Computational frameworks for microRNA functional analysis of inter-kingdom and indirect targeting.

Genes are DNA sequences that encode the information needed to synthesize molecules necessary for the function of the cell. Some genes are called protein-coding genes because they have the code required to manufacture proteins. The expression of a certain gene means its product (protein) is produced. Although some genes are not protein-coding, they regulate the gene expression of other protein-coding genes. Of these, microRNAs (miRNAs) are small RNA molecules that inhibit the expression of other genes by binding to their mRNA transcripts. miRNAs have been shown to be linked to several biological processes like development and diseases like cancer. Recently, researchers have hypothesized that miRNAs are involved in the regulation of the expression of genes from other species. Although tools to predict miRNA target genes are available in the case when miRNAs and target genes belong to the same species, to our knowledge there are no available tools to predict inter-kingdom miRNA target genes (miRNA and target genes belong to two different kingdoms). To address this limitation, we developed an efficient tool to predict potential gut bacterial genes targeted by miRNAs from edible plants. We successfully predicted ginger miRNAs that target two genes from a gut bacterial strain called Lactobacillus rhamnosus GG. To maintain the efficiency of our tool while using a larger number of miRNAs and bacterial strains, we used a hash-table to index the sequences of bacterial genes. To predict the function of a miRNA, we start by compiling the list of direct target genes (ones with binding sites) and then we search for biological process in which these genes are enriched. This approach does not include other genes affected by the miRNA but do not necessarily have a physical binding site (indirect targets). An example of an indirect target is the gene that doesn’t have a binding site and is regulated through other direct targets like transcription factors. To overcome this limitation, we developed miRinGO an interactive web application to include these indirect targets in the functional analysis. Our approach showed better performance compared to the existing approach in predicting biological processes known to be targeted by certain miRNAs

Concentrations of Purine Metabolites Are Elevated in Human Fluids from Adults and Infants and in Livers from Mice Fed Diets Depleted of Bovine Milk Exosomes and their RNA Cargos

Since miRNAs discovery, evidence keeps accumulating on their role in human physiology for homeostasis and implications during disease development. Upon the discovery that miRNAs could be encapsulated in exosomes and provide protection, evidence keeps accumulating on the possibility for miRNAs to be transferred between distant cells and elicit gene expression changes, and clinical trials are being developed to identify miRNAs in body fluids that could predict disease outcomes based on miRNA encapsulation in exosomes making them stable in body fluids. Recently, studies have shown that dietary miRNAs encapsulated in exosomes can be absorbed in mice and humans, and on the other hand, milk consumption has been associated with lower uric acid concentrations and distinct benefits for human health, therefore the aims of this work were to determine whether milk exosome and RNA-depleted (ERD) and exosome and RNA-sufficient (ERS) diets could alter the concentrations of purine metabolites in mouse livers, and to determine whether diets depleted of bovine milk alter the plasma concentration and urine excretion of purine metabolites in adults and infants, respectively. Effects upon ERD diet were observed, hepatic purine metabolites in ERD fed mice were significantly higher compared to ERD controls. Additionally, plasma concentrations and urine excretion of purine metabolites were significantly higher in dairy avoiders and in infants fed soy milk, and finally purine hepatic gene expression in mice was significantly different between ERD and ERS. Comprehensively, diets depleted of bovine milk exosomes and RNA cargos caused increases in hepatic purine metabolites in mice, and in plasma and urine from adults and infants compared to exosome-sufficient controls. These findings are important since purines play a role in intermediary metabolism and cell signaling, therefore there is a possible link between milk benefits and dietary exosomes and their miRNA cargo transfer. Further research will need to address the underlying mechanisms that drive the purine concentration and enzyme gene expression changes upon ERD diets. Advisor: Janos Zemplen

Shedding light on the role of plant miRNAs in DNA damage response (DDR) and trans-kingdom transfer

