Browsing by Author "Enguita, Francisco J."
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- Applications of genome editing by programmable nucleases to the metabolic engineering of secondary metabolitesPublication . Leitão, Ana Lúcia; Costa, Marina C.; Enguita, Francisco J.Genome engineering is a branch of modern biotechnology composed of a cohort of protocols designed to construct and modify a genotype with the main objective of giving rise to a desired phenotype. Conceptually, genome engineering is based on the so called genome editing technologies, a group of genetic techniques that allow either to delete or to insert genetic information in a particular genomic locus. Ten years ago, genome editing tools were limited to virus-driven integration and homologous DNA recombination. However, nowadays the uprising of programmable nucleases is rapidly changing this paradigm. There are two main families of modern tools for genome editing depending on the molecule that controls the specificity of the system and drives the editor machinery to its place of action. Enzymes such as Zn-finger and TALEN nucleases are protein-driven genome editors; while CRISPR system is a nucleic acid-guided editing system. Genome editing techniques are still not widely applied for the design of new compounds with pharmacological activity, but they are starting to be considered as promising tools for rational genome manipulation in biotechnology applications. In this review we will discuss the potential applications of programmable nucleases for the metabolic engineering of secondary metabolites with biological activity.
- Association between miR-146a and tumor necrosis factor alpha (TNF-α) in stable coronary artery diseasePublication . Pereira-da-Silva, Tiago; Napoleão, Patricia; Costa, Marina C.; Gabriel, André F.; Selas, Mafalda; Silva, Filipa; Enguita, Francisco J.; Cruz Ferreira, Rui; Carmo, Miguel MotaBackground and Objectives: Tumor necrosis factor alpha (TNF-α) is proatherogenic and associated with the risk of acute ischemic events, although the mechanisms that regulate TNF-α expression in stable coronary artery disease (SCAD) are not fully understood. We investigated whether metabolic, inflammatory, and epigenetic (microRNA (miRNA)) markers are associated with TNF-α expression in SCAD. Materials and Methods: Patients with SCAD were prospectively recruited and their metabolic and inflammatory profiles were assessed. TNF-α levels were assessed using an enzyme-linked immunosorbent assay. The relative expression of six circulating miRNAs associated with the regulation of inflammation and/or atherosclerosis was determined. Results: Of the 24 included patients with the mean age of 65 (9) years, 88% were male, and 54% were diabetic. The TNF-α levels were (median (interquartile range)) 1.0 (0.7-1.1) pg/mL. The percentage of glycosylated hemoglobin (r = 0.418, p = 0.042), serum triglyceride levels (r = 0.429, p = 0.037), and C-reactive protein levels (r = 0.407, p = 0.048) were positively correlated with TNF-α levels. Of the candidate miRNAs, miR-146a expression levels were negatively correlated with TNF-α levels (as indicated by r = 0.500, p = 0.035 for correlation between delta cycle threshold (ΔCt) miR-146a and TNF-α levels). In multivariate analysis, serum triglyceride levels and miR-146a expression levels were independently associated with TNF-α levels. miR-146 expression levels were not associated with metabolic or other inflammatory parameters and were negatively correlated with the number of coronary vessels with obstructive disease (as indicated by r = 0.556, p = 0.017 for correlation between ΔCt miR-146a and number of diseased vessels). Conclusions: miR-146a expression levels were negatively correlated with TNF-α levels in patients with SCAD, irrespective of other metabolic or inflammatory markers, and with the severity of coronary artery disease. The results add to the knowledge on the role of miR-146a in TNF-α-based inflammation in SCAD and support future research on the potential therapeutic use of miR-146a in such a clinical scenario.
