Introduction
Arsenic trioxide (ATO) is an important clinical therapeutic drug for leukemia [
1] and liver cancer [
2,
3], which also has therapeutic potential for breast cancer [
4,
5], lung cancer [
6,
7], and gastric cancer [
8,
9], etc. However, ATO can induce some toxic or side effects, including cardiotoxicity [
10,
11], liver toxicity [
12], and kidney toxicity [
13]. Among them, cardiotoxicity is the main reason that limited the clinical use of ATO [
14,
15]. However, the mechanism underlying ATO-induced cardiotoxicity has not been fully elucidated.
Noncoding RNAs, such as microRNAs (miRNAs) and long noncoding RNAs (lncRNAs), play pivotal roles in various cardiac diseases, such as cardiac hypertrophy, myocardial infarction, and heart failure [
16‐
18]. MiRNAs can bind to the 3’UTR of target genes, thus regulating gene expression. LncRNAs can exert gene regulatory functions in different ways. In the competing endogenous RNA (ceRNA) mechanism, noncoding RNAs (such as lncRNAs) can interact with miRNAs, thus regulating target mRNA expression. This mechanism is considered the Rosetta Stone of a hidden RNA language [
19]. Many lncRNAs are involved in cardiac apoptosis through competitive binding with miRNAs [
20]. For example, the lncRNA MIRF contributes to cardiac apoptosis through regulation of the miR-26a-Bak1 signaling pathway [
21].
One of the major mechanisms of ATO-induced cardiotoxicity is induction of apoptosis [
22‐
24]. Noncoding RNAs are also involved in ATO-induced cardiotoxicity. LncRNA NEAT1 was found to be downregulated in ATO-treated H9c2 cardiomyocytes. Enhanced expression of lncRNA NEAT1 protected H9c2 cardiomyocytes against ATO-induced injury through the miR-124/NF-κB signaling pathway [
25]. Our previous study demonstrated that ATO-induced QT interval prolongation of electrocardiograms (ECGs) was related to inhibition of lncRNA Kcnq1ot1 [
26]. In addition, recent studies verified that lncRNA Kcnq1ot1 contributes to the apoptosis process [
27,
28]. However, the involvement of lncRNA Kcnq1ot1 in ATO-induced apoptosis of cardiomyocytes remains unclear. Therefore, based on our previous work and the existing findings, the present study aims to clarify the underlying mechanism and to explore the therapeutic potential of lncRNA Kcnq1ot1 in ATO-induced cardiomyocytes apoptosis.
Materials and methods
Animals and treatment
C57BL/6 mice (20–22 g) were obtained from Liaoning Changsheng Biotechnology Co., Ltd. (China). The experimental procedure was approved by the Experimental Animal Ethics Committee of Harbin Medical University, China (No. HMUIRB 20150034). Mice were administered ATO (1.5 mg/kg/day, intraperitoneal injection; Harbin Yida Pharmaceutical Co., Ltd., China) alone or in combination with propranolol (10 mg/kg/day, intragastric administration; YABANG Pharma, China) for 2 weeks.
Primary culture of neonatal mouse cardiomyocytes
Cardiomyocytes were isolated from neonatal mice (1 to 3 days). Myocardial tissues were digested with 0.25% pancreatin (Solarbio, China). After filtering and centrifugation (1500 revolutions per minute at 4 °C for 5 min), the isolated cells were collected and cultured in Dulbecco’s modified Eagle’s medium (DMEM; HyClone, USA) with 10% fetal bovine serum (FBS; BI, Israel) for 1.5 h to remove noncardiomyocytes. Cardiomyocytes were seeded into another culture plate and incubated under 5% CO
2 at 37 °C [
26,
29,
30]. After 48 h, the cardiomyocytes were used for the following experiments.
