Project description:BACKGROUND: Orofacial development is a multifaceted process involving precise, spatio-temporal expression of a panoply of genes. MicroRNAs (miRNAs) constitute the largest family of noncoding RNAs involved in gene silencing, and represent critical regulators of cell and tissue differentiation. MicroRNA gene expression profiling is an effective means of acquiring novel and valuable information regarding the expression and regulation of genes, under the control of miRNA, involved in mammalian orofacial development. RESULTS: To identify differentially expressed miRNAs during mammalian orofacial ontogenesis, miRNA expression profiles from gestation day (GD) -12, -13 and -14 murine orofacial tissue were compared utilizing miRXplore™ microarrays from Miltenyi Biotech GmbH. TaqManTM quantitative Real-Time PCR was utilized for validation of gene expression changes. Cluster analysis of the microarray data was conducted with the clValid R package and the UPGMA (hierarchical) clustering method. Functional relationships between selected miRNAs were investigated using Ingenuity Pathway Analysis. Expression of over 26% of the approximately 588 murine miRNA genes examined was detected in murine orofacial tissues from GD 12, 13 and 14. Among these expressed genes several clusters were seen to be developmentally regulated. Differential expression of genes encoding miRNAs within such clusters were shown to target genes encoding proteins involved in cell proliferation, cell adhesion, differentiation, apoptosis and epithelial-mesenchymal transformation, all processes critical for normal orofacial development. Functional relationships between miRNAs differentially expressed were investigated using Ingenuity Pathway Analysis (IPA; Ingenuity Systems). CONCLUSIONS: Using miRNA microarray technology, unique gene expression signatures of hundreds of miRNAs in embryonic orofacial tissue were defined. Gene targeting and functional analysis revealed that the expression of numerous protein-encoding genes, crucial to normal orofacial ontogeny, may be regulated by specific miRNAs. Time-course experiment (Developmental Stages), ICR mice embryos on gestational days (GD) 12, 13 and 14. Biological replicates: For each day of gestation, 3 independent pools of 15 to 20 staged embryos were used to procure embryonic orofacial tissues for preparation of 3 distinct pools of RNA that were independently processed and applied to individual miRXplore™ microRNA Microarray chips (Miltenyi Biotec GmbH). Technology: 2-color spotted cDNA, Hy5 (experimental sample) vs. Hy3 (control - miRXplore Universal Reference).

Project description:BACKGROUND: Development of the neural tube is a highly orchestrated process relying on precise, spatio-temporal expression of numerous genes as well as hierarchies of signal transduction and gene regulatory networks. Disruption of expression of a number of genes participating in these networks is believed to underlie developmental anomalies such as neural tube defects (NTDs) resulting from anomalous neural tube morphogenesis. MicroRNAs (miRNAs), a large family of noncoding RNAs, have been shown to function as gene silencers, and thus, are key modulators of cell and tissue differentiation. To elucidate potential roles of miRNAs in murine neural tube development, miRNA gene expression profiling has been utilized in the current study, to garner novel and in-depth knowledge on the expression and regulation of genes encoding miRNAs as well as their potential target genes governing maturation of the mammalian neural tube. METHODS: With the aim of identifying differentially expressed miRNAs during mammalian neural tube ontogenesis, miRNA expression profiles from gestation day (GD) -8.5, -9.0 and -9.5 murine neural tube tissue were compared utilizing miRXplore™ microarrays from Miltenyi Biotech GmbH. Gene expression changes observed in microarray analysis were verified by TaqManTM quantitative Real-Time PCR. clValid R package and the UPGMA (hierarchical) clustering method were utilized for cluster analysis of the microarray data. Functional associations among selected miRNAs were examined exploiting Ingenuity Pathway Analysis. RESULTS: Expression of approximately 12% of the 609 murine miRNA genes examined was detected in murine neural tube tissues from GD -8.5, -9.0 and -9.5. Clustering analysis revealed several developmentally regulated clusters among these expressed genes. MicroRNA target analysis enabled identification of a panoply of protein-coding target genes of the differentially expressed miRNAs within such clusters. Interestingly, many of these target genes have been shown to be associated with vital cellular processes such as cell proliferation, cell adhesion, cell migration, differentiation, apoptosis and epithelial-mesenchymal transformation, all of which are essential for normal neural tube development. Utilization of Ingenuity Pathway Analysis (IPA; Ingenuity Systems) allowed identification of interactive biological networks connecting differentially expressed miRNAs and their target genes highlighting functional relationships. CONCLUSIONS: In the present study, a unique gene expression signature of a range of miRNAs in embryonic neural tube tissue was delineated. Analysis of miRNA target genes and gene interaction pathways emphasized that expression of numerous protein-encoding genes, indispensable for normal neural tube morphogenesis, may be regulated by specific miRNAs. Time-course experiment (Developmental Stages), ICR mice embryos on gestational days (GD) 8.5, 9.0 and 9.5. Biological replicates: For each day of gestation, 3 independent pools of 15 to 20 staged embryos were used to procure embryonic orofacial tissues for preparation of 3 distinct pools of RNA that were independently processed and applied to individual miRXplore™ microRNA Microarray chips (Miltenyi Biotec GmbH). Technology: 2-color spotted cDNA, Hy5 (experimental sample) vs. Hy3 (control - miRXplore Universal Reference).

