From Endogenous to Synthetic microRNA-Mediated Regulatory Circuits: An Overview

MicroRNAs are short non-coding RNAs that are evolutionarily conserved and are pivotal post-transcriptional mediators of gene regulation. Together with transcription factors and epigenetic regulators, they form a highly interconnected network whose building blocks can be classified depending on the number of molecular species involved and the type of interactions amongst them. Depending on their topology, these molecular circuits may carry out specific functions that years of studies have related to the processing of gene expression noise. In this review, we first present the different over-represented network motifs involving microRNAs and their specific role in implementing relevant biological functions, reviewing both theoretical and experimental studies. We then illustrate the recent advances in synthetic biology, such as the construction of artificially synthesised circuits, which provide a controlled tool to test experimentally the possible microRNA regulatory tasks and constitute a starting point for clinical applications

Molecular Targets of CNS Tumors

Molecular Targets of CNS Tumors is a selected review of Central Nervous System (CNS) tumors with particular emphasis on signaling pathway of the most common CNS tumor types. To develop drugs which specifically attack the cancer cells requires an understanding of the distinct characteristics of those cells. Additional detailed information is provided on selected signal pathways in CNS tumors

Role of miRNAs in Cancer

MicroRNAs are the best representatives of the non-coding part of the genome and their functions are mostly linked to their target genes. During the process of carcinogenesis, both dysregulation of microRNAs and their target genes can explain the development of the disease. However, most of the target genes of microRNAs have not yet been elucidated. In this book, we add new information related to the functions of microRNAs in various tumors and their associated targetome

Computational Methods for the Analysis of Genomic Data and Biological Processes

In recent decades, new technologies have made remarkable progress in helping to understand biological systems. Rapid advances in genomic profiling techniques such as microarrays or high-performance sequencing have brought new opportunities and challenges in the fields of computational biology and bioinformatics. Such genetic sequencing techniques allow large amounts of data to be produced, whose analysis and cross-integration could provide a complete view of organisms. As a result, it is necessary to develop new techniques and algorithms that carry out an analysis of these data with reliability and efficiency. This Special Issue collected the latest advances in the field of computational methods for the analysis of gene expression data, and, in particular, the modeling of biological processes. Here we present eleven works selected to be published in this Special Issue due to their interest, quality, and originality

Étude de l'expression des microARNs et des enzymes de synthèse des corticostéroïdes dans le développement pulmonaire

