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

VELLON L; VILA AS; MARKS MP; CALVO JC; FLETCHER SJ; PEREYRA-BONNET F; GIMENEZ CA

Congreso; SOCIEDADES DE BIOCIENCIAS. LXII REUNIÓN ANUAL DE LA SOCIEDAD ARGENTINA DE INVESTIGACIÓN CLÍNICA (SAIC).; 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 weredesigned 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 byqRT-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