Regulatory microRNA Network Identification in Bovine Blastocyst Development
Regulatory microRNA Network Identification in Bovine Blastocyst Development
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DOI：
10.1089/scd.2012.0708
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发表时间：
2013-07-01
影响因子：
4
通讯作者：
Peelman, Luc
中科院分区：
文献类型：
--
作者：
Goossens, Karen;Mestdagh, Pieter;Peelman, Luc
哺乳动物胚泡的形成具有两个谱系分离的特征，导致滋养外胚层、下胚层和上胚层细胞谱系的形成。在这些早期的谱系分离过程中，细胞命运的决定与特定转录因子的表达变化有关。除了以转录因子为基础的调控外，microRNAs(MiRNAs)在多能性和分化的转录后调控中也发挥着重要作用。为了阐明miRNAs在早期谱系分离中的作用，我们比较了早期牛囊胚中miRNA的表达与孵化早期囊胚中miRNA的表达。基于逆转录定量PCR的miRNA表达谱显示，在孵化的囊胚中有8个上调的miRNAs(miR-127、miR-130a、miR-155、miR-196a、miR-203、miR-28、miR-29c和miR-376a)和4个下调的miRNAs(miR-135a、miR-218、miR-335和miR-449b)。通过对匹配的miRNA和mRNA表达数据的综合分析，候选miRNA-mRNA相互作用对被优先验证。利用体外荧光素酶报告实验，我们证实了miR-218与CDH2、miR-218与NANOG、miR-449b与NOTCH1之间存在直接相互作用。通过干扰成纤维细胞生长因子信号通路，我们发现主要表达于内细胞团的miR-218调控牛胚泡中NANOG的表达，以响应成纤维细胞生长因子信号。这项研究的结果扩大了我们对牛囊胚的miRNA特征以及miRNAs与调控转录因子的细胞命运之间的相互作用的了解。
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.