- Biomarker potentials of miRNA-associated circRNAs in breast cancer (MCF-7) cells : an in vitro and in silico studyPublication . Misir, Sema; Hepokur, Ceylan; Aliyazicioglu, Yuksel; Enguita, Francisco J.Breast cancer is a heterogeneous disease, which is the most common malignancy in women. The incidence and mortality rates of breast cancer indicate that it is the leading cause of cancer-related with deaths. circRNAs operate as part of competing endogenous RNAs (ceRNAs) mechanisms, which play critical roles in the diferent biological processes of breast cancer such as proliferation, migration, and apoptosis. The goal of the present study is to identify the potential predictive biomarker for breast cancer diagnosis in the circRNA network by in vitro and in silico analyzes. 40 miRNAs were obtained from the miRWalk database and their combinatorial target genes (potential ceRNAs) were identifed with ComiR. We stated that the cancer-specifc circRNA genes in MCF-7 cells using the cancer-specifc circRNA (CSDC) database, and obtained the ones showing potential ceRNA activity in our previous analysis among them. Identifed genes with remarkable expression diferences between BCa and normal breast tissue were determined by the GEPIA database. Moreover, the Spearman correlation test in the GEPIA database was used for the statistical analysis of the relationship between DCAF7 and SOGA1, SOGA1 and AVL 9, DCAF7 and AVL 9 gene pairs. And also, DCAF7, SOGA1, and AVL9 gene expression levels were detected in MCF-7 and MCF-10A cells by RT-qPCR method. DCAF7, SOGA1, and AVL9 gene were signifcantly more expressed to BCa tissue and MCF-7 cells than normal breast tissue and MCF-10 A cells. And also, DCAF7 and SOGA1, SOGA1 and AVL9, DCAF7 and AVL9 genes pairs were found to be signifcantly correlated with BCa. These genes may be considered as potential predictive biomarkers to discriminate BCa patients from healthy persons. Our preliminary results can supply a new perspective for in vitro and vivo studies in the future.
- Catalyzing transcriptomics research in cardiovascular disease: The CardioRNA COST Action CA17129Publication . Gomes, Clarissa; Ágg, Bence; Andova, Andrejaana; Arslan, Serdal; Baker, Andrew; Barteková, Monika; Beis, Dimitris; Betsou, Fay; Wettinger, Stephanie; Bugarski, Branko; Condorelli, Gianluigi; Silva, Gustavo; Danilin, Sabrina; Gonzalo-Calvo, David; Buil, Alfonso; Carmo-Fonseca, Maria; Enguita, Francisco J.; Felekkis, Kyriacos; Ferdinandy, Peter; Gyöngyösi, Mariann; Hackl, Matthias; Karaduzovic-Hadziabdic, Kanita; Hellemans, Jan; Heymans, Stephane; Hlavackova, Markéta; Hoydal, Morten; Jankovic, Aleksandra; Jusic, Amela; Kardassis, Dimitris; Kerkelä, Risto; Kuster, Gabriela; Lakkisto, Päivi; Leszek, Przemyslaw; Lustrek, Mitja; Maegdefessel, Lars; Martelli, Fabio; Novella, Susana; O’Brien, Timothy; Papaneophytou, Christos; Pedrazzini, Thierry; Pinet, Florence; Popescu, Octavian; Potočnjak, Ines; Robinson, Emma; Sasson, Shlomo; Scholz, Markus; Simionescu, Maya; Stoll, Monika; Varga, Zoltan; Vinciguerra, Manlio; Xuereb, Angela; Yilmaz, Mehmet; Emanueli, Costanza; Devaux, YvanCardiovascular disease (CVD) remains the leading cause of death worldwide and, despite continuous advances, better diagnostic and prognostic tools, as well as therapy, are needed. The human transcriptome, which is the set of all RNA produced in a cell, is much more complex than previously thought and the lack of dialogue between researchers and industrials and consensus on guidelines to generate data make it harder to compare and reproduce results. This European Cooperation in Science and Technology (COST) Action aims to accelerate the understanding of transcriptomics in CVD and further the translation of experimental data into usable applications to improve personalized medicine in this field by creating an interdisciplinary network. It aims to provide opportunities for collaboration between stakeholders from complementary backgrounds, allowing the functions of different RNAs and their interactions to be more rapidly deciphered in the cardiovascular context for translation into the clinic, thus fostering personalized medicine and meeting a current public health challenge. Thus, this Action will advance studies on cardiovascular transcriptomics, generate innovative projects, and consolidate the leadership of European research groups in the field.COST (European Cooperation in Science and Technology) is a funding organization for research and innovation networks (www.cost.eu).
