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

Abstract

Embryonic stem cells (ESCs) have the ability to grow indefinitely and retain their pluripotency in culture, and this self-renewal capacity is governed by several crucial molecular pathways controlled by specific regulatory genes and epigenetic modifications. It is reported that multiple epigenetic regulators, such as miRNA and pluripotency factors, can be tightly integrated into molecular pathways and cooperate to maintain self-renewal of ESCs. However, mouse ESCs in serum-containing medium seem to be heterogeneous due to the self-activating differentiation signal of MEK/ERK. Thus, to seek for the crucial miRNA and key regulatory genes that establish ESC properties in MEK/ERK pathway, we performed microarray analysis and small RNA deep-sequencing of J1 mESCs treated with or without PD0325901 (PD), a well-known inhibitor of MEK/ERK signal pathway, followed by verification of western blot analysis and quantitative real-time PCR verification; we found that PD regulated the transcript expressions related to self-renewal and differentiation and antagonized the action of retinoic acid- (RA-) induced differentiation. Moreover, PD can significantly modulate the expressions of multiple miRNAs that have crucial functions in ESC development. Overall, our results demonstrate that PD could enhance ESC self-renewal capacity both by key regulatory genes and ES cell-specific miRNA, which in turn influences ESC self-renewal and cellular differentiation.

SC1 Promotes MiR124-3p Expression to Maintain the Self-Renewal of Mouse Embryonic Stem Cells by Inhibiting the MEK/ERK Pathway

“…The expression of miR296-3p was decreased in SC1-treated mouse ES cells, and was increased during ES cell differentiation [59]. Previous experiments also confirmed that transfection of miR-296 mimics suppressed Nanog levels in J1 mouse ES cells [37]. …”

“…PD0325901(PD) is an inhibitor of the MEK/ERK pathway. Our previous study reported small RNA deep-sequencing results from PD-treated mouse ES cells [37]. Therefore, the general differences in differentially expressed microRNAs in SC1- and PD-treated mouse ES cells were analysed.…”

“…This pathway also suppresses the expression of pluripotency genes to enable endodermal differentiation [54]. As shown in our previous study, the MEK inhibitor PD enhances the self-renewal capacity of mouse ES cells by modulating the expression of the key pluripotent genes and ESC-specific microRNAs [37]. SC1 is an effective small molecule inhibitor of the MEK/ERK pathway that inhibits Erk1/2 activation.…”

“…Each qualified total RNA sample was divided into two aliquots, one for the whole-genome microarray expression profiling experiment and another for the small RNA deep-sequencing experiment. Qualified total RNA samples were used for the microarray experiment, and the specific methods and analytical procedures used for these two independent experiments were described previously [37, 38]. …”

Fine-tuning Nanog expression heterogeneity by altering MicroRNA regulation

“…It turns out that these two inhibitors, in general, even regulate the miR processing significantly by influencing the two important miR processors DGCR8 and Drosha, respectively. Recent reports suggest that MEK/ERK can phosphorylate DGCR8, which increases its intracellular stability and hence induces a pro-growth miR profile [52,53]. On the other hand, GSK3 phosphorylates the Drosha and increases its nuclear localization [54][55][56].…”