MicroRNA‐129‐1‐3p protects cardiomyocytes from pirarubicin‐induced apoptosis by down‐regulating the GRIN2D‐mediated Ca2+ signalling pathway

Abstract Pirarubicin (THP), an anthracycline anticancer drug, is a first‐line therapy for various solid tumours and haematologic malignancies. However, THP can cause dose‐dependent cumulative cardiac damage, which limits its therapeutic window. The mechanisms underlying THP cardiotoxicity are not fully understood. We previously showed that MiR‐129‐1‐3p, a potential biomarker of cardiovascular disease, was down‐regulated in a rat model of THP‐induced cardiac injury. In this study, we used Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genome (KEGG) pathway enrichment analyses to determine the pathways affected by miR‐129‐1‐3p expression. The results linked miR‐129‐1‐3p to the Ca2+ signalling pathway. TargetScan database screening identified a tentative miR‐129‐1‐3p‐binding site at the 3′‐UTR of GRIN2D, a subunit of the N‐methyl‐D‐aspartate receptor calcium channel. A luciferase reporter assay confirmed that miR‐129‐1‐3p directly regulates GRIN2D. In H9C2 (rat) and HL‐1 (mouse) cardiomyocytes, THP caused oxidative stress, calcium overload and apoptotic cell death. These THP‐induced changes were ameliorated by miR‐129‐1‐3p overexpression, but exacerbated by miR‐129‐1‐3p knock‐down. In addition, miR‐129‐1‐3p overexpression in cardiomyocytes prevented THP‐induced changes in the expression of proteins that are either key components of Ca2+ signalling or important regulators of intracellular calcium trafficking/balance in cardiomyocytes including GRIN2D, CALM1, CaMKⅡδ, RyR2‐pS2814, SERCA2a and NCX1. Together, these bioinformatics and cell‐based experiments indicate that miR‐129‐1‐3p protects against THP‐induced cardiomyocyte apoptosis by down‐regulating the GRIN2D‐mediated Ca2+ pathway. Our results reveal a novel mechanism underlying the pathogenesis of THP‐induced cardiotoxicity. The miR‐129‐1‐3p/Ca2+ signalling pathway could serve as a target for the development of new cardioprotective agents to control THP‐induced cardiotoxicity.


| INTRODUC TI ON
Anthracyclines are a fundamental class of antineoplastic drugs that are used to treat more types of cancer than any other form of chemotherapy. 1 These drugs act by intercalating into DNA and interacting with topoisomerase II, thereby blocking DNA replication, RNA transcription and protein synthesis. 2 However, anthracycline administration is often accompanied by dose-dependent and cumulative cardiotoxicity, ranging from transient cardiac dysfunction to congestive heart failure. 3 According to the 2011 report from the China Society of Clinical Oncology (CSCO), 4 more than 50% of patients who have received an anthracycline treatment within the past 6 years have developed subclinical changes in left ventricular function and structure as demonstrated by echocardiographic evaluation. Pirarubicin (THP) is a fourth-generation anthracycline that is less cardiotoxic than the first-generation anthracyclines, including doxorubicin (DOX), 5 and is therefore widely used in clinical practice.
However, patients who receive THP treatment can still suffer significant cardiac injuries. 6,7 Thus, novel therapeutic agents that help combat THP-induced cardiotoxicity are required.
MicroRNAs (miRNAs) are small non-coding RNAs that regulate gene expression by interacting with the 3′-UTR of mRNA molecules. miRNAs have been shown to regulate cardiac physiology and pathology. [17][18][19] Several miRNAs have been implicated in cardiotoxicity caused by anthracyclines. 20,21 For example, miR-21 prevents DOX-induced cardiomyocyte apoptosis by targeting BTG2, 22 while miR-208a mediates DOX-induced cardiotoxicity through regulating GATA4. 23 Additional in vitro and in vivo studies have implicated miR-532-3p, miR-34a-5p and miR-451 in DOX-induced cardiotoxicity. 18,19 We recently performed a microarray analysis on myocardial miR-NAs in a rat model of THP-induced myocardial injury and identified 78 dysregulated miRNAs in the THP group compared with control (P < .05, |log2(fold change) | > 0.585), of which 50 were up-regulated and 28 were down-regulated. 24 In this study, we performed Gene Ontology (GO) enrichment analysis of the 28 miRNAs down-regulated by THP using databases in the public domain (http://www.mirba se.org/, http:// www.micro rna.gr/LncBa se/, https ://david.ncifc rf.gov/, and http:// www.targe tscan.org/vert_71/). The results revealed that miR-129-1-3p, which is a potential biomarker of cardiovascular disease, 25 is highly conserved across species and potentially targets GRIN2D (also known as NMDAR2D, NR2D or GluN2D). GRIN2D is a subunit of the NMDA (N-methyl-D-aspartate) receptor complex, which forms ligand-gated ion channels with high calcium permeability. 26 Considering that calcium overload is a contributing factor in anthracycline-induced myocardial injury, we speculated that miR-129-1-3p might play a role in THP-induced cardiotoxicity by regulating GRIN2D and calcium homoeostasis. In this study, we investigated the function of miR-129-1-3p in THP-induced cardiomyocyte apoptosis and the underlying molecular mechanisms involving GRIN2Dmediated Ca 2+ signalling.