Treatment and transfection of neonatal mouse cardiomyocytes
ATO (5 μM, Harbin Yida Pharmaceutical Co., Ltd., China) and propranolol (10 μM; Sigma, USA) were added to the culture medium for 48 h. MiR-34a-5p mimic, antisense morpholino oligonucleotide targeting miR-34a-5p (AMO-34a-5p) and the corresponding negative controls (miR-NC and AMO-NC) were biosynthesized by RIBOBIO (China), and the working concentration was 5 nmol/250 μL. Small interfering RNA targeting lncRNA Kcnq1ot1 (si-Kcnq1ot1) and the negative control (si-NC) were biosynthesized by GenePharma (Shanghai, China), and the working concentration was 1 OD
260/125 μL [
26]. For the 12-well plate system, each well required 50 μL Opti-MEM (Gibco, USA), 3 μL X-tremeGENE siRNA Transfection Reagent (Roche, Switzerland) and 9 μL miRNA/siRNA. For the 96-well plate system, each well required 20 μL Opti-MEM, 0.5 μL X-tremeGENE siRNA Transfection Reagent and 1.5 μL miRNA/siRNA. After transfection, the culture plates were placed in an incubator containing 5% CO
2 at 37 °C for 48 h.
CCK-8 assay
Cardiomyocytes were cultured in a 96-well plate at a density of 2.5 × 10
4 cells/well. After treatment, the culture medium of cardiomyocytes was removed, and CCK-8 (DOJINDO, China) solution was added. The plate was incubated at 37 °C for 2.5 h. The OD value of each well was measured at 450 nm. The calculation formulas were as follows: Cell viability (%) = (experiment group−blank well)/(control group−blank well) × 100% [
31]; Inhibition rate (%) = (control group−experimental group)/(control group−blank well) × 100% [
32].
TUNEL assay
Briefly, 5 µm frozen heart tissue slides or cultured cardiomyocytes were fixed in 4% paraformaldehyde for 10 min and then washed with phosphate buffered saline (PBS; HyClone, USA) thrice for 5 min each time, followed by incubation with 0.1% Triton X-100 for 2 min and washing with PBS for 5 min. The slides were blocked with goat serum for 20 min and washed with PBS for 10 min, and TUNEL reaction mixture (50 μL; Roche, Switzerland) was added. The slides were incubated at 37 °C for 1 h, washed with PBS for 15 min, incubated with DAPI for 5 min, and washed with PBS for 10 min. A fluorescence microscope (BX53F; OLYMPUS, China) was used to capture images.
Prediction of miRNA targets
Physical interactions between lncRNAs and miRNAs, and between miRNAs and mRNAs were considered to predict the mechanism of lncRNA Kcnq1ot1. MiRNA targets were predicted using RNAhybrid [
33], Miranda [
34], MIREAP[
35], TargetScan [
36] and ENCORI [
37].
Western blot assay
Total protein was extracted from myocardial tissues and cardiomyocytes [
38]. The protein content in each lane of the same membrane is the same. Each lane was loaded with about 30–50 μg cell protein or about 80–120 μg tissues protein. The protein samples were separated via 10% SDS-PAGE, transferred to an NC membrane (Pall Corporation, USA), and incubated with primary antibodies, including antibodies targeting GAPDH (ZSGB-BIO, China), Bcl-2 (ABclonal, China), Bax (ABclonal, China) and Sirt1 (Abcam, Britain), at 4 °C overnight. Afterward, the membrane was washed and incubated with secondary antibody for 1 h at room temperature. An Odyssey infrared fluorescence scanning imaging system was used to obtain the images. The densitometry of the protein bands was quantified using Image Studio software. GAPDH served as an internal control to normalize protein expression levels. The data were normalized to the control group data.
Real-time PCR assay
Total RNA was extracted from myocardial tissues and cardiomyocytes using RNAiso Plus (Takara, Japan). A NanoDrop spectrophotometer (Thermo Fisher Scientific, USA) was used to detect the RNA concentration and the A260/A280 ratio of the samples. An ABI 7500 Fast Real-time PCR System (ABI, USA) was used to perform qRT-PCR analysis with SYBR Green I (Toyobo, Japan) [
38]. The primer sequences were shown in Table
1. U6 and GAPDH served as internal controls for miRNA and mRNA/lncRNA, respectively. The relative gene expression level was analyzed using the 2
−ΔΔCT method.
