DEVELOPMENT OF AN HYDROXYMETHYLGLUTHARYL-COENZYME A REDUCTASE (HMGCR) OVEREXPRESSION SYSTEM WITH CRISPR-ON TECHNOLOGY FOR THE STUDY OF METABOLIC REPROGRAMMING TO STEM-LIKE STATES IN BREAST CANCER

MARKS, MARIA PAULA; GIMENEZ, CARLA ALEJANDRA; FLETCHER, SABRINA JOHANNA; VILA, ANTONELLA SOFÍA; PEREYRA-BONNET, FEDERICO; CALVO, JUAN CARLOS; VELLÓN, LUCIANO

Ciudad Autónoma de Buenos Aires

Congreso; Reunion Conjunta de Sociedades de Biociencias; 2017

Hydroxymethylglutharyl-coenzymeA reductase is deregulated in tumors, increasing the synthesis de novo of cholesterol, critical for the regulation of cell survival and proliferation signals. Therefore, we aimed to induce an HMGCR-on phenotype in the breast cancer (BC)-derived cell line MCF-7, to evaluate whether this phenotype involves 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 CRISPR-on. Five guide RNAs (gRNAs) targeted to the promoter of the human HMGCR gene were designed with the informatics tools Genome Engineering Toolbox from the Zhang Lab (MIT, Cambridge, MA) and CRISPR-ERA. The gRNAs and the CRISPR-on were then co-transfected into MCF-7 cells, and the levels of total HMGCR and its two isoforms (FL-Full Length and DL13-deletion of exon 13) mRNAs were assessed by qRT-PCR at 2 days postransfection. The CRISPR-dCAS9 system increased HMGCR total levels in MCF-7 cells (MCF-7/HMGCR-on) by a 2,26±0,03-fold (X±SD; p