Identification Of Plasma-derived Exosomal Biomarkers Associated With The Anthracycline-Induced Liver Injury In Breast Cancer Patients

Objective:Anthracycline-induced liver injury（AILI）is one of the serious complications of adjuvant chemotherapy for postoperative breast cancer patients.Exosomes,as signaling molecules in intercellular communication,play essential roles in drug-induced liver injury（DILI）.This study investigated the potential of exosomal contents as AILI biomarkers.Methods:A total of 70 post-chemotherapy breast cancer patients were recruited in this study.After isolated plasma-derived exosomes,RNA sequencing revealed the exosomal mRNA,circRNA,and miRNA profiles and differentially expressed mRNA,circRNA,and miRNA were identified between the non-liver injury group and liver injury group.In the study of mRNA,GO and KEGG enrichment analyses of differentially expressed mRNAs were performed using the DAVID database.The protein-protein interaction network（PPI network）of differentially expressed mRNAs was established by the STRING database,and exosomal mRNAs that play an important role in AILI were identified by Cytoscape software.In the study of circRNA,qRT-PCR was applied to verify the differentially expressed circRNAs.The CircInteractome database and the CSCD database were employed to predict the target miRNA of circRNA,and the miRTarBase database was used to query the target mRNA of miRNA.The target mRNAs were intersected with DILIrelated genes in the CTD database,and the circRNA-miRNA-mRNA regulatory networks were constructed according to the interaction relationship.GO and KEGG enrichment analyses were performed on mRNAs using the DAVID database to predict the regulatory function of circRNA.In miRNA research,the miRTarBase database and the miRDB database were used to predict the target genes of differentially expressed miRNAs.The intersection of target genes and DILI-related genes were defined as AILI-related target genes.GO and KEGG enrichment analyses of AILI-related target genes were performed using the DAVID database.The STRING database was used to establish the PPI network of AILI-related target genes,and the Cytoscape software was used to identify exosomal miRNAs that play an important role in AILI.Results:There were 443 mRNAs differentially expressed between the non-liver injury group and the liver injury group.The results of enrichment analysis showed that differentially expressed mRNAs were significantly enriched in Notch signaling pathway,positive regulation of interleukin-10 production,regulation of mitochondrial membrane potential,response to reactive oxygen species,copper ion binding,glutathione metabolism,T cell receptor signaling pathway,metabolism of xenobiotics by cytochrome P450 and other liver injury-related pathways.Among the 10 hub genes screened from the PPI network,ALB,KRT8 and VEGFA were DILI-related genes.There were 34 circRNAs differentially expressed between the non-liver injury group and the liver injury group.PCR results showed that the expressions of circ₀005130 and circ₀094333 were consistent with the sequencing results.A total of 13 circRNA-miRNA-mRNA regulatory networks were predicted by bioinformatics analysis,through which circRNAs can participate in the regulation of response to toxic substance,oxidation-reduction process and other biological functions.There were 30 miRNAs differentially expressed between the non-liver injury group and the liver injury group,and there were 79 AILI-related target genes of miRNA.AILI-related target genes were significantly enriched in NOD-like receptor signaling pathway,HIF-1 signaling pathway and FoxO signaling pathway.In the PPI network,IL-6 and SOD2 are the most important hub genes,which can participate in the occurrence and development of AILI by activating immune response and oxidative stress response,respectively.In the miRNA-hub gene network,miR-1-3p can bind multiple hub genes and play an important regulatory role.Conclusions:ALB,KRT8,VEGFA,circ₀005130,circ₀094333 and miR-1-3p may play an important role in AILI,and these molecules have the potential to be biomarkers for diagnosis or prognosis prediction of AILI.The pathogenesis of AILI may be the activation of biological processes such as oxidative stress,reduced levels of antioxidant enzymes,mitochondrial dysfunction,and inflammation.