Le syndrome de détresse respiratoire du nouveau-né (SDR) est l’une des pathologies les plus fréquentes dont souffrent les bébés prématurés. Le SDR est causé par un déficit dans la synthèse du surfactant pulmonaire en raison de l’immaturité du poumon lors d’une naissance prématurée. Plusieurs éléments régulent le développement pulmonaire notamment les stéroïdes sexuels et les corticostéroïdes. Le sexe est aussi un élément régulateur du développement pulmonaire. En effet, les garçons sont plus atteints que les filles par le SDR. Ce dimorphisme sexuel est attribué aux androgènes. Le traitement anténatal aux glucocorticoïdes est prescrit aux femmes qui sont à risque d’accoucher prématurément. En effet, les corticostéroïdes favorisent la maturation pulmonaire anténatale. Également, il a été démontré que les microARNs sont primordiaux pour le développement pulmonaire. Ceci nous a conduit à étudier l’impact des androgènes sur le profil d’expression des microARNs lors de la transition du stade canaliculaire au stade sacculaire (jour gestationnel (JG)17.0 au JG18.0), période qui coïncide avec la montée de la synthèse et de la sécrétion du surfactant chez la souris. Tout d’abord, nous avons étudié la stabilité des gènes de normalisation (snoRNAs) afin de quantifier les microARNs par qPCR. Cette analyse a été effectuée avec 3 logiciels différents et sur plusieurs stades du développement notamment de la période pseudoglandulaire jusqu’au stade alvéolaire chez les deux sexes. On a identifié les meilleures combinaisons de gènes de normalisation les plus stables pour chaque stade du développement étudié ainsi que pour la période couvrant tous les stades étudiés. Ensuite nous avons analysé à GD17.0 et GD18.0 le profil d’expression des microARNs chez des fœtus mâles dont les mères ont été traitées au flutamide (anti-androgènes pure). Les résultats ont montré que 43 microARNs matures sont modulés par les androgènes à GD17.0 et 35 microARNs à GD18.0. Pour certains microARNs, nous avons identifié des cibles potentielles qui sont inversement modulées par les androgènes par rapport aux microARNs. Ces cibles sont impliquées dans plusieurs processus biologiques tels que le métabolisme des lipides et la prolifération cellulaire ainsi que dans des fonctions moléculaires tels que la liaison des facteurs de transcription. Des expériences de validation ont été effectuées par qPCR. Nos résultats ont montré que les androgènes régulent des processus qui peuvent être impliqués dans la maturation pulmonaire via la régulation des microARNs. En plus de l’intérêt porté aux androgènes dans la maturation pulmonaire, nous avons analysé l’expression d’enzymes de synthèse des corticostéroïdes dans le poumon fœtal humain. L’expression de l’enzyme 21-hydroxylase a été étudiée par qPCR et par immunobuvardage. Également la localisation de l’ARNm de cette enzyme clé de la synthèse des glucocorticoïdes, a été effectuée par hybridation in situ. L’ARNm de CYP21A2 a été détecté par qPCR dans les 34 échantillons analysés et dont les âges variaient entre 17 et 40 semaines de grossesse. Aucune corrélation, avec l’âge gestationnel ou le sexe, n’a été observée. Des niveaux significatifs de la protéine 21-hydroxylase ont été détectés dans nos échantillons. Nous avons investigué l’expression d’autres enzymes impliquées dans la voie de synthèse des glucocorticoïdes notamment CYP11B1, CYP11B2 et CYP17A1. Les ARNm des gènes CYP11B1, CYP11B2 n’ont pas été détectés dans nos échantillons, contrairement à CYP17A1 dont l’ARNm a été détecté dans tous nos tissus fœtaux analysés. La protéine de la 17α-hydroxylase a été détectée à de faibles niveaux. Nos résultats d’hybridation in situ ont montré que l’expression de CYP21A2 est localisée presqu’exclusivement dans l’épithélium pulmonaire distal. Nos résultats suggèrent que les produits de la 21-hydroxylase agiront via une action intracrine sur l’épithélium distal en activant le récepteur des glucocorticoïdes (GR). L’activation du récepteur des minéralocorticoïdes (MR) ne semble pas dépendre de produits de la 21-hydroxylase en raison des quantités importantes d’aldostérone circulante.Respiratory distress syndrome of the newborn (RDS) is one of the most common diseases affecting preterm babies. RDS is caused by a deficiency in the synthesis and secretion of pulmonary surfactant as a result of lung immaturity caused by a premature birth. Several elements and factors regulate lung development including sex steroids and corticosteroids and the sex of the infant. In fact, boys are more affected than girls by RDS. This sexual dimorphism is attributed to the presence of androgens in male lungs. In contrast, corticosteroids are given to mother at higher risk to deliver prematurely to promote antenatal lung maturation of the fetuses. As other factors, it has been shown that microRNAs are essential to lung development. This led us to study the impact of androgen on the expression profile of microRNAs in the transition period between canalicular and saccular stages (gestational day (GD)17.0 and GD18.0). This period overlap the surge of surfactant synthesis in the mouse. First, we studied the stability of normalization genes (snoRNAs) to quantify microRNAs by qPCR. This analysis was performed by 3 methods of calculation at several stages of lung development from the pseudoglandular to the alveolar stages and this for both sexes. We identified the best combinations of the most stable normalization genes for each individual developmental stage studied as well as for the period covering all the studied stages. Then, we analyzed the expression profile of microRNAs on GD17.0 and GD18.0 in male fetuses whose mothers were treated with flutamide (pure anti-androgen). The results showed that 43 mature microRNAs are modulated by endogenous androgens on GD17.0 whereas 35 microRNAs on GD18.0. We have identified some microRNAs and potential targets that are inversely modulated by androgens compared with microRNAs. These targets are involved in several biological processes such as lipid metabolism and cell proliferation as well as in molecular functions such as transcription factor binding. Validation experiments were performed by qPCR. Our results showed that androgens regulate processes that may be involved in lung maturation via the regulation of microRNAs. In addition to the interest in the impact of androgens on lung maturation, we analyzed the expression of corticosteroid synthesis enzymes in the human fetal lung. Expression of the CYP21A2 and the presence of its corresponding 21-hydroxylase enzyme have been studied by qPCR and immunoblot. Also mRNA localization of this key enzyme in the synthesis of glucocorticoids has been also assessed by in situ hybridization. CYP21A2 mRNA was detected by qPCR in all the 34 analyzed samples, whose ages ranged between 17 and 40 weeks of pregnancy. No correlation with gestational age or sex was observed. Significant levels of 21-hydroxylase protein were detected in our samples. We investigated the expression of other enzymes involved in the pathway of glucocorticoid synthesis including CYP11B1, CYP11B2 and CYP17A1. CYP11B1, CYP11B2 mRNA were not detected in our samples, unlike CYP17A1 whose mRNA was detected in all our analyzed fetal tissues. CYP17A1 protein was detected at low levels. In situ hybridization data showed that CYP21A2 expression is localized almost exclusively in the distal epithelium of human fetal lung. Our results suggest that 21-hydroxylase products act via an intracrine action on the distal epithelium by activating the glucocorticoid receptor (GR). Activation of the mineralocorticoid receptor (MR) at this site does not seem to depend on the 21-hydroxylase products due to the large amounts of circulating aldosterone

