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Regulatory microRNA network identification in bovine blastocyst development.

ABSTRACT: Mammalian blastocyst formation is characterized by two lineage segregations resulting in the formation of the trophectoderm, the hypoblast, and the epiblast cell lineages. Cell fate determination during these early lineage segregations is associated with changes in the expression of specific transcription factors. In addition to the transcription factor-based control, it has become clear that also microRNAs (miRNAs) play an important role in the post-transcriptional regulation of pluripotency and differentiation. To elucidate the role of miRNAs in early lineage segregation, we compared the miRNA expression in early bovine blastocysts with the more advanced stage of hatched blastocysts. Reverse transcription-quantitative PCR-based miRNA expression profiling revealed eight upregulated miRNAs (miR-127, miR-130a, miR-155, miR-196a, miR-203, miR-28, miR-29c, and miR-376a) and four downregulated miRNAs (miR-135a, miR-218, miR-335, and miR-449b) in hatched blastocysts. Through an integrative analysis of matching miRNA and mRNA expression data, candidate miRNA-mRNA interaction pairs were prioritized for validation. Using an in vitro luciferase reporter assay, we confirmed a direct interaction between miR-218 and CDH2, miR-218 and NANOG, and miR-449b and NOTCH1. By interfering with the FGF signaling pathway, we found functional evidence that miR-218, mainly expressed in the inner cell mass, regulates the NANOG expression in the bovine blastocyst in response to FGF signaling. The results of this study expand our knowledge about the miRNA signature of the bovine blastocyst and of the interactions between miRNAs and cell fate regulating transcription factors.

SUBMITTER: Goossens K

PROVIDER: S-EPMC3685315 | biostudies-literature | 2013 Jul

REPOSITORIES: biostudies-literature

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Regulatory microRNA network identification in bovine blastocyst development.

Goossens Karen K Mestdagh Pieter P Lefever Steve S Van Poucke Mario M Van Zeveren Alex A Van Soom Ann A Vandesompele Jo J Peelman Luc L

Stem cells and development 20130315 13

Mammalian blastocyst formation is characterized by two lineage segregations resulting in the formation of the trophectoderm, the hypoblast, and the epiblast cell lineages. Cell fate determination during these early lineage segregations is associated with changes in the expression of specific transcription factors. In addition to the transcription factor-based control, it has become clear that also microRNAs (miRNAs) play an important role in the post-transcriptional regulation of pluripotency an ...[more]

PMID: 23398486

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Project description:BackgroundNormal preimplantation embryo development encompasses a series of events including first cleavage division, activation of the embryonic genome, compaction and blastocyst formation. First lineage differentiation starts at the blastocyst stage with the formation of the trophectoderm and the inner cell mass. The main objective of this study was the detection, identification and expression analysis of genes associated with blastocyst formation in order to help us better understand this process. This information could lead to improvements of in vitro embryo production procedures.ResultsA subtractive cDNA library was constructed enriched for transcripts preferentially expressed at the blastocyst stage compared to the 2-cell and 8-cell stage. Sequence information was obtained for 65 randomly selected clones. The RNA expression levels of 12 candidate genes were determined throughout 3 stages of preimplantation embryo development (2-cell, 8-cell and blastocyst) and compared with the RNA expression levels of in vivo "golden standard" embryos using real-time PCR. The RNA expression profiles of 9 (75%) transcripts (KRT18, FN1, MYL6, ATP1B3, FTH1, HINT1, SLC25A5, ATP6V0B, RPL10) were in agreement with the subtractive cDNA cloning approach, whereas for the remaining 3 (25%) (ACTN1, COPE, EEF1A1) the RNA expression level was equal or even higher at the earlier developmental stages compared to the blastocyst stage. Moreover, significant differences in RNA expression levels were observed between in vitro and in vivo produced embryos. By immunofluorescent labelling, the protein expression of KRT18, FN1 and MYL6 was determined throughout bovine preimplantation embryo development and showed the same pattern as the RNA expression analyses.ConclusionBy subtractive cDNA cloning, candidate genes involved in blastocyst formation were identified. For several candidate genes, important differences in gene expression were observed between in vivo and in vitro produced embryos, reflecting the influence of the in vitro culture system on the embryonic gene expression. Both RNA and protein expression analysis demonstrated that KRT18, FN1 and MYL6 are differentially expressed during preimplantation embryo development and those genes can be considered as markers for bovine blastocyst formation.

