Date Published: June 30, 2017
Publisher: Public Library of Science
Author(s): Ping Wang, Xiangping Li, Lihua Cao, Shihai Huang, Haiyan Li, Yan Zhang, Ting Yang, Jianrong Jiang, Deshun Shi, Austin John Cooney.
Incomplete epigenetic reprogramming of donor cell nuclei is one of the main contributors to the low efficiency of somatic cell nuclear transfer (SCNT). To improve the success of SCNT, somatic cell DNA methylation levels must be reduced to those levels found in totipotent embryonic cells. Recent studies have demonstrated that miR-148a can affect DNA methylation via DNMT1 modulation in various cancers. Therefore, the focus of this study was to examine the influence of miR-148a on DNA methylation in donor cells and in SCNT embryo development. Thus, a stable cell line overexpressing miR-148a was established and used to produce SCNT embryos. Upon examination, DNMT1 was found to be a miR-148a target in porcine fetal fibroblasts (PFF). Furthermore, miR-148a overexpression in PFFs significantly decreased DNMT1 expression and global DNA methylation levels (P < 0.05). Moreover, miRNA-148a expression levels in SCNT embryos were significantly lower at the 2-cell and 4-cell stages when compared to IVF and parthenogenetic embryos. The group overexpressing miRNA-148a also showed a significant increase in blastocyst formation and total cell numbers (P < 0.05). Additionally, miR-148a overexpression altered the immunofluorescence signal of 5-mC and H3K9ac, and enhanced pluripotent gene (Oct4 and Nanog) expression levels during embryo development. These results indicate that miR-148a overexpression enhances the developmental potential of SCNT embryos and modifies epigenetic status.
Since the birth of the first cloned animal, Dolly, in 1996, somatic cell nuclear transfer (SCNT) has been successful in a variety of mammalian species . However SCNT technology is still inefficient due to a variety of issues such as low cloning efficiency, fetal abnormalities, and placental deficiency . One of the major causes of SCNT inefficiency is incomplete epigenetic reprogramming of the donor cell nuclei, which subsequently leads to aberrant gene expression during embryo development .
DNA methylation is very important for normal embryonic development and gene expression. In differentiated somatic cell nuclei, methylation levels are high [3, 24]. In order to obtain a high developmental competence, methylation levels must be reduced to those of a totipotent embryonic state . DNA methylation of CpG dinucleotides is mediated by DNMTs. In SCNT-derived embryos, DNMT1 is aberrantly expressed and mainly acts as a maintenance methyltransferase, which may contribute to the aberrant methylation status . In previous studies, DNMT1 knockdown has been shown to decreased DNA hypermethylation levels carried by donor cells in NT embryos . Furthermore, previous studies have indicated that using donor cells with low DNMT1 transcript levels could improve embryonic developmental competence relative to donor cells with high DNMT1 transcript levels. Collectively, these findings suggest that the developmental competence of NT embryos may be improved by reducing donor nuclei hypermethylation.