Abstract
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.
Highlights
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 [1]
The examined DNMT1 30UTR element was found to be partially complementary to human, cattle, mice, and pig miR-148a (Fig 1A), suggesting that miR-148a is highly conserved among various species
In SCNT-derived embryos, DNMT1 is aberrantly expressed and mainly acts as a maintenance methyltransferase, which may contribute to the aberrant methylation status [26]
Summary
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 [1]. SCNT technology is still inefficient due to a variety of issues such as low cloning efficiency, fetal abnormalities, and placental deficiency [2]. 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 [3]. MicroRNA-148a improves the early development of porcine SCNT embryos decision to publish, or preparation of the manuscript
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