Kcnq1ot1 | Forward: 5’-GCACTCTGGGTCCTGTTCTC-3’ Reverse: 5’-CACTTCCCTGCCTCCTACAC-3’ |
Sirt1 | Forward: 5’-GACGCTGTGGCAGATTGTT-3’ Reverse: 5’-GCAAGGCGAGCATAGATACC-3’ |
GAPDH | Forward: 5’-AAGAAGGTGGTGAAGCAGGC-3’ Reverse: 5’-TCCACCACCCTGTTGCTGTA-3’ |
miR-34a-5p | Forward: 5’-GTGGCAGTGTCTTAGCTG-3’ Reverse: 5’-TATCCAGTGCGTGTCGTG-3’ Reverse transcription: 5’-GTCGTATCCAGTGCGTGTCGTGGAGTCGGCAATTGCACTGGATACGACACAACC-3’ |
U6 | Forward: 5’-GCTTCGGCAGCACATATACTAAAAT -3’ Reverse: 5’-CGCTTCACGAATTTGCGTGTCAT-3’ Reverse transcription: 5’-CGCTTCACGAATTTGCGTGTCAT -3’ |
Dual-luciferase reporter assay
HEK-293 cells were seeded in 24-well plates. Transfection was performed using Cellfectin II Reagent (Invitrogen, CA, USA) when the confluence of the cells was approximately 50%−60%. The wild-type (WT) sequence of Kcnq1ot1 is “actgcca”, while the mutation sequence was “ctgattc”. The luciferase density was detected using a Dual-Luciferase Reporter Assay System (GloMax™ 20/20; Promega, WI, USA).
Flow cytometry assay
The cells were digested using trypsin without EDTA, washed with PBS, and resuspended in 1 × binding buffer at a concentration of 2 × 106 cells/mL. The cells were stained using an Annexin V-FITC/PI Apoptosis Detection Kit (4A BIOTECH, Beijing, China) according to the manufacturer’s instructions and detected using a BD FACSCelesta™ flow cytometer. The proportion of apoptotic cells (Annexin V ( +) PI (−)) was analyzed.
Statistical analysis
Data are presented as the mean ± SEM. Each experiment was duplicated at least three times. Comparisons between two groups were analyzed using Student’s t test; and comparisons among three or more groups were analyzed via one-way ANOVA followed by Newman-Keuls multiple comparisons. Statistical significance was defined as P < 0.05.
Discussion
The cardiotoxicity of ATO is still a major problem in its clinical application. However, the involvement of lncRNAs in this process has not been fully clarified. Our previous work discovered that lncRNA Kcnq1ot1 is involved in the cardiotoxicity of ATO [
26]. The present study further explored the role and underlying mechanism of lncRNA Kcnq1ot1 in ATO-induced cardiomyocyte apoptosis.
ATO at 10 mg/day (~ 0.15 mg/kg) is recommended for acute promyelocytic leukemia in the clinic. According to the conversion relationship between humans and mice, 10 mg/day for humans is approximately equal to 1.5 mg/kg for mice. In general, ATO is administered continuously for 2 weeks. Therefore, mice were administered ATO (1.5 mg/kg) for 2 weeks. For in vitro experiments, the dosage of ATO was screened by CCK-8 assay, and the relatively low and effective dose of ATO (5 μM) was selected in our experiments. In ATO-treated mouse cardiomyocytes and myocardial tissues, the number of TUNEL-positive cells was increased, which was associated with increased Bax and decreased Bcl-2 protein expression. These results were in accordance with previous findings that ATO can induce apoptosis of cardiomyocytes [
24,
39,
40]. Then, the target of lncRNA Kcnq1ot1 was predicted based on the ceRNA theory. The results showed that lncRNA Kcnq1ot1 has binding sites with miR-34a-5p. Moreover, Sirt1 is a downstream target of miR-34a-5p. The dual-luciferase reporter assay results showed that mouse-derived miR-34a-5p has a direct binding site with lncRNA Kcnq1ot1. For humans, the direct binding site between lncRNA Kcnq1ot1 and miR-34a-5p was validated using dual-luciferase reporter assay [
41]. In addition, the direct binding site between miR-34a-5p and Sirt1 has previously been validated using dual-luciferase reporter assay [
42,
43]. Therefore, the lncRNA Kcnq1ot1/miR-34a-5p/Sirt1 pathway may be involved in the cardiotoxicity of ATO. The expression of lncRNA Kcnq1ot1 and Sirt1 was downregulated and that of miR-34a-5p was upregulated in ATO-treated mouse myocardial tissues and cardiomyocytes, which is consistent with ceRNA theory.