One of the challenges that living organisms face is to respond promptly to genotoxic stress to avoid DNA damage. To this purpose, they developed complex DNA damage response (DDR) mechanisms. These mechanisms are highly conserved among organisms, including plants, and need to be finely regulated to take place properly. In this scenario, microRNAs are emerging as active players, thus attracting the attention of the research community. The involvement of miRNAs in DDR has been investigated prominently in human cells wherease studies on plants are still scarce. In addition, recently, miRNAs started to be envisioned as trans-kingdom molecules able to exert regulatory functions in evolutionary distant organisms. Particularly, attention is drawn to plant miRNAs ingested with the diet; evidence is accumulating on their ability to regulate genes in organisms other than the one in which they were synthesized, including humans and pathogens.In the present PhD thesis, different bioinformatics approaches have been developed aiming at identifying plant miRNAs along with their endogenous and cross-kingdom targets to pinpoint conserved pathways between evolutionary distant species. Alonside model organisms, the developed pipeline may find application on any species of interest to address species-specific cross-kingdom interactions or to performe large-scale investigations involving several plant/animal species. The emergence of DDR-related miRNAs in plants and humans constitutes fundamental informations obtained from these approaches.To experimentally investigate the involvement of plant miRNAs in the regulation of DDR-associated pathways, an ad hoc system was developed, using the model legume Medicago truncatula. Specific treatments with camptothecin (CPT) and/or NSC120686 (NSC) targeting compoments of DDR, namely topoisomerase I (Top1) and tyrosyl-DNA phosphodiesterase 1 (Tdp1), were used. These treatments, imposed to M. truncatula seeds for a 7-day time period, do not influence the germination process, but result in inhibition of seedling development, causing an increase in cell death and accumulation of DNA damage. To demonstrate that the imposed treatments had an effect on DDR, the expression of SOG1 (suppressor of gamma response 1) master-regulator was investigated by qRT-PCR. Importantly, a phylogenetic study demonstrated that M. truncatula possessed a small SOG1 gene family, composed by MtSOG1A and MtSOG1B genes. The expression of both genes was significantly enhanced in treatment-specific manner. Additionally, the espression of multiple genes playing important roles in different DNA repair pathways, cell cycle regulation and chromatin remodelling, were differentially expressed in a treatment-specific manner. Subsequently, specific miRNAs identifyed from the bioinformatics approach as targeting genes involved in DDR processes, were investigated along side their targets, thus providing a first step in their function validation.To investigate plant miRNAs trans-kingdom potential, additional studies were conducted using apple (M. domestica) since it can be eaten raw and hence, can be a better system for feeding trials. As a proof of concept, artificial miRNAs (amiRNAs) were delivered to human colorectal adenocarcinoma cells and the expression of these microRNAs and their in silico predicted targets were evaluated by qRT-PCR. Specifically, amiRNAs mimicking mdm-miR482a-3p and mdm-miR858 were transfected into HT-29 cell lines. After 72 h, amiRNAs were clearly detected inside the cells and the performed qRT-PCR analysis showed a significant downregulation of the IL4R (Interleukin 4 Receptor) gene, involved in promoting Th2 differentiation, suggesting the possibility of apple miRNAs to regulate the activity of human genes in vitro. Taken together, the results presented in the current PhD thesis demonstrate the involvement of plant miRNAs in DDR-associated processes as well as present evidence on the plant miRNAs trans-kingdom potential.One of the challenges that living organisms face is to respond promptly to genotoxic stress to avoid DNA damage. To this purpose, they developed complex DNA damage response (DDR) mechanisms. These mechanisms are highly conserved among organisms, including plants, and need to be finely regulated to take place properly. In this scenario, microRNAs are emerging as active players, thus attracting the attention of the research community. The involvement of miRNAs in DDR has been investigated prominently in human cells wherease studies on plants are still scarce. In addition, recently, miRNAs started to be envisioned as trans-kingdom molecules able to exert regulatory functions in evolutionary distant organisms. Particularly, attention is drawn to plant miRNAs ingested with the diet; evidence is accumulating on their ability to regulate genes in organisms other than the one in which they were synthesized, including humans and pathogens.In the present PhD thesis, different bioinformatics approaches have been developed aiming at identifying plant miRNAs along with their endogenous and cross-kingdom targets to pinpoint conserved pathways between evolutionary distant species. Alonside model organisms, the developed pipeline may find application on any species of interest to address species-specific cross-kingdom interactions or to performe large-scale investigations involving several plant/animal species. The emergence of DDR-related miRNAs in plants and humans constitutes fundamental informations obtained from these approaches.To experimentally investigate the involvement of plant miRNAs in the regulation of DDR-associated pathways, an ad hoc system was developed, using the model legume Medicago truncatula. Specific treatments with camptothecin (CPT) and/or NSC120686 (NSC) targeting compoments of DDR, namely topoisomerase I (Top1) and tyrosyl-DNA phosphodiesterase 1 (Tdp1), were used. These treatments, imposed to M. truncatula seeds for a 7-day time period, do not influence the germination process, but result in inhibition of seedling development, causing an increase in cell death and accumulation of DNA damage. To demonstrate that the imposed treatments had an effect on DDR, the expression of SOG1 (suppressor of gamma response 1) master-regulator was investigated by qRT-PCR. Importantly, a phylogenetic study demonstrated that M. truncatula possessed a small SOG1 gene family, composed by MtSOG1A and MtSOG1B genes. The expression of both genes was significantly enhanced in treatment-specific manner. Additionally, the espression of multiple genes playing important roles in different DNA repair pathways, cell cycle regulation and chromatin remodelling, were differentially expressed in a treatment-specific manner. Subsequently, specific miRNAs identifyed from the bioinformatics approach as targeting genes involved in DDR processes, were investigated along side their targets, thus providing a first step in their function validation.To investigate plant miRNAs trans-kingdom potential, additional studies were conducted using apple (M. domestica) since it can be eaten raw and hence, can be a better system for feeding trials. As a proof of concept, artificial miRNAs (amiRNAs) were delivered to human colorectal adenocarcinoma cells and the expression of these microRNAs and their in silico predicted targets were evaluated by qRT-PCR. Specifically, amiRNAs mimicking mdm-miR482a-3p and mdm-miR858 were transfected into HT-29 cell lines. After 72 h, amiRNAs were clearly detected inside the cells and the performed qRT-PCR analysis showed a significant downregulation of the IL4R (Interleukin 4 Receptor) gene, involved in promoting Th2 differentiation, suggesting the possibility of apple miRNAs to regulate the activity of human genes in vitro. Taken together, the results presented in the current PhD thesis demonstrate the involvement of plant miRNAs in DDR-associated processes as well as present evidence on the plant miRNAs trans-kingdom potential

