Maintenance of Self-Renewal and Pluripotency in J1 Mouse Embryonic Stem Cells through Regulating Transcription Factor and MicroRNA Expression Induced by PD0325901

Figure 5

MEK/ERK signal-related miRNAs promote homogeneous ESC. (a–c) Venn diagram shows the differential expression of miRNA in PD- and CHIR-treated ESCs. Venn diagram showed the upregulated miRNAs (a) and the downregulated miRNAs (b) in PD- and CHIR-treated ESCs, respectively. The global differential miRNAs between CHIR- and PD-treated ESCs are shown in (c). (d) Schematic diagram of the miRNA biosynthesis and functions in maintaining the undifferentiated state of mESCs. PD and CHIR influence Dgcr8-Drosha complex activity. → means active and ⊥ means inactive. (e) miR-296 mimics regulated Nanog expression in a posttranscriptional regulation manner. Schematic representation of the 3′-UTR reporter constructs in the upper panel. TK, hluc+, SV40, and hRluc represent HSV-TK promoter, firefly luciferase gene, SV40 early enhancer/promoter, and Renilla luciferase gene, respectively. In the lower panel, psiCHECK2-Nanog-CDS or psiCHECK2 control plasmid was cotransfected with mimics NC or miR-296 mimics/inhibitor into 293T cells. At 24 h after incubation, 1 μM PD or an equal volume of DMSO was added to cell medium for another 24 h. Luciferase activity is presented relative to negative control pTA-luc. Data are presented as mean ± SD of three independent experiments, . (f) miR-296 mimics regulated Nanog expression. J1 mESCs were transfected with mimics NC or miR-296 mimics. At 5 h after transfection, fresh medium was added and 1 μM PD or an equal volume of DMSO was added to the transfected cells for another 24 h. The expression level of Nanog was detected by RT-qPCR. Error bars indicate mean ± SD of three independent experiments, compared with controls. (g) miR-296 regulates Nanog expression. ESCs were transfected with mimics NC or miR-296 mimics for 24 h; then the protein expression level of Nanog was analyzed by western blot. Gapdh was used as a normalization control.

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