BECAS
VILA Antonella SofÍa
congresos y reuniones científicas
Título:
DEVELOPMENT OF AN HYDROXYMETHYLGLUTHARYL-COENZYME A REDUCTASE (HMGCR) OVEREXPRESSION SYSTEM FOR THE STUDY OF REPROGRAMMING TO STEM-LIKE STATES IN HUMAN BREAST CANCER
Autor/es:
MARKS, MARIA PAULA; GIMENEZ, CARLA ALEJANDRA; ROMORINI, LEONARDO; FLETCHER, SABRINA JOHANNA; VILA, ANTONELLA SOFÍA; PEREYRA-BONNET, FEDERICO; CALVO, JUAN CARLOS; CHASSEING, NORMA ALEJANDRA; VELLÓN, LUCIANO
Lugar:
Melbourne
Reunión:
Congreso; ISSCR Anual Meeting; 2018
Resumen:
The rate-limiting enzyme in the mevalonic acid (MVA) pathway, hydroxymethylglutharyl- coenzyme A reductase (HMGCR), is deregulated in tumors, increasing the synthesis de novo of cholesterol, critical for cell survival and proliferation. However, the role of HMGCR in the induction and maintenance of stemness in both transformed and non-transformed cells is still unclear. Therefore, we set out to induce an HMGCR-on phenotype in the breast cancer (BC)-derived cell line MCF-7, to evaluate whether this phenotype facilitates the acquisition of stem-like traits in BC. With this purpose, we developed an HMGCR overexpression model taking advantage of a CRISPR-on system (dCas9-VP160), which includes expression plasmids for guide RNAs (pSPgRNAs) and a plasmid carrying the sequence coding for the dCAS9. Five guide RNAs (gRNAs) targeted to the promoter of the human HMGCR gene were designed ith the informatics tools Genome Engineering Toolbox from the Zhang Lab (MIT, Cambridge, MA) and CRISPR-ERA. The gRNAs and the dCAS9 were then co-transfected into MCF-7 cells, and the levels of total HMGCR was assessed by qRT-PCR at 2 days post-transfection. The CRISPR- dCAS9 system increased HMGCR total levels in MCF-7 cells (MCF-7/CR) a 2,46±0,4-fold (X±SD; p