MiR-25 Regulates Wwp2 and Fbxw7 and Promotes Reprogramming of Mouse Fibroblast Cells to iPSCs

Dong Lu,Julia Siede,Lia S Campos,Matthew P A Davis,Pentao Liu,Ian Dunham,William C Skarnes,Elena Vigorito,Wei Wang,Cei Abreu-Goodger,Anton J Enright

doi:10.1371/journal.pone.0040938

Abstract

BackgroundmiRNAs 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).MethodsWe 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.Results and conclusionWe 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.

Highlights

Mouse and human fibroblast cells can be reprogrammed to induced pluripotent stem cells (iPSCs) by expressing the four Yamanaka factors: Oct4, Sox2, Klf4 and c-Myc [1,2,3,4,5,6,7]
Pluripotent iPSCs reprogrammed from these Oct4-reporter mouse embryonic fibroblasts (MEFs) are resistant to 2.0 mg/ml puromycin (Puro) selection and identifiable as GFP+ in flow cytometry [51]
In an effort to identify new genetic factors in reprogramming, we screened 52 miRNAs/clusters that are expressed in embryonic stem (ES) cells or over-expressed in cancer

Summary

Introduction

Mouse and human fibroblast cells can be reprogrammed to iPSCs by expressing the four Yamanaka factors: Oct, Sox, Klf and c-Myc [1,2,3,4,5,6,7]. The long primary miRNAs (primiRNAs) are processed by the microprocessor complex, composed of the double-stranded RNA-binding protein DGCR8 and the RNase III enzyme DROSHA, into short hairpins called precursor miRNAs (pre-miRNAs) [30,31,32,33]. These hairpins are exported to the cytoplasm and are processed by Dicer into mature miRNAs [34,35]. In a dramatic show of miRNA’s function in reprogramming, Morrisey and colleagues recently demonstrated that expressing the miR-302/367 cluster alone is sufficient to reprogramme mouse and human fibroblast cells to iPSCs [43]. Recent studies demonstrate that some miRNAs have important roles in reprogramming somatic cells to induced pluripotent stem cells (iPSCs)

Methods

Results

Conclusion