Establishment of MageC2-knockout cells through CRISPR/CAS9 technology

ESCALADA, MICAELA; PASCUCCI, FRANCO A.; MONTE, MARTÍN; LADELFA, M FÁTIMA

Congreso; LXIV Reunión Científica de la Sociedad Argentina de Investigación Clínica (SAIC).; 2019

MageC2 is a member of the melanoma antigen gene (MAGE) family, specifically expressed in a wide variety of human cancers and associated with a non-favourable clinical course. It has been reported that MageC2 oncoprotein downregulates p53 and activates STAT3 transcription factors. Recently, we identified the Ras oncogene as responsible for MageC2 stability increase and phosphorylation through the MEK/ERK pathway. Then, our hypothesis is that Ras oncogene could regulate p53 and STAT3 by enhancing MageC2 protein levels. In order to analyze the functional consequences of endogenous MageC2 expression, we established a MageC2 knockout (KO) cell line through the CRISPR/Cas9 system in human melanoma A375 cells. All the obtained clones were probed for MageC2 protein expression by Western blot. Three clones were selected and sequenced for indel detection (KO1, gRNA1; KO2, gRNA1 and KO3, gRNA2). To validate the A375 MageC2 KO (A375-C2KO) clone behaviour, we quantified the mRNA levels of genes regulated by p53 or STAT3. Analysis of RT-qPCR data carried out in three pairs of biological replicates in A375-C2KO cells indicated enhanced transcription of p53 targets (p21waf1 and bax, p