Unknown,Transcriptomics,Genomics,Proteomics

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DEVELOPMENTAL MicroRNA EXPRESSION PROFILING OF MURINE EMBRYONIC OROFACIAL TISSUE

ABSTRACT: 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).

ORGANISM(S): Rattus norvegicus

SUBMITTER: Guy Brock

PROVIDER: E-GEOD-20880 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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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:MicroRNAs (miRNAs) are small non-coding RNAs that associate with Argonaute 2 protein to regulate gene expression at the post-transcriptional level in the cytoplasm. However, recent studies have reported that some miRNAs localize to and function in other cellular compartments. Mitochondria harbour their own genetic system that may be a potential site for miRNA-mediated post-transcriptional regulation. We aimed at investigating whether nuclear-encoded miRNAs can localize to and function in human mitochondria. To enable identification of mitochondrial-enriched miRNAs, we profiled the mitochondrial and cytosolic RNA fractions from the same HeLa cells by miRNA microarray analysis. Mitochondria were purified using a combination of cell fractionation and immunoisolation, and assessed for the lack of protein and RNA contaminants. We found 57 miRNAs differentially expressed in HeLa mitochondria and cytosol. Of these 57, a signature of 13 nuclear-encoded miRNAs was reproducibly enriched in mitochondrial RNA and validated by RT-PCR for hsa-miR-494, hsa-miR-1275 and hsa-miR-1974. This study provides the first comprehensive view of the localization of RNA interference components to the mitochondria. Our data outline the molecular bases for a novel layer of crosstalk between nucleus and mitochondria through a specific subset of human miRNAs that we termed ‘mitomiRs’. To assess whether nuclear-encoded miRNA are detectable in human mitochondria, we performed the following four steps approach. First, cultured HeLa cells were allowed to reach 80-100% confluence and subjected to fractionation in order to isolate the cytosolic fraction. From the same HeLa cells, mitochondria were isolated by immunomagnetic Anti-TOM22 MicroBeads from the Mitochondria Isolation Kit (Miltenyi Biotec). In total, six mitochondria preparations were perfromed, three of these were additionally treated with RNase A. Second, total RNA was extracted from the mitochondrial and cytosolic fractions. Third, mitochondrial and cytosolic RNA were respectively profiled by microRNA microarray analysis. Last, data were analyzed and normalized.Three independent assays were performed.

Project description:microRNAs, important regulators of cell proliferation and apoptosis, have been shown to be involved in the pathogenesis of acute myeloid leukemia in adulthood AML. However, comprehensive studies in AML of children and adolescents are missing so far. We investigated the miRNA expression profiles of different AML subtypes from 102 pediatric patients in comparison to CD34+ cells from healthy donors and adult AML patients, in order to identify differentially expressed miRNAs. Pediatric samples with core factor binding acute myeloid leukemia and promyelocytic leukemia could be distinguished from each other and MLL rearranged AML subtypes by 9 and 18 miRNAs, respectively. miR-126, -146a, -181a/b, -100, and miR-125b were identified as highest differentially expressed with marked difference of expression between pediatric and adulthood samples of the same cytogenetic subgroup. We next isolated the miRNA targeting complex from t(8;21) and t(15;17) cell line models and comprehensively identified bound miRNAs and targeted mRNAs by a newly devised immunoprecipitation assay followed by rapid microarray detection. Our findings indicate separate binding preferences for the four human Argonaute proteins. Subsequent bioinformatic analysis revealed a concerted action of different Ago proteins in the regulation of AML-relevant pathways, providing an experimental based database of miRNA-mRNA target interaction in Argonaute proteins. Ago-associated microRNAs: Co-immunoprecipitation in the acute myeloid leukemia cell line models, KASUMI-1 and NB4, of the four human Argonaute complexes using monoclonal antibodies and stringent washing conditions. We performed photo-activated UV cross-linking using 4M-bM-^@M-^Y-thioruidine before cell lysis. Unspecific binding to the bead matrix and to the Fc part of the monoclonal rat antibody were recorded and corrected by empty bead controls and an antibody isotype control. Argonaute-associated miRNAs/mRNAs and unspecific bound miRNAs/mRNAs of the isotype control were identified by microarray hybridization. These experiments were performed in triplicates. Ago-associated mRNAs: Co-immunoprecipitation in the acute myeloid leukemia cell line models, KASUMI-1 and NB4, of the four human Argonaute complexes using monoclonal antibodies and stringent washing conditions. We performed photo-activated UV cross-linking using 4M-bM-^@M-^Y-thioruidine before cell lysis. Unspecific binding to the bead matrix and to the Fc part of the monoclonal rat antibody were recorded and corrected by empty bead controls and an antibody isotype control. Argonaute-associated miRNAs/mRNAs and unspecific bound miRNAs/mRNAs of the isotype control were identified by microarray hybridization. These experiments were performed in triplicates. AML-patient microRNAs: RNA extraction using the TRIzol protocol (Invitrogen), labeling of RNA with Cy3- (universal reference) and Cy5-Dyes (samples) using a truncated and mutated RNA ligase, hybridization of miRNAs over night at 42M-BM-0C using miRXplore microarrays and the a-Hyb Hybridization Station (MACS molecular Miltenyi Biotec)

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DEVELOPMENTAL MicroRNA EXPRESSION PROFILING OF MURINE EMBRYONIC OROFACIAL TISSUE

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