoma Development.

Ewing's sarcoma family tumors (ESFT) are the second most common bone malignancy in children and young adults, characterized by unique chromosomal translocations that in 85% of cases lead to expression of the EWS-FLI-1 fusion protein. EWS-FLI-1 functions as an aberrant transcription factor that can both induce and suppress members of its target gene repertoire. We have recently demonstrated that EWS-FLI-1 can alter microRNA (miRNA) expression and that miRNA145 is a direct EWS-FLI-1 target whose suppression is implicated in ESFT development. Here, we use miRNA arrays to compare the global miRNA expression profile of human mesenchymal stem cells (MSC) and ESFT cell lines, and show that ESFT display a distinct miRNA signature that includes induction of the oncogenic miRNA 17-92 cluster and repression of the tumor suppressor let-7 family. We demonstrate that direct repression of let-7a by EWS-FLI-1 participates in the tumorigenic potential of ESFT cells in vivo. The mechanism whereby let-7a expression regulates ESFT growth is shown to be mediated by its target gene HMGA2, as let-7a overexpression and HMGA2 repression both block ESFT cell tumorigenicity. Consistent with these observations, systemic delivery of synthetic let-7a into ESFT-bearing mice restored its expression in tumor cells, decreased HMGA2 expression levels and resulted in ESFT growth inhibition in vivo. Our observations provide evidence that deregulation of let-7a target gene expression participates in ESFT development and identify let-7a as promising new therapeutic target for one of the most aggressive pediatric malignancies.

Bicc1 links the regulation of cAMP signaling in polycystic kidneys to microRNA-induced gene silencing.

Genetic defects in autosomal-dominant polycystic kidney disease (ADPKD) promote cystic growth of renal tubules, at least in part by stimulating the accumulation of cAMP. How renal cAMP levels are regulated is incompletely understood. We show that cAMP and the expression of its synthetic enzyme adenylate cyclase-6 (AC6) are up-regulated in cystic kidneys of Bicc1(-)(/-) knockout mice. Bicc1, a protein comprising three K homology (KH) domains and a sterile alpha motif (SAM), is expressed in proximal tubules. The KH domains independently bind AC6 mRNA and recruit the miR-125a from Dicer, whereas the SAM domain enables silencing by Argonaute and TNRC6A/GW182. Bicc1 similarly induces silencing of the protein kinase inhibitor PKIα by miR-27a. Thus, Bicc1 is needed on these target mRNAs for silencing by specific miRNAs. The repression of AC6 by Bicc1 might explain why cysts in ADPKD patients preferentially arise from distal tubules.