| Cell culture, transfection and THP treatment
The H9C2 rat cardiomyoblast cell line and the mouse HL-1 cardiac muscle cell line were obtained from Shanghai Institute of Cell Biology and Otwo Biotech Inc, respectively. The cells were maintained in Dulbecco's Modified Eagle Medium (DMEM; Hyclone) with 10% foetal bovine serum (FBS; Hyclone) and penicillin-streptomycin.
All cells were cultured at 37°C in a humidified atmosphere containing 5% CO 2 .
The miRNAs were transfected into H9C2 and HL-1 cells for 8 hours in serum-free medium using Lipofectamine 2000 (Beijing TransGen Biotech Co., Ltd.) following the manufacturer's instructions. The serum-free medium was replaced with fresh normal medium after transfection was completed. The transfection efficacy was calculated as the ratio of the number of positive nuclei to the total number of nuclei.

| Cell viability assay
To test the effects of THP on cell viability, THP working solutions were prepared in DMEM. The cells were seeded in 96-well plates (5 × 10 4 cells/well) and cultured for 24 hours. The cells were subsequently treated with THP (0-10 μmol/L) for 24 hours. Cell viability was evaluated using the CCK-8 assay kit on a Bio-Rad POLARstar microplate reader.

| Measurement of intracellular ROS levels
Intracellular ROS levels were determined using the DCFH-DA ROS assay kit. In brief, the cells were plated in six-well plates at a density of 5 × 10 4 cells/well and cultured for 24 hours. The cells were subsequently treated with THP (5 μmol/L) for 24 hours. After that, the medium was removed, and 1.5 mL of DCFH-DA (10 μmol/L) was added.
The cells were incubated at 37°C for 30 minutes and subjected to analysis under a BX43 fluorescence microscope (Olympus; Tokyo, Japan) at 100 × magnification.

| TUNEL assay
Following treatment, cells were fixed in 4% paraformaldehyde and permeabilized in 0.1% Triton-X 100. Cell apoptosis was assessed using the TUNEL Apoptosis Detection kit. The cells were counterstained with DAPI and analysed under a BX43 fluorescence microscope at 100× magnification. The apoptosis rate was calculated as the ratio of TUNEL-positive nuclei to total nuclei.

| Bioinformatics analysis
GO and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analyses were performed using public domain databases (https ://david.ncifc rf.gov/). Pathway regulation networks were visualized using Cytoscape software mapping. Potential targets for rat or mouse miR-129-1-3p were identified using TargetScan database screening.

| Measurement of intracellular calcium levels
Intracellular calcium levels were measured using the fluorescent Ca 2+ -sensitive dye Fluo-3 AM. Briefly, after washing with PBS, the cells were loaded with 5 μmol/L Fluo-3 AM at 37°C for 30 minutes and examined under a BX43 fluorescence microscope at 100× magnification. Intracellular Ca 2+ accumulation was evaluated as the ratio of Fluo-3 AM-positive nuclei to total nuclei.

| Dual-luciferase reporter assay
The wild-type 3′-untranslated region (UTR) of GRIN2D containing putative binding sites for miR-129-1-3p was PCR-amplified using genomic DNA from HL-1 cells. The corresponding mutant 3′-UTR was created by altering the seed regions of the miR-129-1-3p binding sites. The wild-type and mutant 3′-UTRs were subcloned into the psiCHECK-2 luciferase vector downstream of the luciferase gene. Both constructs were verified by DNA sequencing. HL-1 cells were cotransfected with the miR-129-1-3p mimics or miR-129-1-3p inhibitor and the luciferase plasmid comprising the wild-type or mutant 3′-UTR in 24-well plates. Luciferase activity was determined 48 hours after transfection using the Dual-Luciferase Reporter Assay System (Promega) and normalized to Renilla activity.
Each RNA sample was reverse transcribed into cDNA using the TransStart Top Green qPCR SuperMix kit (Beijing TransGen Biotech).
The levels of GRIN2D mRNA and GAPDH mRNA were determined by qRT-PCR. Total miRNA was isolated using the SanPrep  Tables   2 and 3. Relative expression levels of GRIN2D and miRNA-129-1-3p were calculated using the 2 −ΔΔCt method and normalized to GAPDH and U6, respectively.

| Western blot analysis
Cells were lysed in RIPA lysis buffer, and the protein concentrations were determined using the BCA method. The samples were subjected to SDS-PAGE and transferred to PVDF membranes (Millipore). After blocking in 5% skim milk, the membranes were probed with primary antibodies (Table 4) overnight at 4°C, followed by anti-rabbit secondary antibody (

| Data analysis
All results are presented as mean ± standard deviation (SD). Data analysis was performed with the SPSS 19.0 and GraphPad 8.0 software.
The Student's t test or one-way ANOVA was applied to compare data from different groups. Statistical significance was defined as P < .05.

