Cardiovascular Toxicology (2019) 19:264–275 https://doi.org/10.1007/s12012-018-9495-6 MiR-15b-5p is Involved in Doxorubicin-Induced Cardiotoxicity via Inhibiting Bmpr1a Signal in H9c2 Cardiomyocyte Guo‑xing Wan1 · Lan Cheng1 · Hai‑lun Qin1 · Yun‑zhang Zhang1 · Ling‑yu Wang1 · Yong‑gang Zhang1 Published online: 7 December 2018 © Springer Science+Business Media, LLC, part of Springer Nature 2018 Abstract The wide use of anthracyclines represented by doxorubicin (DOX) has benefited cancer patients, yet the clinical applica- tion is limited due to its cardiotoxicity. Although numerous evidences have supported a role of microRNAs (miRNAs) in DOX-induced myocardial damage, the exact etiology and pathogenesis remain largely obscure. In this study, we focused on the role of miR-15b-5p in DOX-induced cardiotoxicity. We employed a public miRNA and gene microarray to screen differentially expressed miRNAs (DEMs) and differentially expressed genes (DEGs) in rat cardiomyocytes, and 33 DEMs including miR-15b-5p and 237 DEGs including Bmpr1a and Gata4 were identified. The Gene ontology (GO) and pathway enrichment analysis of 237 DEGs indicated that the DEGs were mainly enriched in heart development and ALK pathway in cardiomyocyte which included the main receptor Bmpr1a and transcription factor Gata4. The up-regulated miR-15b-5p and down-regulated Bmpr1a and Gata4 mRNA expressions were further validated in H9c2 cardiomyocytes exposed to DOX. Moreover, the results showed overexpression of miR-15b-5p or inhibition of Bmpr1a may enhance the DOX-induced apop- tosis, oxidative stress and mitochondria damage in H9c2 cardiomyocytes. The Bmpr1a was suggested as a potential target of miR-15b-5p by bioinformatics prediction. We further verified the negatively regulatory effect of miR-15b-5p on Bmpr1a signaling. Moreover, we also confirmed that overexpression of miR-15b-5p may exacerbate the DOX-induced apoptosis of H9c2 cardiomyocytes by affecting the protein expression ratio of Bcl-2/Bax and Akt activation, while this pro-apoptotic effect was able to be suppressed by Bmpr1a agonist. Collectively, the results suggest that miR-15b-5p is likely involved in doxorubicin-induced cardiotoxicity via inhibiting Bmpr1a signaling in H9c2 cardiomyocytes. Our study provides a novel insight for investigating DOX-induced cardiotoxicity. Keywords MiR-15b-5p · Bmpr1a · Doxorubicin · Cardiotoxicity Introduction m2, the incidence of heart failure or left ventricular dys- function rises from 5 to 48% [2], which may impair the sur- Doxorubicin (DOX) is one of the most widely used and vival benefit DOX treatment brings. Current evidences have forceful anthracycline agents, delivering anti-cancer activ- proposed several mechanisms accounting for DOX-induced ity against numerous types of malignancy. However, the cardiotoxicity, including oxidative stress, inflammation, lipid clinical benefit of DOX treatment has been limited by its peroxidation, mitochondrial impairment, intracellular cal- cumulative and dose-dependent cardiotoxicity [1]. With an cium dysregulation and inhibitory topoisomerase II (Top2), accumulation dose of DOX increased from 400 to 700 mg/ which may be involved in irreversible myocardial damage and apoptosis [3–5]. Nevertheless, the exact mechanisms remain poorly understood. Handling Editor: Gen Suzuki. MicroRNAs (miRNAs) as key players in gene expres- sion regulation by altering the translation or stability of tar- * Yong-gang Zhang get mRNAs have been found functionally participated in [email protected] heart development, function, and diseases including DOX- 1 Department of Cardiology, The Second Affiliated induced cardiotoxicity [6]. Recently, miR-15b was found up- Hospital of Shantou University Medical College, regulated in the mice ischemia reperfusion (I/R) model [7]. Dongxia North Road, Shantou 515041, Guangdong, Similarly, miR-15b was also reported to enhance hypoxia/ People’s Republic of China Vol:.(1234567890)1 3 Cardiovascular Toxicology (2019) 19:264–275 265 reoxygenation (H/R)-induced apoptosis of cardiomyocytes Gene Ontology (GO) and Pathway Enrichment via a mitochondrial apoptotic pathway [8]. Conversely, the Analysis of DEGs suppression of miR-15b was able to prevent rapid loss of cardiac function in an induced cardiac dysfunction model GO enrichment analysis of DEGs was implemented with the in adult mice [9]. Moreover, the inhibition of miR-15b was clusterProfiler package [14]. GO terms (molecular function found capable of decreasing apoptosis in neonatal rat cardiac and biological processes) with adjust p value < 0.05 were myocytes by modulating cellular adenosine triphosphate considered significantly enriched by DEGs. The clusterPro- (ATP) level and mitochondrial integrity [10]. Consequently, filer involving Kyoto