Vergleich der miRNA-Expressionsmuster in Primärtumoren und Fernmetastasen des klarzelligen Nierenzellkarzinoms

Das NZK ist der dritthäufigste Tumor des Urogenitaltraktes und das kzNZK mit 75% der häufigste Subtyp des NZKs. Patienten mit einem NZK besitzen zu 30% bereits zum Diagnosezeitpunkt Metastasen und 30-50% entwickeln im weiteren Verlauf Metastasen. Die Metastasierung stellt den wichtigsten prognoselimitierenden Faktor dar. Um das individuelle Metastasierungsrisiko bestimmen zu können, sind bisher keine hinreichenden klinischen und histopathologischen Parameter vorhanden. MiRNAs sind nicht kodierende einzelsträngige RNA-Moleküle mit einer Länge von ca. 22 nt. Den miRNAs wird eine wichtige Rolle bei der Tumorentstehung, dem -progress und für die Metastasierung zugesprochen. Somit sind miRNAs potentielle Biomarker für Diagnostik und Prognosebewertung von Tumoren. Zu Beginn der Promotionsarbeit war für verschiedene Karzinome die metastasierungsassoziierte Funktion von miRNAs bereits belegt. Allerdings lagen für das NZK zur Bedeutung von miRNAs im Prozess der Metastasierung wenige Daten vor. Es konnte gezeigt werden, dass die Expression von verschiedenen miRNAs in metastasierten und nicht metastasierten Primärtumoren different ist. Allerdings wurden die Expressionsprofile von miRNAs nicht in Fernmetastasen untersucht, sodass das Ziel der vorliegenden Arbeit darin bestand, die Hauptmetastasierungsorte hinsichtlich der miRNA-Expression spezifischer miRNAs zu untersuchen und ortsspezifische Gemeinsamkeiten und Unterschiede zu explorieren, um somit ein besseres Verständnis über den Prozess der Metastasierung zu gewinnen

Interacción entre las vías de HGF y TGF-ß en células progenitoras adultas hepáticas y células de hepatocarcinoma

