Identification of microRNAs regulated by E2F transcription factors in human pluripotent stem cells

Human pluripotent stem cells (hPSCs), which include embryonic and induced pluripotent stem cells (hESCs and hiPSCs, respectively), have an unusual cell cycle structure which consists of a short G1 phase and the absence of the G1/S checkpoint regulation. E2F transcription factors (E2Fs) play an important role in the G1/S transition. G1 duration contributes to hPSC fate determination, and microRNAs (miRNAs) play critical roles in this commitment. As little is known about the interplay between E2Fs and miRNAs in hPSCs, we aimed to identify miRNAs that are regulated by E2Fs in these cells. We first found that mRNA expression levels of canonical E2F repressors were more expressed than most E2F activators in G1-arrested hPSCs. Moreover, we observed higher mRNA and protein expression levels of canonical E2F2, E2F3A, and E2F5 in G1 synchronized hPSCs compared to human fibroblasts (HF). However, E2F1 and E2F4 protein expression levels were higher in HF. We next found that E2F inhibition with HLM006474 induced an increase in the G1 cell population without affecting hPSC viability, concomitantly with a decrease in OCT-4 mRNA levels and the percentage of OCT-4+ hPSCs. Next, by RNA-seq analysis we found 52 differentially expressed (DEGs) miRNAs in HLM006474-treated hESCs. RT-qPCR validation of some of the DEGs let us conclude that miR-19a-3p, miR-19b-3p, miR-4454, miR-1260a, miR-1260b, miR-454-3p and miR-301a-3p are regulated by E2Fs in hPSCs. Interestingly, gene target and ontology analysis of these miRNAs revealed a possible implication in proliferation and cell cycle regulation, development, and neural differentiation.