INVESTIGADORES
ROMORINI Leonardo
congresos y reuniones científicas
Título:
Identification of miRNAs regulated by E2F transcription factors in human embryonic stem cells
Autor/es:
MARIA SOLEDAD RODRIGUEZ VARELA; SOFIA MUCCI; LUCIANA ISAJA; GUSTAVO E. SEVLEVER; MARÍA E. SCASSA; LEONARDO ROMORINI
Lugar:
Viena
Reunión:
Simposio; SY-Stem, 3rd Symposium on Stem Cell Research; 2021
Resumen:
Human embryonic or induced human pluripotent stem cells (hESCs and hiPSCs) have an unusual cell cycle structure which consists of a short G1 phase and absence of the G1/S checkpoint regulation. MicroRNAs (miRNAs) are small non-coding RNAs that play an important role in many key processes. E2F transcription factors (E2Fs) regulate G1/S transition. G1 duration contributes to hPSCs fate determination and miRNAs play a key role in achieving this cell cycle regulation. In hPSCs, the role of RB/E2F complexes remains uncertain and their expression profiles during cell cycle progression have not been fully studied yet. Due to this, the aim of this work was to explore if E2Fs mRNAs are constitutively or periodically expressed in hPSCs and to identify miRNAs that are regulated by E2Fs in hESCs. First, by RT-qPCR analysis, we observed high mRNA expression levels of the canonical E2Fs in hPSCs compared to somatic cells (human fibroblasts). At the same time, to determine if E2Fs are periodically or constitutively expressed we synchronized hPSCs in G1/S with PD0332991 (30h 5 µM), and in G2/M with Nocodazole (24h 100 ng/ml). RT-qPCR analysis of synchronized cells revealed a periodic gene expression profile of E2Fs transcripts. Then, we treated H9 hESCs line 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 viability and cell cycle profile of hESCs-treated cells determined by XTT assay and BrdU-7AAD flow cytometry analysis. A 20µM HLM006474 concentration and 24 hours treatment was chosen for further experiments as it induced an increase in G1 cell population in H9 hESCs without affecting cell death rate. Next, we performed a RNA-seq analysis of small RNAs of H9 hESCs treated or not with HLM006474 inhibitor. From 52 miRNAs differentially expressed we selected 20 miRNAs candidates, some of which were already 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 these 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 transcriptionally regulated by canonical E2Fs.