Tesis inédita de la Universidad Complutense de Madrid, Facultad de Farmacia, Departamento de Bioquímica y Biología Molecular, leída el 11-10-2019Chronic liver diseases (CLDs) are associated with fibrosis, which eventually progress to cirrhosis and ultimately to hepatocellular carcinoma (HCC) development, constituting a major global health problem. In the context of chronic liver injury where the proliferative capacity of adult hepatic cells is impaired, the population of adult hepatic progenitor cells (HPCs), also known as oval cells in rodents, takes over the regenerative process. Upon activation, HPCs/oval cells expand, proliferate and migrate into liver parenchyma and due to their bipotential nature differentiate into hepatocytes and cholangiocytes compensating the cellular loss and maintaining liver functionality. However, some authors give HPCs/oval cells a pro-fibrotic role, establishing a direct relationship between the HPCs/oval cell expansion and the severity of thefibrosis. They could also be the cells of origin of a subset of HCC. It is therefore evident that the signals and mechanisms regulating HPC/oval cell biology and function need to be clarified not only because of their potential utility in regenerative medicine, but also because of their still uncertain role in the aforementioned diseases..Las enfermedades hepáticas crónicas (CLD) están asociadas con fibrosis, que eventualmente progresa a cirrosis, y en último término al desarrollo de un carcinoma hepatocelular (HCC), y constituyen un importante problema de salud global. En este contexto de daño hepático crónico en el que la capacidad regenerativa de las células maduras hepáticas se ve comprometida, es la población de células progenitoras adultas (HPCs), también conocidas como células ovales en modelos murinos, la que va a tomar las riendas del proceso de regeneración hepática. Tras su activación, las HPCs/células ovales se expanden,proliferan y migran en el parénquima hepático y gracias a su naturaleza bipotencial se diferencian a hepatocitos y colangiocitos, compensando así la pérdida de masa hepática y manteniendo la funcionalidad hepática. Sin embargo, algunos autores dotan a las HPCs/células ovales de un papel profibrótico, estableciendo una relación directa entre su expansión y la severidad de la fibrosis. Estas células también pueden ser el origen celular de algunos subtipos de HCC. Resulta por tanto evidente la necesidad de estudiar las señales y mecanismos que regulan la biología y la función de estas células, no solo por su potencial utilidad en medicina regenerativa sino también por su papel aún no claro en las ya mencionadas enfermedades hepáticas...Sección Deptal. de Bioquímica y Biología Molecular (Farmacia)Fac. de FarmaciaTRUEunpu

Oncogene and Cancer

This book describes a course of cancer growth starting from normal cells to cancerous form and the genomic instability, the cancer treatment as well as its prevention in form of the invention of a vaccine. Some diseases are also discussed in detail, such as breast cancer, leucaemia, cervical cancer, and glioma. Understanding cancer through its molecular mechanism is needed to reduce the cancer incidence. How to treat cancer more effectively and the problems like drug resistance and metastasis are very clearly illustrated in this publication as well as some research result that could be used to treat the cancer patients in the very near future. The book was divided into six main sections: 1. HER2 Carcinogenesis: Etiology, Treatment and Prevention; 2. DNA Repair Mechanism and Cancer; 3. New Approach to Cancer Mechanism; 4. New Role of Oncogenes and Tumor Suppressor Genes; 5. Non Coding RNA and Micro RNA in Tumorigenesis; 6. Oncogenes for Transcription Factor

In silico and molecular validation of identified putative genes and functional analysis of a N K G2D ligand as a breast cancer biomarkers

