miR-145, miR-296 and miR-302 family expression fluctuate periodically allong the cell cycle of human pluripotent stem cells

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

Mar del Plata

Congreso; LXIV Reunión Científica Anual de la Sociedad Argentina de Investigación Clínica SAIC; 2019

SAIC

an unusual mode of cell cycle regulation, reflected by a cell cycle structure where G1 and G2 phases are truncated. hPSCs are primed to initiate cell fate decisions during their transition through G1. MicroRNAs (miRNAs) are small non-coding RNA molecules involved in the regulation of gene expression. Several miRNAs have been shown to target transcripts that directly or indirectly coordinate the cell cycle of pluripotent cells. miR-145 and miR-296 are induced during differentiation and silence the self-renewal and pluripotency program. miR-302 family is essential for hPSCs stemness and its expression decreases during differentiation. Although, it is clear that miRNAs can regulate components of the cell-cycle machinery, its temporal expression profile along hPSCs cell cycle remains poorly characterized. Thus, we synchronized hPSCs populations using three pharmacological inhibitors: PD0332991, Aphidilcolin and Nocodazole to arrest cells in early G1, G1/S and G2/M phases, respectively. Analysis of cell cycle by DNA content was performed using propidium iodide staining followed by flow cytometry analysis. Then, by RT-qPCR, using specific stem loop primers, we analyzed the expression levels of miR-145, miR-296 and miR-302 family in early G1, G1/S and G2/M arrested hPSCs and determined that all studied miRNAs were periodically expressed. Finally, we performed Aphidilcolin- block and release experiments, and measured the levels of these miRNAs throughout hPSCs cell cycle progression at 0, 4, 8, 12, 14, 16, 20 and 24 hours after release. Importantly, we confirmed the periodic expression of all studied microRNAs and observed that miR-302 family expression is induced at 14-16 h post release, which coincides with G1/S transition, presumably to impede differentiation onset.