MiR-34a-5p has been verified to be increased in cardiomyocytes undergoing apoptosis induced by different factors, such as ischemia, hypoxia, and doxorubicin. This enhanced expression can aggravate cardiomyocyte apoptosis, while inhibition of miR-34a-5p can protect against cardiomyocyte apoptosis [
44‐
47]. In our study, miR-34a-5p was overexpressed or inhibited in cardiomyocytes to observe its effect on Sirt1 expression. The upregulation of miR-34a-5p inhibited Sirt1 expression, while the downregulation of miR-34a-5p increased Sirt1 expression. The involvement of the miR-34a-5p/Sirt1 pathway was detected by inhibition of miR-34a-5p in ATO-treated cardiomyocytes. The inhibition of miR-34a-5p alleviated ATO-induced apoptosis in mouse cardiomyocytes and attenuated the inhibitory effect of ATO on Sirt1 expression. Sirt1 is an NAD + -dependent deacetylase that is involved in the regulation of cellular senescence and apoptosis[
48‐
50]. Enhanced Sirt1 expression exerts a protective effect on cardiomyocytes [
50,
51]. In addition, the miR-34a-5p/Sirt1 pathway contributes to doxorubicin-induced cardiomyocyte apoptosis [
47].
The effect of lncRNA Kcnq1ot1 on cardiomyocytes and the miR-34a-5p/Sirt1 pathway was then detected. The results showed that knockdown of lncRNA Kcnq1ot1 promoted apoptosis of cardiomyocytes. In addition, miR-34a-5p was upregulated and Sirt1 was downregulated after knockdown of lncRNA Kcnq1ot1 in cardiomyocytes. While, administration of AMO-34a-5p attenuated the effect of lncRNA Kcnq1ot1 knockdown. The above findings suggest that the lncRNA Kcnq1ot1/miR-34a-5p/Sirt1 pathway is involved in ATO-induced cardiotoxicity.
Subsequently, we explored the potential of the lncRNA Kcnq1ot1/miR-34a-5p/Sirt1 pathway as a therapeutic target for ATO-induced cardiotoxicity. Cardioprotective drugs may alleviate ATO-induced cardiotoxicity through the lncRNA Kcnq1ot1/miR-34a-5p/Sirt1 pathway. The beta-blocker propranolol is a widely used cardioprotective agent [
52]. We explored the effect of propranolol on ATO-induced cardiotoxicity. Coadministration of propranolol alleviated ATO-induced apoptosis in mouse myocardial tissues and cardiomyocytes. Similarly, propranolol has also been shown to alleviate clozapine-induced cardiac oxidative stress injury and cardiomyocyte apoptosis[
53]. Finally, we detected the effect of propranolol on the lncRNA Kcnq1ot1/miR-34a-5p/Sirt1 pathway. The results showed that propranolol increased lncRNA Kcnq1ot1 and Sirt1 expression, and decreased miR-34a-5p expression in ATO-treated mouse cardiomyocytes and myocardial tissues. These findings suggest that propranolol alleviated ATO-induced cardiomyocyte apoptosis both in vitro and in vivo, which at least partially through the lncRNA Kcnq1ot1/miR-34a-5p/Sirt1 pathway.
Conclusions
In conclusion, the lncRNA Kcnq1ot1/miR-34a-5p/Sirt1 pathway is involved in ATO-induced cardiotoxicity. Propranolol can attenuate ATO-induced cardiotoxicity at least partially through the lncRNA Kcnq1ot1/miR-34a-5p/Sirt1 pathway. Combined administration with propranolol may be a new strategy for alleviating the cardiotoxicity of ATO. This study revealed a new mechanism of ATO-induced cardiotoxicity and a molecular basis of combined application of ATO and propranolol, which provides new insight into the study and rational use of ATO in the clinic.
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