Vitamin C induces a pluripotent state in mouse embryonic stem cells by modulating microRNA expression

MicroRNAs (miRNAs), a group of noncoding RNAs, function as post-transcriptional gene regulators and control the establishment, self-renewal and differentiation of stem cells. Vitamin C has been recognized as a reprogramming enhancer because of its ability to induce a blastocyst-like state in embryonic stem cells (ESCs). However, knowledge on the regulation of miRNAs by vitamin C in ESCs is limited. In this study, we found that vitamin C induced miRNA expression, particularly of ESC-specific miRNAs. Moreover, vitamin C maintained the miRNA expression of the Dlk1-Dio3 imprinting region. The miRNAs in this region contain identical seed sequences, which target a class of genes, including Kdm6b, Klf13, and Sox6, and are mainly related to cell differentiation and development. These genes were significantly downregulated by vitamin C. Notably, miR-143 promoted self-renewal of mouse ESCs and suppressed expression of the de novo methyltransferase gene Dnmt3a. Knockdown of miR-143 by use of its inhibitor counteracted the vitamin C-induced reduction in Dnmt3a expression, showing that vitamin C repressed Dnmt3a expression via miR-143. Vitamin C also promoted DNA demethylation, including of pluripotency gene promoters (Tbx3, Tcl1, and Esrrb) and ESC-specific miRNA promoters (miR-290-295 and miR-17-92 clusters), and DNA hydroxymethylation, including of the intergenic differentially methylated region of the Dlk1-Dio3 region. These results strongly suggested that vitamin C promoted widespread DNA demethylation in gene promoters by modulating epigenetic modifiers, including Dnmt3a, which activated pluripotency genes and ESC-specific miRNAs. Then, differentiation and development genes were repressed by ESC-enriched miRNAs, which maintained the stem cell state.