MicroRNA-29 impairs the early phase of reprogramming process by targeting active DNA demethylation enzymes and Wnt signaling

Abstract

Somatic cell reprogramming by transcription factors and other modifiers such as microRNAs has opened broad avenues for the study of developmental processes, cell fate determination, and interplay of molecular mechanisms in signaling pathways. However, many of the mechanisms that drive nuclear reprogramming itself remain yet to be elucidated. Here, we analyzed the role of miR-29 during reprogramming in more detail. Therefore, we evaluated miR-29 expression during reprogramming of fibroblasts transduced with lentiviral OKS and OKSM vectors and we show that addition of c-MYC to the reprogramming factor cocktail decreases miR-29 expression levels. Moreover, we found that transfection of pre-miR-29a strongly decreased OKS-induced formation of GFP-colonies in MEF-cells from Oct4-eGFP reporter mouse, whereas anti-miR-29a showed the opposite effect. Furthermore, we studied components of two pathways which are important for reprogramming and which involve miR-29 targets: active DNA-demethylation and Wnt-signaling. We show that inhibition of Tet1, Tet2 and Tet3 as well as activation of Wnt-signaling leads to decreased reprogramming efficiency. Moreover, transfection of pre-miR-29 resulted in elevated expression of β-Catenin transcriptional target sFRP2 and increased TCF/LEF-promoter activity. Finally, we report that Gsk3-β is a direct target of miR-29 in MEF-cells. Together, our findings contribute to the understanding of the molecular mechanisms by which miR-29 influences reprogramming.

Metformin Increases Proliferative Activity and Viability of Multipotent Stromal Stem Cells Isolated from Adipose Tissue Derived from Horses with Equine Metabolic Syndrome

“…Overexpression of miR-29a was correlated to reduced levels of Slit glycoprotein 2 (SLIT2) and its receptor Roundabout 1 (ROBO1) which, in turn, resulted in inhibition of mesenchymal stem cell viability and proliferation [78]. Moreover, it was shown that transfection of miR-29 family members at an early stage of somatic cell reprogramming may decrease the number of colonies expressing pluripotent markers, such as Oct4 [79]. These results may explain the mir-29a-3p expression profile observed in EqASC EMS treated with metformin.…”

High-content screen in human pluripotent cells identifies miRNA-regulated pathways controlling pluripotency and differentiation

“…In fact, contrasting findings arising from different methods used were previously pointed out by our group in the study of the miR-29 family. Using transfection of synthetic miRs or inhibitors, our group and others showed that miR-29a hampers reprogramming by different mechanisms, including activation of WNT/beta-catenin signaling (by targeting GSK3B) and targeting of active DNA demethylation enzymes of the TET family [71, 78, 79]. Strikingly in contrast, expression of miR-29 throughout the process using a retroviral vector promotes reprogramming, an effect resulting from passive global demethylation following constitutive knockdown of the targets Dnmt3a and Dnmt3b [80].…”

Arrayed functional genetic screenings in pluripotency reprogramming and differentiation

“…Based on these conflicting results, and also based on a work from our group, which identified Tet1, 2, and 3 (components of active DNA demethylation) as targets of miR-29 [118], we further explored miR-29’s role in reprograming using synthetic miRs and anti-miRs. We showed that by targeting Tet1, 2, and 3 and Gsk3-beta (the phosphatase involved in B-catenin degradation), miR-29a hampers reprograming, likely by interfering with hydroximethylation and by activating Wnt signaling, respectively [119].…”