Bone Marrow Mesenchymal Stem Cell Derived Exosomes Alleviate Doxorubicin-induced Cardiotoxicity Via Regulating LncRNA-mediated Inflammation

Objective: To determine the therapeutic effect of bone marrow mesenchymal stem cellderived exosomes(BMSC-exos)on doxorubicin-damaged cardiac cells by mediating lncRNA,and to clarify the underlying mechanism.To provide a novel exosome-based strategy for alleviating doxorubicin-induced cardiotoxicity(DIC).Methods:(1)The neonatal primary cardiomyocytes were treated DOX for 1 day,which was defined as DOX group;BMSC and DOX injured cardiomyocytes were co-cultured for 2days,which was defined as BEC group.RNA of the two groups was extracted and converted into c DNA by reverse transcription,and high-throughput sequencing and transcriptome bioinformatics analysis of lncRNA were performed.(2)Transcriptome library quality assessment and gene expression analysis were performed using bioinformatics software to determine the number,classification,expression level and conservatism of lncRNAs in cardiomyocytes of the two groups;(3)Overall assessment of inter-group and intra-group gene correlation of two groups was conducted by Pearson’s Correlation Coefficient,and differences of intracellular long non-coding RNA(lncRNA)between the two groups were analyzed using R language.Fold Change(FC)≥ 1.5 and P < 0.5 were selected as the differential screening criteria,and elevated lncRNA(ElncRNA)in BEC compared with DOX group was selected as the target.(4)Based on the location and co-expression relationship between lncRNA and its target genes,we predicted two target genes of ElncRNA,named cis＿targets and trans＿targets.The predicted target genes were analyzed by Gene Ontology(GO)and the Kyoto Encyclopedia of Genes and Genomes(KEGG).(5)Based on the GO and KEGG analysis results,we identified the key cellular biological pathways and related signaling pathways during the function of BMSC-Exos.(6)CCK-8assay was used to detect the activity of cardiomyocytes in the DOX and BEC group,ROS assay was used to detect the oxidative stress level of cardiac cells in DOX and BEC group.Real-time fluorescence quantitative polymerase chain reaction(q RT-PCR)was used to detect the expression levels of inflammation-related m RNA(IL-1β,IL-6 and TNF-α)in cardiomyocytes in the DOX and BEC group.The top 10 elevated lncRNA(ElncRNAs)in the BEC group were selected compared with the DOX group,and the expression trend of lncRNA in DOX and BEC groups was verified by q RT-PCR.(7)The selected lncRNA target genes were predicted and the lncRNA-m RNA network was constructed,and the intersection of target genes was determined to identify common target genes for subsequent research.Results:(1)A total of 34,874 lncRNAs were predicted in the two groups,of which linc-and intra-lncRNAs were the main types of predicted lncRNAs.(2)Compared with DOX group,169 lncRNAs in the BEC group were up-regulated and 132 lncRNAs were downregulated;(3)GO enrichment analysis showed that cis＿targets of ElncRNA were mainly enriched in inflammatory biological processes,while trans＿targets were mainly involved in apoptosis,intracellular transport and chemotaxis.(4)KEGG enrichment results show that cis＿targets of ElncRNAs are mainly enriched in oxidative stress,inflammation and myocardial cell proliferation pathways,while trans＿targets are mainly involved in cancerrelated pathways,apoptosis,inflammation and fatty acid metabolism pathways.(5)Compared with DOX group,viability of cardiomyocytes in the BEC group was recovered,oxidative stress level was decreased and IL-6 expression was down-regulated.Two lncRNAs,MSTRG.98097.4 and MSTRG.58791.2,were down-regulated in DOX group but increased in BEC group(P < 0.05).Conclusions: Bone marrow mesenchymal stem cell-derived exosomes inhibit the inflammatory response of cardiomyocytes by mediating lncRNA,thereby alleviating DIC.In addition,two lncRNAs,MSTRG.98097.4 and MSTRG.58791.2,which may be derived from exosomes and regulate DIC,were preliminarily screened out in this study.