Protein arginine methyltransferase 7-mediated microRNA-221 repression maintains Oct4, Nanog, and Sox2 levels in mouse embryonic stem cells

The stemness maintenance of embryonic stem cells (ESCs) requires pluripotency transcription factors, including Oct4, Nanog, and Sox2. We have previously reported that protein arginine methyltransferase 7 (PRMT7), an epigenetic modifier, is an essential pluripotency factor that maintains the stemness of mouse ESCs, at least in part, by down-regulating the expression of the anti-stemness microRNA (miRNA) miR-24-2. To gain greater insight into the molecular basis underlying PRMT7-mediated maintenance of mouse ESC stemness, we searched for new PRMT7-down-regulated anti-stemness miRNAs. Here, we show that miR-221 gene-encoded miR-221-3p and miR-221-5p are anti-stemness miRNAs whose expression levels in mouse ESCs are directly repressed by PRMT7. Notably, both miR-221-3p and miR-221-5p targeted the 3'untranslated regions of mRNA transcripts of the major pluripotency factors Oct4, Nanog, and Sox2 to antagonize mouse ESC stemness. Moreover, miR-221-5p silenced also the expression of its own transcriptional repressor PRMT7. Transfection of miR-221-3p and miR-221-5p mimics induced spontaneous differentiation of mouse ESCs. CRISPR-mediated deletion of the miR-221 gene, as well as specific antisense inhibitors of miR-221-3p and miR221-5p, inhibited the spontaneous differentiation of PRMT7depleted mouse ESCs. Taken together, these findings reveal that the PRMT7-mediated repression of miR-221-3p and miR221- 5p expression plays a critical role in maintaining mouse ESC stemness. Our results also establish miR-221-3p and miR221- 5p as anti-stemness miRNAs that target Oct4, Nanog, and Sox2 mRNAs in mouse ESCs.