The Mechanism Research Of Let-7i-3p Regulated Doxorubicin-induced Cardiotoxicity By Targeting RPS6KA2

Objective:The clinical application of Doxorubicin(DOX)has been limited by its cardiotoxicity.Many studies have shown that microRNA(miRNA)is critical in DOX-induced cardiotoxicity.The aim of this study was to investigate the role of let-7i-3p in DOX-induced cardiotoxicity and its potential mechanism.Methods:Anthropogenic AC16 cardiomyocytes were treated with an appropriate concentration of DOX,and the pre-treated AC16 cells were treated as the control group.The differentially expressed genes(DEGs)were identified by RNA sequencing(RNA-Seq);The DEGs enrichment results were analyzed based on Gene ontology(GO)and Kyoto Encyclopedia of Genes and Genomes(KEGG).The STRING online database combined with Cytoscape software was used to analyze protein-protein interactions(PPI)and draw a visual network diagram.The quantitative real-time polymerase chain reaction(qRT-PCR)was used to verify the differential genes and select the genes with the most significant differences for subsequent study.AC16 cells were transfected with miRNA mimic and inhibitor,and DOX treatment group was set up after transfection.By measuring cell viability,reactive oxygen species(ROS)production,malondialdehyde(MDA)content and superoxide dismutase(SOD)activity in cells,as well as apoptosis and autophagy,the effects of miRNA on cell viability,oxidative stress,apoptosis and autophagy of DOX-treated AC16 cells were evaluated.The target gene of let-7i-3p was screened by bioinformatics prediction software combined with the results of RNA-seq,and the expression level of the target gene was determined by qRT-PCR,and the correlation between let-7i-3p and the target gene was analyzed.The effect of the target genes on DOX-induced cardiotoxicity has been verified with small interfering RNA(siRNA)transfection techniques.The qRT-PCR and Western blot were used to further verify the regulation effect of miRNA on target genes.The expression levels of key proteins in mammalian target of rapamycin(mTOR)signaling pathway were determined by Western blot assay to clarify the role of let-7i-3p/mTOR signaling pathway in DOX-induced autophagy disorders.Finally,t test and one-way analysis of variance were used for statistical analysis.The data were expressed as mean ± standard error(SEM),and P<0.05 was considered statistically significant.Results:1.The RNA-seq results showed that a total of 24 differentially expressed miRNAs(Differentially expressed miRNA,DE miRNA)and 1619 differentially expressed m RNAs(Differentially expressed m RNA,DE m RNA),of which 20 miRNA expressions were up-regulated and 4 miRNA expressions were down-regulated;the first 6 miRNA molecules with significant differences were further verified by q RT-PCR,and the results showed that DOX significantly up-regulated the expression of let-7i-3p in AC16 cells compared with the control group(P<0.05),and the above results were consistent with the sequencing results;2.Up-and down-regulation of let-7i-3p expression in AC16 cells was successfully achieved using let-7i-3p mimic and inhibitor transfections.Compared with the un-transfected group and transfection-negative control group,AC16 cells treated with DOX and up-regulated let-7i-3p expression showed reduced viability,increased ROS levels and MDA content in cells,while SOD activity decreased and apoptosis and autophagy increased(P<0.05).AC16 cells with down-regulated let-7i-3p expression showed increased viability,decreased ROS levels and MDA content in cells,while SOD activity increased and apoptosis and autophagy decreased(P<0.05);3.We tentatively predicted that ribosomal protein S6 kinase2(RPS6KA2)is the target gene of let-7i-3p via using bioinformatics software combined with RNA-seq results.qRT-PCR and Western blot experiments showed that after upregulating the expression of let-7i-3p,the m RNA expression and protein levels of RPS6KA2 were significantly down-regulated,while the protein levels of RPS6KA2 were significantly up-regulated after down-regulating the expression of let-7i-3p.Furthermore,correlation analysis showed that let-7i-3p was significantly negatively correlated with RPS6KA2(r=-0.80,p<0.01);4.We found that let-7i-3p could not only negatively regulate RPS6KA2 expression in AC16 cells,but also mediated the DOX-induced autophagy in AC16 cells by downregulating RPS6KA2 protein expression and inhibiting the m TOR signaling pathway.Conclusions:1.let-7i-3p is significantly differentially expressed in AC16 cardiomyocytes before and after DOX treatment;2.let-7i-3p reduced the viability of AC16 cells under DOX treatment conditions and promoted oxidative stress,apoptosis and autophagy in AC16 cells;3.let-7i-3p exacerbated DOX-induced cardiotoxicity by negatively regulating RPS6KA2,which was achieved in part by promoting oxidative stress,apoptosis,and m TOR signaling pathway-mediated autophagy in cardiomyocytes.