Project description:BACKGROUND: Development of the neural tube is a highly orchestrated process relying on precise, spatio-temporal expression of numerous genes as well as hierarchies of signal transduction and gene regulatory networks. Disruption of expression of a number of genes participating in these networks is believed to underlie developmental anomalies such as neural tube defects (NTDs) resulting from anomalous neural tube morphogenesis. MicroRNAs (miRNAs), a large family of noncoding RNAs, have been shown to function as gene silencers, and thus, are key modulators of cell and tissue differentiation. To elucidate potential roles of miRNAs in murine neural tube development, miRNA gene expression profiling has been utilized in the current study, to garner novel and in-depth knowledge on the expression and regulation of genes encoding miRNAs as well as their potential target genes governing maturation of the mammalian neural tube. METHODS: With the aim of identifying differentially expressed miRNAs during mammalian neural tube ontogenesis, miRNA expression profiles from gestation day (GD) -8.5, -9.0 and -9.5 murine neural tube tissue were compared utilizing miRXplore™ microarrays from Miltenyi Biotech GmbH. Gene expression changes observed in microarray analysis were verified by TaqManTM quantitative Real-Time PCR. clValid R package and the UPGMA (hierarchical) clustering method were utilized for cluster analysis of the microarray data. Functional associations among selected miRNAs were examined exploiting Ingenuity Pathway Analysis. RESULTS: Expression of approximately 12% of the 609 murine miRNA genes examined was detected in murine neural tube tissues from GD -8.5, -9.0 and -9.5. Clustering analysis revealed several developmentally regulated clusters among these expressed genes. MicroRNA target analysis enabled identification of a panoply of protein-coding target genes of the differentially expressed miRNAs within such clusters. Interestingly, many of these target genes have been shown to be associated with vital cellular processes such as cell proliferation, cell adhesion, cell migration, differentiation, apoptosis and epithelial-mesenchymal transformation, all of which are essential for normal neural tube development. Utilization of Ingenuity Pathway Analysis (IPA; Ingenuity Systems) allowed identification of interactive biological networks connecting differentially expressed miRNAs and their target genes highlighting functional relationships. CONCLUSIONS: In the present study, a unique gene expression signature of a range of miRNAs in embryonic neural tube tissue was delineated. Analysis of miRNA target genes and gene interaction pathways emphasized that expression of numerous protein-encoding genes, indispensable for normal neural tube morphogenesis, may be regulated by specific miRNAs.

Project description:Schmitz2014 - RNA triplex formation The model is parameterized using the parameters for gene CCDC3 from Supplementary Table S1. The two miRNAs which form the triplex together with CCDC3 are miR-551b and miR-138. This model is described in the article: Cooperative gene regulation by microRNA pairs and their identification using a computational workflow. Schmitz U, Lai X, Winter F, Wolkenhauer O, Vera J, Gupta SK. Nucleic Acids Res. 2014 Jul; 42(12): 7539-7552 Abstract: MicroRNAs (miRNAs) are an integral part of gene regulation at the post-transcriptional level. Recently, it has been shown that pairs of miRNAs can repress the translation of a target mRNA in a cooperative manner, which leads to an enhanced effectiveness and specificity in target repression. However, it remains unclear which miRNA pairs can synergize and which genes are target of cooperative miRNA regulation. In this paper, we present a computational workflow for the prediction and analysis of cooperating miRNAs and their mutual target genes, which we refer to as RNA triplexes. The workflow integrates methods of miRNA target prediction; triplex structure analysis; molecular dynamics simulations and mathematical modeling for a reliable prediction of functional RNA triplexes and target repression efficiency. In a case study we analyzed the human genome and identified several thousand targets of cooperative gene regulation. Our results suggest that miRNA cooperativity is a frequent mechanism for an enhanced target repression by pairs of miRNAs facilitating distinctive and fine-tuned target gene expression patterns. Human RNA triplexes predicted and characterized in this study are organized in a web resource at www.sbi.uni-rostock.de/triplexrna/. This model is hosted on BioModels Database and identified by: BIOMD0000000530. To cite BioModels Database, please use: BioModels Database: An enhanced, curated and annotated resource for published quantitative kinetic models. To the extent possible under law, all copyright and related or neighbouring rights to this encoded model have been dedicated to the public domain worldwide. Please refer to CC0 Public Domain Dedication for more information.

