Wellcome Trust Sanger Institute, Wellcome Trust Genome Campus, Hinxton, Cambridge, United Kingdom.
PLoS One. 2012;7(8):e40938. doi: 10.1371/journal.pone.0040938. Epub 2012 Aug 17.
miRNAs are a class of small non-coding RNAs that regulate gene expression and have critical functions in various biological processes. Hundreds of miRNAs have been identified in mammalian genomes but only a small number of them have been functionally characterized. Recent studies also demonstrate that some miRNAs have important roles in reprogramming somatic cells to induced pluripotent stem cells (iPSCs).
We screened 52 miRNAs cloned in a piggybac (PB) vector for their roles in reprogramming of mouse embryonic fibroblast cells to iPSCs. To identify targets of miRNAs, we made Dgcr8-deficient embryonic stem (ES) cells and introduced miRNA mimics to these cells, which lack miRNA biogenesis. The direct target genes of miRNA were identified through global gene expression analysis and target validation.
We found that over-expressing miR-25 or introducing miR-25 mimics enhanced production of iPSCs. We identified a number of miR-25 candidate gene targets. Of particular interest were two ubiquitin ligases, Wwp2 and Fbxw7, which have been proposed to regulate Oct4, c-Myc and Klf5, respectively. Our findings thus highlight the complex interplay between miRNAs and transcription factors involved in reprogramming, stem cell self-renewal and maintenance of pluripotency.
miRNAs 是一类小的非编码 RNA，可调节基因表达，并在各种生物过程中发挥关键作用。在哺乳动物基因组中已经鉴定出数百种 miRNAs，但只有少数具有功能特征。最近的研究还表明，一些 miRNAs 在体细胞重编程为诱导多能干细胞（iPSCs）中具有重要作用。
我们筛选了 52 个克隆在 piggybac（PB）载体中的 miRNAs，以研究它们在重编程小鼠胚胎成纤维细胞为 iPSCs 中的作用。为了鉴定 miRNAs 的靶标，我们制造了缺乏 Dgcr8 的胚胎干细胞（ES）细胞，并将 miRNA 模拟物引入这些细胞中，这些细胞缺乏 miRNA 生物发生。通过全基因组表达分析和靶标验证鉴定 miRNA 的直接靶基因。
我们发现过表达 miR-25 或引入 miR-25 模拟物可增强 iPSC 的产生。我们鉴定了许多 miR-25 的候选靶基因。特别有趣的是两个泛素连接酶，Wwp2 和 Fbxw7，它们分别被认为分别调节 Oct4、c-Myc 和 Klf5。我们的研究结果因此强调了 miRNA 和转录因子在重编程、干细胞自我更新和多能性维持中的复杂相互作用。
