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Abstract
Objective:To investigate the mechanism underlying miR146a participating in the cardiotoxicity of doxorubicin. Methods:Rat cardiomyocytes H9c2 were treated with doxorubicin (DOX). After that, the cell viability was detected by CCK-8, the miR146a variation was quantified by qPCR and the change of cleaved Caspase 3 was measured by Western blot (WB). Using CRISPR, two sgRNAs were designed to knockout (KO) the expression of miR146a. The cell viability and cleaved Caspase 3 of miR146a KO cells were respectively detected by CCK-8 and WB. The potential target of miR146a was predicted and validated by luciferase system. The change of target gene after DOX treatment, and the influence of miR146a KO on target gene were detected using WB. Results:After DOX treatment, cell viability decreased, the level of cleaved Caspase 3 elevated and miR146a expression increased 3.6 folds. Using CRISPR, miR146a expression was successfully suppressed, with a much higher inhibitory rate than microRNA decoy (88.6% vs 57.6%). miR146a KO significant impeded DOX induced miR146a elevation, cell viability decrement and cleaved Caspase 3 increment. Smad family member 4(Smad4) was proved to be a target of miR146a by informatics prediction and luciferase experiment. The treatment of cardiomyocytes by DOX led to a decrease of Smad4 expression. In miR146a KO cells, the expression of Smad4 increased, which was not changed by DOX treatment. Conclusions:In rat cardiomyocytes, miR146a participated in doxorubicin cardiotoxicity through Smad4.
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