Avaliação da expressão de miRNAs exossomais na toxicidade hepática induzida pelo clopidogrel em células HepG2

Abstract

Clopidogrel is an essential therapy for prevention of thrombosis and atherosclerosis. However, clopidogrel-induced hepatotoxicity is a potential adverse effect related to liver damage and antiplatelet response. Considering the lack of diagnostic for this adverse effect, new exosomes-derived miRNAs may be useful for improve the monitoring of response and hepatotoxicity risk. Therefore, we first aimed to investigate the miRNA-mRNA interactions with drug toxicity by in silico using available microarray data and Ingenuity Pathways Analysis 6 (IPA) software. After, the exosomal-expression profile of miR-26a-5p, miR-145-5p, miR-15b-5p and miR-4701-3p, as well as the cell-derived mRNAs target PLOD2, SENP5, EIF4G2, HMGA2, STRADB and TLK1 were evaluated in vitro, once they were the molecular targets mainly associated with the adverse effect. Thus, HepG2 cells were incubated with clopidogrel at 6.25, 12.5, 25, 50, and 100 μM for 24 and 48 h. The cytotoxicity was evaluated by flow cytometry to analyze DNA fragmentation and cell cycle. Profile expression of exosomes-derived miRNAs obtained by column methods and cell-derived mRNAs was evaluated by RT-qPCR. Cells treated with higher concentrations of 50 and 100 μM caused an increased DNA fragmentation after 24 and 48 h suggesting a toxic concentration for the cells. The miR-26a-5p upregulation in toxic concentration of 100 μM of clopidogrel and a downregulation of miR-15b-5p in comparison to control were observed in both period of 24 and 48 h. HMGA2, EIF4G2, STRADB and SNP5 targets of miR-26a-5p were downregulated in toxic concentrations at 24 h and HMGA2 remains downregulated after 48 h of clopidogrel treatment. TLK1, a target of miR-15b-5p, was downregulated at 24 h in toxic concentration. The results are suggestive that toxic concentrations of clopidogrel may modulate the miR-26a-5p and miR-15b-5p expression and their mRNA targets. Moreover, miR-26a may represent an important marker to predict clopidogrel-induced hepatotoxicity.