mir-520a induce re-enter in S phase of pluripotent stem cell derived cardiomyocytes

NATALIA LUCÍA SANTÍN VELAZQUE; XIMENA GARATE; MARIA AGUSTINA SCARAFIA; ALAN MIQUEAS MÖBBS; CAROLINA COLLI; ARIEL WAISMAN; LUCIA NATALIA MORO; GUADALUPE AMIN; CARLOS DANIEL LUZZANI; GUSTAVO EMILIO SEVLEVER; ALEJANDRO DAMIÁN LA GRECA; SANTIAGO GABRIEL MIRIUKA

Mar del Plata

Congreso; LXIII Annual Meeting of the Argentine Society for Clinical Investigation (SAIC); 2018

SAIC - Sociedad Argentina de Investigacion Clinica

Productionof induced pluripotent stem cells derived cardiomyocytes (iPSC-CM)and controlling their proliferation is a potentially promisingstrategy for regenerative therapies. MicroRNAs (miRNAs) are keyregulators of gene expression at the post-transcriptional level andplay essential roles in diverse biological processes. A role formiRNA in cardiomyocyte proliferation and regeneration was revealed inseveral studies. Forced overexpression of certain synthetic miRNAscan promote CM proliferation. The aim of this study was to analyzethe ability of iPSC-CMs to re-enter cell cycle after exposure tomiR520a-mimic. We generated iPSC with cell cycle indicator Fucci.Fucci-iPSCs were constructed by a transposable recombinationapproach, using a system that employs both a red (mCherry) and agreen (mVenus) fluorescent protein fused to different regulators ofthe cell cycle: cdt1 (G1) and geminin (S/G2/M), respectively.iPSC-CMs were obtained by the implementation of a previously reportedprotocol (Lian et al. 2012). We performed an Ago-IP in iPSC-CMstransfected with 10nM miR-520a mimic or 10nM negative control-mimic.That experiment showed 152 genes differentially expressed.Gene-Ontology analysis revealed mRNAs involved in the regulation ofmicrotubule polymerization or depolymerization and mitotic cell cycle(GO:0000278). For example, SUGT1 and SKA2 are genes that encodeproteins involved in kinetochore function and required for the G1/Sand G2/M transitions. Then, iPSC-CMs were transfected withmiR-520a-mimic or a control-mimic and cells proliferation wasobserved at 48hs. Our results show that only a small proportion ofiPSC-CMs were cycling in control condition (~3%), but this percentageincreased when exposed to miR-520a-mimic (>10%). iPSC-CMs wereable to duplicate their DNA and re-enter S phase, but generatedmultinucleated cells. With control-mimic we observed 6% ofmultinucleated cells, while with miR-520a-mimic they increase to 12%.Taken together, our data indicate that miR-520a-mimic let IPSC-CMsre-enter cell cycle without cytokinesis.p { margin-bottom: 0.25cm; line-height: 115%; }