MicroRNA-10a-5p Negatively Regulates Doxorubicin-induced Cardiomyocyte Pyroptosis And Its Mechanism

Background: Doxorubicin(DOX)is an anthracycline widely used in clinic,but its long-term application is limited by adverse reactions,especially cardiotoxicity.Pyroptosis is an important mode of programmed cell death,which is related to drug-induced cardiotoxicity.MicroRNAs(Micro-ribonucleic acids(miRNAs)play an important role in the process of pyroptosis,and have also been reported to play a very important role in the pathogenesis of cardiotoxicity.Objective: To investigate the role of miR-10a-5p and KLF11(Kruppel-like factor 11,KLF11)in DOX-induced cardiomyocyte pyroptosis.Methods:(1)DOX was used to treat cardiomyocytes with different concentrations and set different treatment times to explore the best modeling method for cardiomyocyte pyroptosis;(2)By RNA sequencing,miRNAs most likely to play a role in cardiomyocyte pyroptosis were screened from cardiac tissue samples of DOX induced myocardial injury in mice;(3)To explore the function of overexpressed miRNA in cardiomyocyte pyroptosis,real-time quantitative polymerase chain reaction(real-time quantitative polymerase chain reaction,qRT-PCR was used to detect the expression of miRNA,western blot was used to detect the expression level of pyroptosis-related proteins,microscope was used to take pictures of pyroptosis-related cells,and cell Counting kit-8 was used to determine the expression level of miRNA.CCK-8)To detect the vitality of cells,and to detect the release of LDH from cells with Lactic Dehydrogenase(LDH)release experiments;(4)Search for downstream targets directly binding to miRNA based on bioinformatics prediction and verification by double luciflucase assay;(5)After silencing the downstream target of miRNA,qRT-PCR,western blot,pictures of pyroptosis cells taken by microscopy,CCK-8 assay and LDH release assay were used to study the function of miRNA in pyroptosis cardiomyocytes.Results:(1)The optimal modeling method was DOX treated cardiomyocytes with 5 μM for 9 h,DOX induced decreased cardiomyocyte activity,increased LDH release,and pyroptosis morphology;(2)miR-10a-5p was significantly differentially expressed in DOX-induced myocardial injury model and down-regulated in cardiomyocyte pyroptosis model;(3)Overexpression of miR-10a-5p can inhibit the activation of pyroptosis-related proteins,inhibit the pyroptosis-related morphology of cardiomyocytes,restore cell viability,and inhibit the release of LDH.(4)KLF11 is a direct downstream target of miR-10a-5p;(5)Silencing KLF11 inhibited the activation of pyroptosis-related proteins,inhibited the pyroptosis morphology of cardiomyocytes,restored cell viability,and inhibited the release of LDH from cardiomyocytes.After DOX induction,the expression of miR-10a-5p in HL-1 cells was significantly decreased,while the expression of KLF11 was significantly increased in HL-1 cells.We also found that DOX-induced cardiomyocyte pyroptosis was significantly reduced by microRNA-transfected mimics or small molecule interfering RNA(Si-RNA)overexpression of miR-10a-5p or silencing of KLF11.Subsequently,we found that miR-10a-5p could bind to KLF11 and negatively regulate KLF11 expression.Conclusions: The cardiotoxicity of DOX may be related to the down-regulation of miR-10a-5p,the up-regulation of KLF11,and the induction of cardiomyocyte pyroptosis.