MicroRNA-302 Increases Reprogramming Efficiency via Repression of NR2F2

Abstract

MicroRNAs (miRNAs) have emerged as critical regulators of gene expression through translational inhibition and RNA decay, and have been implicated in the regulation of cellular differentiation, proliferation, angiogenesis, and apoptosis. In this study, we use global bioinformatics analysis of miRNA and mRNA microarrays to predict novel miRNA-mRNA interactions in human embryonic stem cells (hESCs) and induced pluripotent stem cells (iPSCs). In particular, we demonstrate a regulatory feedback loop between the miR-302 cluster and two transcription factors, NR2F2 and OCT4. Our data show high expression of miR-302 and OCT4 in pluripotent cells, while NR2F2 is expressed exclusively in differentiated cells. Target analysis predicts that NR2F2 is a direct target of the miR-302, which we experimentally confirm by reporter luciferase assays and real-time PCR. We also demonstrate that NR2F2 directly inhibits the activity of the OCT4 promoter and thus diminishes the positive feedback loop between OCT4 and the miR-302. Importantly, higher reprogramming efficiencies were obtained when we reprogrammed human adipose-derived stem cells (hASCs) into iPSCs using four factors (KLF4, C-MYC, OCT4, and SOX2) plus miR-302 (this reprogramming cocktail is hereafter referred to as “KMOS3”) when compared to using four factors (“KMOS”). Furthermore, shRNA knockdown of NR2F2 mimics the over-expression of miR-302 by also enhancing reprogramming efficiency. Interestingly, we were unable to generate iPSCs from miR-302a/b/c/d alone, which is in contrast to previous publications that have reported that miR-302 by itself can reprogram human skin cancer cells and human hair follicle cells. Taken together, these findings demonstrate that miR-302 inhibits NR2F2 and promotes pluripotency through indirect positive regulation of OCT4. This feedback loop represents an important new mechanism for understanding and inducing pluripotency in somatic cells.

MicroRNAs in regulation of pluripotency and somatic cell reprogramming

“…67 However, Hu et al failed to reprogram human adipose stem cells to iPSCs by miR-302, which indicates that miRNA-induced pluripotent stem cells could only be derived from certain types of adult cell types. 68 It is worth noting that since miRNA-mediated reprogramming are mostly dependent on endogenous pathways and, hence, maintain a smooth epigenetic modification, it shows certain advantages in producing better, safer, and more ES-like iPSCs over the conventional methods. Therefore, more studies about reprogramming different adult cell types and transduction of other miRNA families need to be performed to tackle this issue.…”

Reviewing and Updating the Major Molecular Markers for Stem Cells

“…This cluster is highly expressed in ESCs [88], and as observed for other miRNA clusters, its expression is rapidly downregulated when the cells are induced to differentiate [92].…”

Activation of Aryl Hydrocarbon Receptor (AhR) by Tranilast, an Anti-allergy Drug, Promotes miR-302 Expression and Cell Reprogramming

“…The expression level of miR-302 correlates well with the expression level of Oct4 following induction of ESC differentiation (14,18). On the other hand, miR-302 functions to regulate cell cycle regulators CDKN1A and RBL2 (19), TGF-␤ signaling negative regulator Lefty1/2 (20), and pluripotency factors Oct4 and Sox2 (21) and thereby contributes to the maintenance of ESC pluripotency. Recent studies show that ectopic expression of miR-302 can promote somatic cell reprogramming (22,23).…”

“…show that expression of miR-302 facilitates somatic cell reprogramming (21)(22)(23)(24). We then tested whether tranilast could promote iPSCs generation through miR-302.…”