Philosophiae Doctor - PhDThe current diagnostic, prognostic, predictive and therapeutic monitoring methods used for breast cancer are limited. Thus, research into more specific, sensitive and effective strategies is required. Breast cancer is the most prevalent form of cancer in women worldwide and accounts for the most common cause of death in women every year. Cancer development is characterized by a wide spread of genetic abnormalities of gene sequences that can be used in detecting and monitoring treatment of the disease as a result of altered gene expression patterns which leave a trail of biomarkers. Seven candidate genes (Gene 1-7) were identified from a previous in silico study and their gene products (BRG 1-7) were annotated to be good candidate breast cancer biomarkers. Differential gene expression analysis using quantitative real-time PCR (qRT-PCR) validated the over-expression of Gene 3, Gene 4 and Gene 7 in a breast cancer cell line (MCF7), of which Gene 7, annotated as a Natural killer group 2, member D (NKG2D) ligand, was observed to be the most over-expressed gene. The innate immune system is the first line of the body's physiological defense against diseases and the natural killer (NK) cells, are central to mediating this type of immunity. NK cells are activated when a specific surface receptor such as the NKG2D receptor binds its ligands expressed by tumor cells. To evade being detected by the immune system, cancer cells are reported to shed off the NKG2D ligands and are expected to be present in the bodily fluids of cancer patients. Also, chemotherapeutics have been reported to suppress the natural anti-tumour immune response, thus should be taken into account when designing optimal therapy for cancer patients. The aim of this research was to validate these candidate genes as effective breast cancer biomarkers using several in silico methods as well as molecular techniques and study the effect of Gene 7 on modulating the effect of several pro-apoptotic compounds. The in silico part of the study investigated the functional, protein interaction, pathways, and tissue expression specificity of the candidate biomarkers using computational software such as DAVID, STRING, KEGG, Genecards and GEA. Also an in silico validation of the prognostic/predictive values of the genes was analysed using SurvExpress, KMplot, and GOBO. Protein expression of selected genes was analysed by Western blot, and immunofluorescence analysis. BRG 7 gene was cloned into pcDNA3.1 vector using recombinant DNA technology while commercial shRNA construct was used to 'knock-down' Gene 7 expression. The two constructs were used to transfect MCF-7 and MCF-12A cells. Over-expression and 'knock down' Gene 7 in transfected cells was confirmed using western blot analysis. Stably transfected cells were then treated with three pro-apoptotic compounds (Camptothecin, Doxorubicin and DMSO) for 24 hours. The apoptotic cells were stained with 3, 4, 5, 6-tetrachloro-2', 4', 5', 7' tetraiodofluorescein (TCTF) and then analysed using flow cytometry. Functional analysis linked Gene 1, Gene 2, Gene 4, Gene 6 and Gene 7 to different cancer related processes. The pathway analysis showed Gene 1, Gene 2, Gene 4 and Gene 7 were involved in pathways that can be linked to cancer modulation. The protein-protein interaction analysis showed only BRG 2 was directly linked to two major hallmarks of cancer (Apoptosis and Autophagy). Breast cancer associated Transcription factors were shown to regulate these genes. Gene 1 and Gene 5 as well as the three genes observed to be highly expressed in the qRT-PCR study were validated to differentially express in breast cancer. An additional protein (BRG 8) was identified and postulated to be a good biomarker candidate for breast cancer based on its direct interaction with BRG 7 and estrogen receptor protein (ESR). The prognostic value of the candidate genes were monitored in two datasets (DATA1 and DATA2) in SurvExpress. DATA1 showed that Gene 6 and Gene 8 while DATA2 showed that Gene 3, Gene 6 and Gene 7 were valuable candidate genes in breast cancer prognosis. The survival curves from the two datasets showed the combined genes could predict the outcome of breast cancer patients undergoing treatments. A plot box output from SurvExpress showed most of the genes were differentially expressed comparing two risk groups. The Kaplan Meier plotter confirmed, Gene 1, Gene 3, Gene 4 and Gene 7 have a significant P-value in predicting the survival outcome based on gene differential expression value. GOBO analysis showed the genes may accurately predict the survival outcome of estrogen positive subtype, ERBB2 subtype of estrogen receptor negative and lymph node negative subtype of ER- tumours, but not all subtype of ER- tumours. Western blot analysis showed BRG 7 may be highly expressed in MCF-7 as compared to MCF-12A, BRG 8 was found to be expressed in all cancer cell types analyzed except for MCF-7 and HT29. BRG 2 was found to be expressed in all cancer types analyzed. immunofluorescence analysis showed BRG 3, BRG 4 and BRG 7 are differentially expressed in breast cancer cell line and are more localized on the cell membrane when compared to the breast non-cancer cell line. Over-expression and gene knock down in cells were successfully confirmed with Western blot analysis. Stably transfected MCF-12A cell for over-expression of BRG7 protein, resulted in cell senescent and the cell stopped growing while stably transfected MCF-7 over-expressing BRG7 did not show any morphological changes. Apoptosis was enhanced in cells treated with camptothecin, doxorubicin and DMSO overexpressing BRG7. Apoptosis was reduced in camptothecin and DMSO treated gene 'knock-down' cells but not doxorucin treated. BRG7 gene 'knock down' in transfected cells showed varying response to all three pro-apoptotic compounds. From this study Gene 3, 5, 7 and 8 and their protein levels were confirmed to be differentially expressed in breast cancer cells and could serve as putative biomarkers for breast cancer. However the variance in the effectiveness of individual genes suggests that the set of genes would perform better than individual gene. The modulating role of BRG7 in drug induced apoptosis, suggest it could probably play an important role in personalised medicine and could serve as a biomarker to monitor the prognosis and/or therapeutic outcome of pro-apoptotic drugs in breast cancer patients. These findings will be further investigated in human breast tissues to validate these data