MicroRNAs en el proceso de aclimatación aguda y crónica a la altitud

Los microRNAs (miRNAs) son transcritos cortos (22-25 nt) no codificantes que regulan la expresión génica mediante la unión a secuencias de mRNA complementarias. Un miRNA actúa sobre varios genes o varios miRNAs pueden regular el mismo gen, lo cual genera efectos cooperativos en la respuesta celular. Los “hipoxamiRs” son un grupo de miRNAs cuya expresión está relacionada con la condición de hipoxia. A la fecha, se han reportado aproximadamente 30 hipoxamiRs encargados de regular procesos relacionados con el metabolismo energético, diferenciación, proliferación, crecimiento y muerte celular. Aunque se ha estudiado el papel de los hipoxamiRs en patologías como el cáncer y las isquemias, su papel durante la aclimatación a la hipoxia hipobárica aún es incierto. En este trabajo se evaluaron algunas variables hematológicas y el patrón de expresión de 5 hipoxamiRs circulantes en plasma de individuos expuestos a hipoxia hipobárica (2600 msnm) durante un periodo de 4 meses. Para cada miRNA se establecieron las condiciones óptimas para la amplificación mediante la técnica de reacción en cadena de polimerasa en tiempo real (RT-qPCR). También se realizó el análisis bioinformático de cada miRNA para establecer su posible relación con los cambios presentes durante la aclimatación mediante redes de regulación. Con relación a las variables hematológicas se encontraron diferencias interindividuales y entre género, que son consistentes con los cambios presentes durante el proceso de aclimatación. Por otra parte, los miRNAs presentaron variaciones interindividuales, sin embargo, la tendencia de algunos miRNAs es similar entre los individuos a lo largo del seguimiento. El análisis bioinformático reveló que los 5 hipoxamiRs evaluados se encuentran estrechamente relacionados y tienen como blanco genes involucrados en procesos como la ubiquitinación de proteínas, la regulación metabólica, la inflamación y procesos derivados del incremento en las especies reactivas de oxígeno. Así mismo, se estableció que el componente central de regulación de estos hipoxamiRs es la vía de respuesta a estrés (MAPKs). Los resultados indican que los niveles plasmáticos de estos hipoxamiRs tienen relación con las adaptaciones fisiológicas que se presentan durante la aclimatación a la altitud, al tener como blanco genes que codifican para proteínas relacionadas con el control de la hipertensión pulmonar, la angiogénesis, la regulación de la función de HIF y de los líquidos corporales.Abstract: MicroRNAs (miRNAs) are short (22-25 nt) non-coding transcripts that regulate gene expression by binding to complementary mRNA sequences. A miRNA acts on several genes or several miRNAs can regulate the same gene, which generates cooperative effects in the cellular response. The "hipoxamiRs" are a group of miRNAs whose expression is related to the hypoxia condition. To date, approximately 30 hypoxamiRs have been reported to regulate processes related to energy metabolism, differentiation, proliferation, growth and cell death. Although the role of hypoxamids in pathologies such as cancer and ischemia has been studied, their role during acclimation to hypobaric hypoxia is still uncertain. In this work some hematological variables and the expression pattern of 5 circulating hypoxamyRs in plasma of individuals exposed to hypobaric hypoxia (2600 masl) during a period of 4 months were evaluated. For each miRNA optimal conditions for amplification were established by the real-time polymerase chain reaction (RT-qPCR) technique. The bioinformatic analysis of each miRNA was also performed to establish its possible relationship with the changes present during acclimation through regulation networks. Regarding the hematological variables, interindividual and gender differences were found, which are consistent with the changes present during the acclimation process. On the other hand, the miRNAs presented interindividual variations, however, the tendency of some miRNAs is similar among individuals throughout the follow-up. The bioinformatic analysis revealed that the 5 evaluated hypoxamiRs are closely related and target genes involved in processes such as protein ubiquitination, metabolic regulation, inflammation and processes derived from the increase in reactive oxygen species. Likewise, it was established that the central regulatory component of these hypoxamids is the stress response pathway (MAPKs). The results indicate that the plasma levels of these hypoxamiRs could be related to the physiological adaptations that occur during altitude acclimation, by targeting genes that code for proteins related to the control of pulmonary hypertension, angiogenesis, the regulation of the function of HIF and body fluids.Maestrí

