Targeting G-quadruplex motifs interferes with differentiation of adipose-derived mesenchymal stem cells

G-quadruplex (G4) motifs are nucleic acid secondary structures observed in mammalian genomes and transcriptomes able to regulate various cellular processes. Several small molecules have been developed so far to modulate G4 stability, frequently associated with anticancer activity. However, how G4 structures are regulated over homeostatic conditions is mostly unexplored. Here, we used human adipose-derived mesenchymal stem cells (ASCs) to address the role of G4 motifs during adipogenic differentiation.

Adipocyte differentiation of ASCs was investigated in the presence or absence of a well-known G4 ligand, Braco-19. Cell viability was determined by sulforhodamine B assay. Cell dimension and granularity, DNA G4 motifs and cell cycle were detected by flow cytometry. Lipid droplet accumulation was assessed by Oil Red O staining. Cell senescence was evaluated by β-galactosidase staining. Gene expression was measured by qPCR. Protein release in the extracellular medium was quantified by ELISA.

Braco-19 used at non-cytotoxic concentrations induced morphological changes in mature adipocytes partially restoring an undifferentiated-like status. Braco-19 reduced lipid vacuolization and PPARG, AP2, LEP and TNFA mRNA levels in terminally differentiated cells. No effect was observed in cell senescence, fibrotic markers, IL-6 and IL-8 production, while the secretion of VEGF was dose-dependently reduced. Interestingly, G4 structures were increased in differentiated adipocytes compared to their precursors. Braco-19 treatment reduced G4 content in mature adipocytes.

Our data highlight a new role of G4 motifs as genomic structural elements related to human ASC differentiation into mature adipocytes, with potential implications in physio-pathological processes.