Project description:Currently, micro RNAs (miRNAs) constitute a promising models for cell-to-cell communication since they are transferred between cells to execute essential roles in many processes. Their structure and size, in addition to various transport mechanisms, allow them to remain stable within various biological fluids, surviving in extremely adverse conditions, including low pH, boiling, and freezing. The presence of miRNAs in reproductive fluids, such as follicular, uterine and seminal fluid, indicate potential roles in the reproductive system. These extracellular miRNAs can be transported by lipoproteins (both HDL and LDL) or other proteins, including Argonaute2 (AGO2) and nucleophosmin1 (NPM1). Another transport system is mediated by extracellular vesicles (EVs), such as apoptotic bodies, microvesicles (MVs) and/or exosome-like vesicles. EVs protect miRNAs from degradation and contribute to their stability within biological fluids. Furthermore, EVs can transport a wide range of components packaged in a selective way. For instance, Squadrito et al. (2014), used macrophages and endothelial cells to demonstrate that the sorting of miRNAs into EVs for heterotrophic cell communication is altered by both, the presence of target transcripts and the self-presence of the respectively miRNA. In addition, the signature of miRNAs found in the exosomes significantly differed for those detected in the parent cells. To date, mechanisms controlling the specific loading of miRNAs into exosomes remain unclear. Indeed, several mechanisms may govern exosome sorting of specific subsets of miRNAs. MiRNA sorting appears to be influenced by different pathways and molecules in different cell types and tissues, and miRNAs contain well defined motifs (i.e., EXOmotifs), that direct the miRNA allocation into exosomes before delivery into recipient cells. A recent study showed that this RNA sequence can be recognized by the sumoylated form of the heterogeneous ribonucleoprotein A2B1 (hnRNPA2B1). Moreover, a terminal 30 nucleotide addition in miRNAs affects their selective sorting in B cells. Another hypothesis suggests that RNAs are selectively sorted depending on the differential affinity of RNA motifs towards the raft-like region of the cytoplasmic surface of microvesicular body (MVB) limiting membranes. Current data indicate that cells can communicate with each other through the transfer of miRNA-loaded exosomes. For example, monocyte-derived exosomes deliver miR-150 to endothelial cells and enhance endothelial cell migration by reducing c-myb expression. The miRNA content of exosomes plays a critical role in such cell-to-cell communication and determines the fate of recipient cells. Thus, exosomes derived from the bone marrow mesenchymal stromal cells of myeloma patients promote tumor growth depending on the content of miR-15a in exosomes. Our group has described a novel cell-to-cell communication mechanism involving the delivery of endometrial miRNAs from the maternal endometrium to the trophectoderm cells of preimplantation embryos. Specifically, in B6C3 derived mouse embryos, we found EV-associated and free miR-30d to cause overexpression of genes involved in embryonic adhesion processes, including Itb3, Itga7 and Cdh5. Furthermore, supplementing murine embryos with miR-30d significantly improved embryo adhesion, suggesting that external miRNAs may have a functional role as transcriptomic modifiers of preimplantation embryos. Based on profiling of miRNAs in endometrial fluid, maternally-derived miRNAs are present within EVs in the uterine microenvironment. The internalization of maternally-derived exosomes has been visualized, but the mechanism by which miR-30d becomes incorporated into exosomes remains unknown. The present study aimed to elucidate the underlying mechanism of hsa-miR-30d transfer from human endometrial epithelial cells (hEECs) to the interior of exosomes and eventually to early-stage blastocysts, using a mir-30d knockout murine mode.

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DEVELOPMENTAL MicroRNA EXPRESSION PROFILING OF MURINE EMBRYONIC OROFACIAL TISSUE

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