小腸有機アニオン輸送体OATP2B1に及ぼす食品作用の多様性

13301甲第4990号博士（創薬科学）金沢大学博士論文本文Full 以下に掲載：Molecular Pharmaceutics 15(12) pp.5772-5780 2018. The American Chemical Society. 共著者：Daichi Fujita,Toshiki Arai, Hisakazu Komori, Yuma Shirasaki, Tomohiko Wakayama, Takeo Nakanishi, Ikumi Tama

Introduction to RNAi and miRNA pathways

Dráhy malých RNA jsou skupinou drah využívajících malé RNA k sekvenčně specifické represi. Tento soubor článků o drahách malých RNA má původ ve zprávě vypracované pro European Food and Safety Authority (EFSA) v letech 2016 a 2017. Text byl nově rozčleněn do dvanácti kapitol a doplněn úvody; vypuštěn byl naopak materiál podléhající autorskému právu třetích stran. Devět kapitol je věnováno drahám malých RNA ve zvířatech a rostlinách, zbývající tři připadají na obecný úvod a problematiku extracelulární RNA.Small RNA pathways or RNA silencing is a group of pathways, which utilize small RNAs as guides for sequence-specific repression. This collection of texts has an origin in a report prepared for the EFSA in 2016 and 2017. The original text was reorganized into twelve chapters, which were reformatted and revised in order to remove copyrighted material from third parties and provide a introductory parts for stand alone chapters. Nine of the chapters focus on small RNA pathways in animals and plants. The remaining three chapters include a general introduction and reviews of important phenomena – off-targeting and extracellular small RNAs

Additional file 5: of Role of plant MicroRNA in cross-species regulatory networks of humans

The result of grouping generic GO categories for Arabidopsis and human individually by CateGOrizer. (XLS 23 kb

Additional file 2: Figure S1. of Role of plant MicroRNA in cross-species regulatory networks of humans

Node weight assignment. (a) Node weight distribution of all 531 nodes. (b) Weight changes during iterations. The change was reduced to within 0.003 after 15 iterations. (PDF 8264 kb

Additional file 6: Figure S2. of Role of plant MicroRNA in cross-species regulatory networks of humans

Comparison between plant and human target distributions after the filtering process. (a) Original Arabidopsis targets. (b) Arabidopsis targets after screening. (c) Original Human targets. (d) Human targets after screening. There is a remarkable reduction of the noisy points between (a) and (b), and between (c) and (d), which strongly supports the effectiveness of our method and parameters, and provides a valid guide that can help explore the mechanism of cross-species miRNA targets. (PDF 8744 kb