- Cigarette smoking, miR-27b downregulation, and peripheral artery disease : insights into the mechanisms of smoking toxicityPublication . Pereira-da-Silva, Tiago; Napoleão, Patricia; Costa, Marina C.; Gabriel, André F.; Selas, Mafalda; Silva, Filipa; Enguita, Francisco J.; Ferreira, Rui Cruz; Carmo, Miguel MotaCigarette smoking is a risk factor for the development of peripheral artery disease (PAD), although the proatherosclerotic mediators of cigarette smoking are not entirely known. We explored whether circulating microRNAs (miRNAs) are dysregulated in cigarette smokers and associated with the presence of PAD. Ninety-four participants were recruited, including 58 individuals without and 36 with PAD, 51 never smokers, 28 prior smokers, and 15 active smokers. The relative expression of six circulating miRNAs with distinct biological roles (miR-21, miR-27b, miR-29a, miR-126, miR-146, and miR-218) was assessed. Cigarette smoking was associated with the presence of PAD in multivariate analysis. Active smokers, but not prior smokers, presented miR-27b downregulation and higher leukocyte, neutrophil, and lymphocyte counts; miR-27b expression levels were independently associated with active smoking. Considering the metabolic and/or inflammatory abnormalities induced by cigarette smoking, miR-27b was independently associated with the presence of PAD and downregulated in patients with more extensive PAD. In conclusion, the atheroprotective miR-27b was downregulated in active smokers, but not in prior smokers, and miR-27b expression was independently associated with the presence of PAD. These unreported data suggest that the proatherogenic properties of cigarette smoking are mediated by a downregulation of miR-27b, which may be attenuated by smoking cessation.
- circRNA-miRNA cross-talk in the transition from paroxysmal to permanent atrial fibrillationPublication . Costa, Marina C.; Cortez-Dias, Nuno; Gabriel, André F.; De Sousa, João; Fiuza, Manuela; Gallego, Javier; Nobre, Ângelo; Pinto, Fausto J.; Enguita, Francisco J.Background: Atrial fibrillation (AF) is the most prevalent cardiac arrhythmia in western countries. The factors governing the progression of AF to a permanent chronic condition are still not well characterized. Among epigenetic factors, non-coding RNAs (ncRNAs) such as miRNAs and lncRNAs have been recently described as important players involved in the AF progression. We hypothesize about the existence of additional regulatory layers in AF involving an intricate cross-talk between different ncRNA species, namely miRNAs and circRNAs for the establishment of a chronic AF condition. Methods and results: We have performed an unbiased study analyzing the expression profile for miRNAs and circRNAs in left-atrial biopsies from patients with paroxysmal and permanent AF by RNA-seq. The transition from paroxysmal to permanent AF is characterized by a pattern of down-regulated miRNAs, concomitant to the appearance of specific circRNA species. The analysis of the sponging activities of the circRNAs exclusively expressed in permanent AF samples, allowed us to determine that they could be responsible for the downregulation of specific miRNAs in establishment of a permanent AF condition. Conclusion: Sponging activity of circRNAs sequestering specific miRNAs is an important factor to be considered for the determination of the molecular mechanisms involved in AF progression.
- Circular RNA-centered regulatory networks in the physiopathology of cardiovascular diseasesPublication . Gabriel, André F.; Costa, Marina C.; Enguita, Francisco J.Non-coding regulatory RNAs are generated as a core output of the eukaryotic genomes, being essential players in cell biology. At the organism level, they are key functional actors in those tissues and organs with limited proliferation capabilities such as the heart. The role of regulatory networks mediated by non-coding RNAs in the pathophysiology of cardiovascular conditions is starting to be unveiled. However, a deeper knowledge of the functional interactions among the diverse non-coding RNA families and their phenotypic consequences is required. This review presents the current knowledge about the functional crosstalk between circRNAs and other biomolecules in the framework of the cardiovascular diseases.