| THP induces cardiomyocyte injury
In accordance with reported THP cardiotoxicity, 24-hour THP treatment dose-dependently reduced H9C2 and HL-1 cell viability as indicated in the CCK-8 assay ( Figure 1A

| THP down-regulates miR-129-1-3p in cardiomyocytes
A recent miRNA microarray analysis performed in our laboratory revealed that miR-129-1-3p was down-regulated by THP in a rat model of THP-induced myocardial injury. 24 We further examined the effects of THP on miR-129-1-3p expression in cardiomyocytes using qRT-PCR. After 24-hour treatment with 5 μmol/L THP, miR-129-1-3p levels in H9C2 and HL-1 cells were reduced to 41% and 32% of control, respectively ( Figure 1D,E). Together, these in vitro and in vivo results implicate miR-129-1-3p in the pathogenesis of THP-induced cardiomyocyte injury.

| MiR-129-1-3p is linked to the Ca 2+ pathway by directly targeting GRIN2D
To uncover the molecular mechanisms underlying the protective effects of miR-129-1-3p in cardiomyocytes, we performed GO enrichment analysis of miR-129-1-3p and thereby revealed a potential link  Figure 4D). Subsequent TargetScan database screening identified a potential miR-129-1-3p-binding site at the 3′-UTR of GRIN2D mRNA ( Figure 4B). Considering that GRIN2D is a subunit of the NMDA receptor Ca 2+ channel, we speculated that miR-129-1-3p regulates Ca 2+ influx in THP-treated cardiomyocytes by directly targeting GRIN2D.

| MiR-129-1-3p inhibits THP-induced calcium overload in cardiomyocytes
To test the regulatory function of miR-129-1-3p in Ca 2+ signalling in cardiomyocytes, we assessed intracellular calcium levels using fluorescence microscopy combined with the Ca 2+ -sensitive dye Fluo-3 AM. As shown in Figure 6A (Figures 3 and 6C,D). Together, these results demonstrate that miR-129-1-3p mitigates THP-induced cardiomyocyte injury by inhibiting THP-induced calcium overload and activation of Ca 2+ signalling in cardiomyocytes.  29 Hence, efforts to target STAT3 in breast cancer had little success in the past because of the potential adverse effects, such as cardiotoxicity. THP, a new generation anthracycline antineoplastic drug, is frequently used to treat various solid tumours and haematologic malignancies. 30 Although THP is more effective and less cardiotoxic than DOX, 5 its cardiotoxicity still severely limits  Despite decades of intense research, the pathophysiology associated with anthracycline cardiotoxicity is not fully understood. The leading hypothesis is related to iron-mediated ROS production in cardiac tissues. 33 Indeed, dexrazoxane, an EDTA derivative, is believed to protect against anthracycline cardiotoxicity by chelating iron and inhibiting iron-anthracycline complex formation, consequently decreasing ROS regeneration. 34 Disruption of mitochondrial calcium homoeostasis has also been implicated as a contributing mechanism for anthracycline-induced cardiac injury. 35,36 Mitochondrial calcium overload can lead to mitochondrial permeability transition pore (mPTP) opening, further aggravating oxidative stress. 37 In microscopic studies, Ca 2+ -triggered mitochondria swelling was observed in cardiac tissues that suffered anthracycline-induced damage. 38 Additionally, calcium overload in cardiomyocytes may cause degradation of the myofilament protein titin, leading to sarcomere disruption and cell necrosis, 39 further highlighting the role of calcium overload in the pathogenesis of anthracycline cardiotoxicity.

| D ISCUSS I ON
However, the molecular mechanisms involved in anthracycline-induced calcium overload in cardiomyocytes remain largely unknown.
In the present study, we found that miR129-1-3p, a potential biomarker of cardiovascular disease, 24 is down-regulated by THP in H9C2 and HL-1 cardiomyocytes. Our GO and KEGG pathway enrichment analyses linked miR129-1-3p to the Ca 2+ signalling pathway.
Encouraged by these early results, we searched for potential targets of miR129-1-3p using TargetScan database screening. We identified a tentative miR129-1-3p-binding site at the 3′-UTR of GRIN2D, a subunit of the NMDA receptor calcium channel that is critical for calcium influx. 26 By studying the effects of miR129-1-3p overexpression and knock-down, we confirmed that miR129-1-3p directly regulates GRIN2D to ameliorate calcium overload and apoptosis of cardiomyocytes induced by THP challenge. These bioinformatics

ACK N OWLED G EM ENTS
This work was supported by funding from the National Natural Science Foundation of China (No. 81773934). We thank Yanhou Liu for technical assistance in flow cytometry.

CO N FLI C T O F I NTE R E S T
The authors declare no conflicts of interest. QL wrote the manuscript. LR supervised this study.

DATA AVA I L A B I L I T Y S TAT E M E N T
The data used to support the findings of this study are included within the article.