63 Bovine embryo-secreted microRNA-30c negatively regulates cell cycle progression through downregulation of CDK12
X. Lin A , E. Beckers A , S. Mc Cafferty A , J. P. Catani A , K. J. Szymanska B , A. Van Soom C and L. Peelman AA Department of Nutrition, Genetics and Ethology, Faculty of Veterinary Medicine, Ghent University, Merelbeke, Belgium;
B Physiology group, Department of Basic Medical Sciences, Ghent University, Ghent, Belgium;
C Reproduction, Obstetrics and Herd Health, Ghent University, Merelbeke, Belgium
Reproduction, Fertility and Development 31(1) 157-157 https://doi.org/10.1071/RDv31n1Ab63
Published online: 3 December 2018
Abstract
Identifying microRNA (miRNA) differentially secreted by intermediate (cleaved between 26.6-31.4 hpi) and slow cleaving bovine embryos (cleaved after 31.4 hpi), investigating how they influence embryo development and consequently if they can be used as biomarkers for bovine embryo development. MicroRNA collected from conditioned media (CM) of 167 individually cultured embryos were sequenced on an Illumina Miseq (Illumina, San Diego, CA, USA) after small RNA library construction with the Tailormix v2 kit (SeqMatic, Fremont, CA, USA) and compared with miRBase, using cow as primary organism, to identify known miRNA in all samples. To predict potential novel miRNA, the sequences were aligned against the annotated cow genome (GCA_000003055.3), structurally analysed, and compared with all other mammalian data in miRBase. Quantitative RT-PCR was used to validate miRNA sequencing results. Apoptosis staining was performed for functional analysis of the differentially expressed miRNA-30c (miR-30c). Luciferase reporter assay, Western blotting, and quantitative RT-PCR were used to validate one of the predicted target genes of miR-30c, CDK12. The DNA content analysis using propidium iodide staining followed by flow cytometry and a water-soluble tetrazolium-1 proliferation assay were performed together with ELISA to assess the effect of miR-30c on cell progression. Quantitative RT-PCR was used to assess mRNA levels of DNA damage response (DDR) genes. In total, 114 known and 180 potentially novel miRNA were found in bovine embryo CM. Seven differentially expressed miRNA were identified in the CM from embryos with different cleavage patterns and different quality: miR-30c, miR-10b, and miR-novel-66 were differentially expressed between slow and intermediate developing embryos; miR-10b, miR-novel-113, miR-novel-44, miR-novel-45, and miR-novel-139 were differentially expressed between blastocysts and degenerate embryos. We further focused on functional analysis of miR-30c. Exogeneous delivery of miR-30c mimics to cultured embryos resulted in an increased cell apoptosis. Additionally, transfer of miR-30c mimics to Madin-Darby bovine kidney cells (MDBK) reduced cell cycle progression/cell proliferation, while inhibition of miR-30c resulted in the opposite. In addition, knockdown of CDK12 caused significant decreases in mRNA levels of DDR genes BRCA1, FANCD2, FANCI, and ATR. These data were derived from 3 independent experiments and were considered significant with a P-value < 0.05. In vitro-cultured bovine embryos secrete miRNA in the culture medium depending on their developmental capacity. One of these miRNA, miR-30c, increases bovine embryo apoptosis when exogeneously delivered. In addition, miR-30c directly targets and down-regulates CDK12, indicating the possibility that delivery of miR-30c mimics influences cell cycle progression by inhibiting DDR pathways through regulating CDK12. These findings contribute to a better understanding of the mechanisms of secreted miRNA participation in intercellular communication.