Project description:BackgroundOocyte maturation and preimplantation embryo development are controlled by array of genes that are post-transcriptionally regulated by microRNAs. With respect to this, previously, we identified altered expression of microRNA-130b (miR-130b) during oocyte maturation. Here, we aimed to investigate the role of miR-130b in bovine granulosa and cumulus cell function, oocyte maturation and preimplantation embryo development using gain- and loss-of- function approach.MethodsFor this study, the granulosa cells, cumulus cells and the oocytes were collected from ovaries obtained from slaughterhouse. The genes targeted by miR-130b were identified using dual-luciferase reporter assay. The role of miR-130b in granulosa and cumulus cell function was investigated by increasing and inhibiting its expression in in vitro cultured cells using miR-130b precursor and inhibitor, respectively while the role of miR-130b on oocyte development, immature oocytes were microinjected with miR-130b precursor and inhibitor and the polar body extrusion, the proportion of oocytes reaching to metaphase II stage and the mitochondrial were determined in each oocyte group 22 h after microinjection. Moreover, to investigate the role of miR-130b during preimplantation embryo development, zygote stage embryos were microinjected with miR-130b precursor or inhibitor and the cleavage rate, morula and blastocyst formation was analyzed in embryos derived from each zygote group after in vitro culture.ResultsThe luciferase assay showed that SMAD5 and MSK1 genes were identified as the direct targets of miR-130b. Overexpression of miR-130b increased the granulosa and cumulus cell proliferation, while inhibition showed the opposite phenotype. Apart from these, modulation of miR-130b altered the lactate production and cholesterol biosynthesis in cumulus cells. Furthermore, inhibition of miR-130b expression during oocyte in vitro maturation reduced the first polar body extrusion, the proportion of oocytes reaching to metaphase II stage and the mitochondrial activity, while inhibition of miR-130b during preimplantation embryo development significantly reduced morula and blastocyst formation.ConclusionThis study demonstrated that in vitro functional modulation of miR-130b affected granulosa and cumulus cell proliferation and survival, oocyte maturation, morula and blastocyst formation suggesting that miR-130b is involved in bovine oocyte maturation and preimplantation embryo development.

Project description:BackgroundCircular RNA (circRNA) is a noncoding RNA that forms a closed-loop structure, and its abnormal expression may cause disease. We aimed to find potential network for circRNA-related competitive endogenous RNA (ceRNA) in atrial fibrillation (AF).MethodsThe circRNA, miRNA, and mRNA expression profiles in the heart tissue from AF patients were retrieved from the Gene Expression Omnibus database and analyzed comprehensively. Differentially expressed circRNAs (DEcircRNAs), differentially expressed miRNAs (DEmiRNAs), and differentially expressed mRNAs (DEmRNAs) were identified, followed by the establishment of DEcircRNA-DEmiRNA-DEmRNA regulatory network. Functional annotation analysis of host gene of DEcircRNAs and DEmRNAs in ceRNA regulatory network was performed. In vitro experiment and electronic validation were used to validate the expression of DEcircRNAs, DEmiRNAs, and DEmRNAs.ResultsA total of 1611 DEcircRNAs, 51 DEmiRNAs, and 1250 DEmRNAs were identified in AF. The DEcircRNA-DEmiRNA-DEmRNA network contained 62 circRNAs, 14 miRNAs, and 728 mRNAs. Among which, two ceRNA regulatory pairs of hsa-circRNA-100053-hsa-miR-455-5p-TRPV1 and hsa-circRNA-005843-hsa-miR-188-5p-SPON1 were identified. In addition, six miRNA-mRNA regulatory pairs including hsa-miR-34c-5p-INMT, hsa-miR-1253-DDIT4L, hsa-miR-508-5p-SMOC2, hsa-miR-943-ACTA1, hsa-miR-338-3p-WIPI1, and hsa-miR-199a-3p-RAP1GAP2 were also obtained. MTOR was a significantly enriched signaling pathway of host gene of DEcircRNAs. In addition, arrhythmogenic right ventricular cardiomyopathy, dilated cardiomyopathy, and hypertrophic cardiomyopathy were remarkably enriched signaling pathways of DEmRNAs in DEcircRNA-DEmiRNA-DEmRNA regulatory network. The expression validation of hsa-circRNA-402565, hsa-miR-34c-5p, hsa-miR-188-5p, SPON1, DDIT4L, SMOC2, and WIPI1 was consistent with the integrated analysis.ConclusionWe speculated that hsa-circRNA-100053-hsa-miR-455-5p-TRPV1 and hsa-circRNA-005843-hsa-miR-188-5p-SPON1 interaction pairs may be involved in AF.

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Regulatory microRNA network identification in bovine blastocyst development.

Publications

Regulatory microRNA network identification in bovine blastocyst development.

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