MicroRNA expression profiling involved in doxorubicin-induced cardiotoxicity using high-throughput deep-sequencing analysis

Abstract

MicroRNAs (miRNAs/miRs) are sensitive biomarkers and endogenous repressors of gene expression by decreasing mRNA stability and interfering with mRNA translation. Despite a number of investigations revealing the dysregulation of miRNA expression associated with cardiotoxicity induced by doxorubicin (Dox), perturbation of miRNAs directly resulting from Dox at early stage in cardiomyocytes and the target gene interaction remain largely unknown. In the present study, high-throughput deep-sequencing was used to analyze changes in global miRNA expression in H9c2 cardiomyocytes exposed to 5 µg/ml Dox for 0, 12 or 24 h. Compared with the 0-h time point, the expression levels of 386 unique miRNAs were altered. Based on miRNA expression and fold-change, the target genes of 76 selected miRNAs were further analyzed using gene interaction networks and pathway enrichment analysis. These miRNAs were involved in the regulation of different pathways, whose functions included apoptosis, cell proliferation, extracellular matrix remodeling, oxidative stress and lipid metabolism. These differentially expressed miRNAs included let-7 family, miR-29b-3p, miR-378-3/5p, miR-351-3p, miR-664-3p, miR-455-3p, miR-298-3p, miR-702-5p, miR-128-1-5p, miR-671 and miR-421-5p. The present data indicated that global wide miRNA profiling in Dox-induced cardiomyocytes may provide a novel mechanistic insight into understanding Dox-induced heart failure and cardiotoxicity, as well as novel biomarkers and therapeutic targets.

Keywords: cardiomyocyte; cardiotoxicity; doxorubicin; microRNA.

Figure 1.

Characteristics of miRNAs in Dox-induced H9c2 cells. (A) Procedure of Dox treatment in H9c2 cells. (B) Venn diagram of miRNA expression at each time point. (C) Heatmap of the 198 pairs of miRNAs with-3p and −5p mature forms in each sample. Normalized reads per million values were used. 0A/B/C, 12A/B/C and 24A/B/C represent samples collected at 0, 12 and 24 h of Dox treatment, respectively. miRNA, microRNA; Dox, doxorubicin.

Figure 2.

Dynamics of miRNAs in H9c2 cells during Dox treatment. Cluster analysis of miRNAs with altered miRNA expression, determined using high-throughput sequencing, were analyzed at 0, 12 and 24 h after Dox treatment. (A) A total of 76 differentially expressed miRNAs (RPM >10) were identified, whose expression levels changed ≥1.5-fold compared with the 0-h time point. The miRNA RPM values were normalized with log10-transformation before using cluster analysis. The 76 miRNAs were classified into 4 clusters (clusters 1–4) using the Hierarchical Clustering Explorer v3.5 software. (B) Dynamic miRNA expression curves for each cluster. miR/miRNA, microRNA; RPM, reads per million; Dox, doxorubicin.

Figure 3.

Representative GO and KEGG pathway enrichment analysis of differentially expressed miRNAs. (A) GO and (B) KEGG pathway analysis of upregulated miRNAs after Dox treatment, with the top 9 biological processes and 6 pathways presented, respectively. (C) GO and (D) KEGG pathway analysis of downregulated miRNAs after Dox treatment, with the top 15 biological processes or the top 15 pathways presented. GO, Gene Ontology; KEGG, Kyoto Encyclopedia of Genes and Genomes; miRNA, microRNA; Dox, doxorubicin.

Figure 4.

Interaction network of targeted genes downstream of upregulated miRNAs. A total of 7 miRNAs and their targets were included in the interaction network. The interaction network was visualized using the Cytoscape software. Rectangle nodes denote miRNAs, and oval nodes denote genes. miR/miRNA, microRNA.

Figure 5.

Interaction networks of targeted genes downstream of downregulated miRNAs. A total of 22 miRNAs and their targets were included in the interaction network. The interaction network was visualized using the Cytoscape software. Rectangle nodes denote miRNAs, and oval nodes denote genes. miR/miRNA, microRNA.

Figure 6.

Model of Dox-induced cardiotoxicity through profiles of miRNA expression in cardiomyocyte cells. In cardiomyocyte cells, Dox may directly disturb the homeostasis of miRNA expression. Differentially expressed miRNAs, such as miR-145-5p and miR-29b-3p, may be enriched in several biological processes associated with autophagy, ROS accumulation and ECM remodeling. miR/miRNA, microRNA; ROS, reactive oxygen species; ECM, extracellular matrix; Dox, doxorubicin.