ACYL-COA SYNTHETASE 4 EXPRESSION MODIFIES microRNA EXPRESSION PROFILE IN BREAST CANCER CELL LINE.

QUEVEDO, LUCIANO; SEMINARA CUNTO, ZOE; BIGI, MERCEDES; PRADA, JESICA ; MALOBERTI, PAULA; YANG, SEONG WOOK; CASTILLO, ANA FERNANDA

Rosario

Congreso; Reunión anual de la Sociedad Argentina de Investigaciones en Bioquímica y Biología Molecular, SAIB.; 2023

Acyl-CoA synthetases are essential enzymes that activate fatty acids by converting them to acyl-CoA esters. Particularly, acyl-CoA synthetase 4 (ACSL4) catalyzes the esterification of long-chain fatty acids, with a preference for arachidonic acid as a substrate. ACSL4 expression is increased in breast, prostate, colon, and hepatocellular carcinomas. Its expression levels correlate with the phenotype of breast and prostate cancer cells. Previous results have shown that an increase in ACSL4 expression leads to the induction of a highly aggressive phenotype of breast and prostate cancer cells and to the growth of breast tumors in vivo.It is relevant to know which molecular events are triggered by this enzyme to change the normal cellular phenotype to a highly aggressive one. We focused on the study of microRNAs (miRNAs) as mediators of its effects on the tumor phenotype. miRNAs are small non-coding RNAs that carry out the silencing of target genes by targeting mRNAs inducing their transcriptional repression. Their involvement in cancer has been demonstrated, and their expression patterns are associated with tumor type and grade. Using large-scale mRNA sequencing (RNA-Seq) data, we have preliminarily shown that ACSL4 expression alters the expression of several miRNA precursor transcripts. In this study, we investigated the expression of functional and mature miRNAs in response to changes in ACSL4 expression. We performed mature miRNA sequencing (miR-Seq) using an MCF-7 breast cancer cell model in which ACSL4 was stably transfected under the repressible Tet-Off system (MCF-7 Tet-Off/ACSL4). The differential expression profile was analyzed comparing ACSL4-overexpressing cells with control cells using a cutoff of Log2 fold change > |1|. This doxycycline-repressable overexpression system allowed us to adjust the miRNA profile to those miRNAs for which decreasing ACSL4 levels in MCF-7 Tet-Off/ACSL4 resulted in an expression pattern similar to that of control cells. Thus, we selected those miRNAs whose levels were specifically altered by the expression of the enzyme. A total of 56 differentially expressed miRNAs were obtained, of which only 3 miRNAs were found to be upregulated by ACSL4. Based on the miRNA signature obtained, a predictive analysis was performed for each miRNA regarding its role in biological pathways, tissue expression, physiological conditions or related pathologies in which it might be involved. For this purpose, databases such as KEGG Pathway, WikiPathways and miRPathDB were used. Several miRNAs were selected for further validation of their expression by RT-qPCR, considering not only their differential expression levels, but also the information provided by the databases.Therefore, we describe and validate the profile of miRNAs whose expression levels are modified by ACSL4. These results suggest that these small RNAs may be involved in mediating the effects of this enzyme on the cellular phenotype of breast cancer cells.