MicroRNA-148a overexpression improves the early development of porcine somatic cell nuclear transfer embryos.
MicroRNA-148a overexpression improves the early development of porcine somatic cell nuclear transfer embryos.
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MicroRNA-148a过表达改善猪体细胞核移植胚胎的早期发育
DOI：
10.1371/journal.pone.0180535
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发表时间：
2017
期刊：
影响因子：
3.7
通讯作者：
Shi D
中科院分区：
文献类型：
--
作者：
Wang P;Li X;Cao L;Huang S;Li H;Zhang Y;Yang T;Jiang J;Shi D
供体细胞核的表观遗传重编程不完全是体细胞核移植（SCNT）效率低下的主要原因之一。为了提高SCNT的成功率，体细胞DNA甲基化水平必须降低到全能胚胎细胞中的水平。最近的研究表明，miR-148a可以通过DNMT1调节多种癌症中的DNA甲基化。因此，本研究的重点是检查miR-148a对供体细胞和SCNT胚胎发育中DNA甲基化的影响。因此，建立了过表达miR-148a的稳定细胞系并用于产生SCNT胚胎。经检查，发现DNMT1是猪胎儿成纤维细胞（PFF）中的miR-148 a靶标。miR-148a过表达可显著降低PFF中DNMT1的表达和DNA甲基化水平（P <0.05）。此外，与IVF和孤雌生殖胚胎相比，SCNT胚胎中的miRNA-148 a表达水平在2细胞和4细胞阶段显著较低。过表达miRNA-148a组的囊胚形成率和总细胞数也显著增加（P <0.05）。此外，miR-148a过表达改变了5-mC和H3K9ac的免疫荧光信号，并增强了胚胎发育过程中多能基因（Oct4和Nanog）的表达水平。这些结果表明，miR-148a过表达增强了SCNT胚胎的发育潜力，并改变了表观遗传状态。
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.