Identification of miRNAs regulated by E2F transcription factors in human embryonic stem cells

RODRÍGUEZ VARELA, MARÍA SOLEDAD; MUCCI, SOFÍA; ISAJA, LUCIANA; SEVLEVER, GUSTAVO EMILIO; SCASSA, MARÍA ELIDA; ROMORINI, LEONARDO

Buenos Aires

Congreso; Reunión de Sociedades Biociencias 2020, SAIC.SAI.SAFIS; 2020

SAIC SAI SAFIS

Human embryonic stem cells (hESCs) exhibit an unusual cell cy- cle structure with an abbreviated G1 phase. MicroRNAs (miRNAs) are small non-coding RNAs that play an important role in many key processes; including cell cycle regulation and cell differentiation, as post-transcriptional regulators of gene expression. E2F transcription factors (E2Fs) regulate G1/S transition. G1 duration contributes to hESCs fate determination and miRNAs play a key role in achieving this cell cycle regulation. Due to this, we aimed to identify miRNAs that are regulated by E2Fs in hESCs. Importantly, we had previous- ly reported high mRNA expression levels of the canonical E2Fs in hESCs line H9 in comparation to somatic cells (human fibroblasts). First, we treated H9 hESCs with the general inhibitor of E2Fs (pan- E2F inhibitor) HLM006474. Concentration and incubation time used for HLM006474 treatment was fine-tuned by studying the cell cycle profile of hESCs-treated cells determined by measuring DNA con- tent by propidium iodide staining followed by flow cytometry anal- ysis. A 20mM HLM006474 concentration and 24 hours treatment was chosen for further experiments as it induced an increase in G1 cell population in H9 hESCs. Next, we performed a RNA-seq anal- ysis of small RNAs of H9 hESCs treated or not with HLM006474 inhibitor (20mM, 24h). We found that 52 miRNAs were differentially expressed upon E2Fs inhibition (FDR: 0.1), some of which were al- ready related with E2Fs family and others whose relationship with these factors or with hESCs-cell cycle has not yet been reported. Finally, upon validation of the expression levels of 20 selected miR- NAs candidates by RT-qPCR with specific stem loop primers, we concluded that miR-19a-3p, miR-19b-3p, miR-4454, miR-1260a, miR-1260b, miR-454-3p and miR-301a-3p would be transcriptional- ly regulated by canonical E2Fs.