- Circular RNAs serve as miRNA sponges in breast cancerPublication . Misir, Sema; Hepokur, Ceylan; Aliyazicioglu, Yüksel; Enguita, Francisco J.Circular RNAs are a large group of non-coding RNAs with a closed-loop structure. circRNAs play significant roles in many biological processes as miRNA sponges, regulators for gene transcription, combining with RNA-binding proteins and translation of protein. Nowadays, circRNAs have become a research hotspot in the field of cancer and molecular biology. Accumulating evidences have indicated that circRNAs participate in the initiation and development of various cancers such as breast cancer. Breast cancer is a heterogeneous disease, which is the most common malignancy in women. The incidence and mortality rates of breast cancer indicate that it is the leading cause of cancer-related deaths. The goal of the present review is to introduce biogenesis, function characteristics and types of circRNAs, and also their biological functions on breast cancer, especially as miRNA sponges. Additionally, we discuss their use as a new therapeutic target for the treatment of breast cancer.
- Circulating mir-122-5p/mir-133b ratio is a specific early prognostic biomarker in acute myocardial infarctionPublication . Dias, Nuno Cortez; Costa, Marina C.; Silva, Doroteia; Jorge, Cláudia; Calisto, Carina; Pessoa, Teresa; Martins, Susana Robalo; Sousa, João Carvalho de; Silva, Pedro Canas da; Fiúza, Manuela; Diogo, António Nunes; Pinto, Fausto J.; Enguita, Francisco J.; Carrilho-Ferreira, PedroBackground: MicroRNAs (miRNAs) are key players in cardiovascular development and disease. However, not only miRNAs of a cardiac origin have a critical role in heart function. Recent studies have demonstrated that miR-122-5p, a hepatic miRNA, increases in the bloodstream during ischemic cardiogenic shock and it is upregulated in the infarcted myocardium. The aim of the present study was to determine the potential of circulating miR-122-5p as a biomarker for early prognostic stratification of ST-segment elevation acute myocardial infarction (STEMI) patients. Methods and Results: One hundred and forty-two consecutive STEMI patients treated with primary angioplasty were included in the study. Serum levels of miR-1-3p, -122-5p, -133a-3p, -133b, -208b-3p and -499a-5p were measured at the time of cardiac catheterization by quantitative polymerase chain reaction and related to in-hospital and long-term outcome. During a follow up of 20.8 months, 9 patients died, 6 had recurrence of myocardial infarction, and 26 patients suffered an adverse cardiovascular event. Event-free survival was significantly worse in patients with a higher miR-122-5p/133b ratio (3rd tertile distribution, above 1.42 Log(10)), having almost a 9-fold higher risk of death or myocardial infarction and a 4-fold higher risk of adverse cardiovascular events. Conclusions: This study showed that the miR-122-5p/133b ratio is a new prognostic biomarker for the early identification of STEMI patients at a higher risk of developing major adverse events after undergoing primary percutaneous coronary intervention.
- Circulating miRNAs are associated with the systemic extent of atherosclerosis : novel observations for miR-27b and miR-146Publication . Pereira da Silva, Tiago; Napoleão, Patricia; Costa, Marina C.; Gabriel, André F.; Selas, Mafalda; Silva, Filipa; Enguita, Francisco J.; Ferreira, Rui Cruz; Carmo, Miguel MotaThe mechanisms that regulate the systemic extent of atherosclerosis are not fully understood. We investigated whether the expression of circulating miRNAs is associated with the extent of stable atherosclerosis to a single territory or multiple territories (polyvascular) and with the severity of atherosclerosis in each territory. Ninety-four participants were prospectively recruited and divided into five age- and sex-matched groups: presenting no atherosclerosis, isolated coronary atherosclerosis, coronary and lower extremity atherosclerosis, coronary and carotid atherosclerosis, and atherosclerosis of the coronary, lower extremity, and carotid territories. The expression of six circulating miRNAs with distinct biological roles was assessed. The expression of miR-27b and miR-146 differed across groups (p < 0.05), showing a decrease in the presence of atherosclerosis, particularly in the three territories. miR-27b and miR-146 expression decreased in association with a higher severity of coronary, lower extremity, and carotid atherosclerosis. Polyvascular atherosclerosis involving the three territories was independently associated with a decreased miR-27b and miR-146 expression. Both miRNAs presented an area under the curve of ≥0.75 for predicting polyvascular atherosclerosis involving the three territories. To conclude, miR-27b and miR-146 were associated with the presence of severe polyvascular atherosclerosis and with the atherosclerosis severity in each territory. Both are potential biomarkers of severe systemic atherosclerosis.