Encyclopedia of Genes and Genomes the current findings suggested that the manipulation of miR- (KEGG), Reactome and Molecular Signatures Database 15b was likely developed for therapeutic approaches [9]. (MSigDb) pathway datasets was used to perform pathway However, the role of miR-15b in DOX-induced cardiotoxic- enrichment analysis to expound promising signaling path- ity remains largely unknown. ways correlated with the DEGs, and p value < 0.05 with In this study, the differentially expressed miRNAs in adjust p value < 0.05 were defined as the cut-off criteria. DOX-treated cardiomyocytes were identified by using a public microarray dataset. We focused on the role of miR- Bioinformatics Prediction of miR‑15b‑5p Targets 15b in the cardiomyocytes exposed to DOX, and explored the underlying mechanism, which may provide a valuable First, to list the putative targets of miR-15b-5p, a vast insight to protect against DOX-induced cardiotoxicity. search in mirWalk database (http://zmf.umm.unihe idelb erg.de/apps/zmf/mirwa lk2/) was performed, in which the miRwalk, miRanda, miRNAMap and Targetscan databases Materials and Methods were included. Each target gene must be searched in at least three databases. Commonly, miRNAs negatively regulated Chemicals and Materials the expression of its targets. The candidate targets were nar- rowed down by an intersection with the DEGs and were DOX was obtained from Hisun-Pfizer Pharmaceutical Co., further selected by a negative correlation with miR-15b-5p Ltd. (Zhejiang, China), which was dissolved in PBS. Protein expression. Extraction Kit was obtained from KEYGEN Biotech. Co., Ltd. (Nanjing, China). The ROS assay kit, mitochondrial Cell Culture and Transfection Experiments membrane potential assay kit with JC-1, cell lysis buffer and phenylmethanesulfonyl fluoride (PMSF) were obtained The H9c2 cardiomyocytes were obtained from the Shang- from Beyotime Institute of Biotechnology (Jiangsu, China). hai Institutes for Biological Sciences (Shanghai, China), The Annexin V-FITC Apoptosis Detection Kit was pur- routinely grown in Dulbecco’s Modified Eagle (DMEM) chased from BestBio Science (Shanghai, China). 20 ng/ml medium supplemented with 12% fetal bovine serum in a recombinant human bone morphogenetic protein (rhBMP, humidified atmosphere of 5% CO at 37 °C. The cardiomyo- Bmpr1a agonist) was purchased from PrimeGene Biotech- 2 cytes were exposed to DOX at the concentration of 6 µM for nology (Shanghai, China), and the 30 nM Bmpr1a inhibitor 24 h as previously described [2, 15]. H9c2 cardiomyocytes LDN193189 was obtained from MedChemExpress China at 80% confluence were transfected with rno-miR-15-5p (Shanghai, China). mimics (5′-UAG CAG CAC AUC AUG GUU UACA-3′) or rno-miR-15-5p inhibitors (5′-UGU AAA CCA UGA UGU Microarray Analysis GCU GCUA-3′), or scrambled controls (Gene Pharma, Shanghai, China), respectively, at a concentration of 20 µM The miRNA(GSE36239) and gene expression with the use of Lipofectamine 2000(Invitrogen) according profile(GSE42177) were downloaded from the public GEO to the manufacturer’s protocol. All the transfections were database (www. ncbi.nlm.gov/geo/) [1, 11], The selected performed subsequent to the rhBMP (Bmpr1a agonist) or array data in GSE36239 dataset consisted of three primary LDN193189 (Bmpr1a inhibitor) treatment for 30 min. rat cardiomyocytes samples treated with DOX and three controls, and similar design and treatment were given in Apoptosis Assay, Measurement of Mitochondrial GSE42177. After data processing as previous [12], dif- Membrane Potential (MMP), and Intracellular ROS ferentially expressed miRNAs (DEMs) and differentially expressed genes (DEGs) were identified by significance The apoptosis, oxidative stress, and mitochondria dam- analysis of the microarrays with the limma package [13], age were common hallmarks of DOX-induced myocardial following the criteria p < 0.05 and |logFC| > 1. 1 3 266 Cardiovascular Toxicology (2019) 19:264–275 damage. To examine the influence of interfering miR- Western Blotting Assay 15b-5p or Bmpr1a on apoptosis, MMP and ROS, the experiment was grouped as follows: (1) for interfering To examine the effect of miR-15b-5p targeting Bmpr1a miR-15b-5p: control, DOX, DOX + miR-15b-5p mimic signal on cell apoptosis and survival of H9c2 cardiomyo- (20 µM), DOX + miR-15b-5p inhibitor(20 µM); (2) for cytes, the Bcl-2/Bax protein expression ratio (for anti- interfering Bmpr1a: control, DOX, DOX + rhBMP (20 ng/ apoptosis) and Akt activation (for survival) was detected ml), DOX + LDN193189(30 nM). The H9c2 cardiomyo- by western blotting, and the experiment was designed as cytes were plated in 6-well culture plates at a density of that in the RT-qPCR array. The extraction and detection 5 × 104 cells/mL and then treated with DOX