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MicroRNA-22 inhibition prevents doxorubicin-induced cardiotoxicity via upregulating SIRT1
Biochemical and Biophysical Research Communications xxx (xxxx) xxx
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MicroRNA-22 inhibition prevents doxorubicin-induced cardiotoxicity via upregulating SIRT1 Can Xu a, Chang-Hui Liu a, Da-Li Zhang b, * a b
Department of Cardiology, The First Affiliated Hospital of University of South China, Hengyang, Hunan, 421001, PR China Department of Emergency, The First Affiliated Hospital of University of South China, Hengyang, Hunan, 421001, PR China
a r t i c l e i n f o
a b s t r a c t
Article history: Received 30 September 2019 Accepted 19 October 2019 Available online xxx
Oxidative stress and cardiomyocyte apoptosis contributed to the progression of doxorubicin (Dox)induced cardiotoxicity. Recent studies identified microRNA-22 (miR-22) as a cardiac- and skeletal muscle-enriched microRNA that functioned as a key regulator in stress-induced cardiac injury. The present study aimed to investigate the role and possible mechanism of miR-22 on Dox-induced oxidative stress and cardiomyocyte apoptosis. Mice were exposed to reduplicative injections of Dox (i.p., 4 mg/kg) weekly for consecutive 4 weeks to generate Dox-induced cardiotoxicity. Herein, we found that miR-22 level was significantly increased in murine hearts subjected to chronic Dox treatment. MiR-22 inhibition attenuated oxidative stress and cardiomyocyte apoptosis in vivo and in vitro, thereby preventing Doxinduced cardiac dysfunction. Mechanistically, we observed that miR-22 directly bound to the 30 -UTR of Sirt1 and caused SIRT1 downregulation. Conversely, miR-22 antagomir upregulated SIRT1 expression and SIRT1 inhibitor abolished the beneficial effects of miR-22 antagomir. In conclusion, miR-22 inhibition prevented oxidative stress and cardiomyocyte apoptosis via upregulating SIRT1 and miR-22 might be a new target for treating Dox-induced cardiotoxicity. © 2019 Elsevier Inc. All rights reserved.
Keywords: MicroRNA-22 Doxorubicin Oxidative stress Cardiomyocyte apoptosis SIRT1
1. Introduction Doxorubicin (Dox) is regarded as the first-line drug in human cancer chemotherapy, however, its clinical use is hampered by the cumulative cardiotoxicity [1]. Previous studies prove that Dox targets topoisomerase-II alpha (Top2a) and therefore forms the ternary Top2a-Dox-DNA cleavage complex to kill tumor cells. Top2b, which is extensively expressed in mammalian cardiomyocytes, is also a target for Dox. The formation of Top2b-DoxDNA ternary cleavage complex is essential for Dox-induced structural and functional damage in cardiomyocyte mitochondria, which ultimately leads to reactive oxygen species (ROS) overproduction, cardiomyocyte apoptosis and the occurrence of Dox-induced cardiotoxicity [2e4]. Therefore, a better understanding about the pathogenesis of Dox-induced oxidative stress and cardiomyocyte apoptosis and the identification of novel therapeutic targets are of great significance. Silent information regulator 1 (SIRT1) is implicated in kinds of
* Corresponding author. Department of Emergency, The First Affiliated Hospital of University of South China, Chuanshan road 69, Hengyang, 421001, PR China.. E-mail address: [email protected] (D.-L. Zhang).
pathophysiological processes, including oxidative stress and cell apoptosis [5,6]. Growing interests have been laid on SIRT1 for its protective roles in cardiovascular diseases [7]. In the myocardium, SIRT1 is essential for normal growth of cardiomyocytes, and conversely, Sirt1 knockout mice exhibited cardiac malformation [8]. In addition, SIRT1 activation exerted beneficial effects on cardiac ischemia, remodeling and electrical disorder in mice [9]. Recent studies identified SIRT1 as the potential node in regulating Doxinduced cardiotoxicity. Zhang et al. determined that SIRT1 activation by resveratrol suppressed p53 acetylation and p53-dependent transcription of Bax in Dox-treated murine hearts, and Yuan et al. found that SIRT1 upregulation prevented Dox-induced cell loss and cardiac dysfunction [10,11]. These data implied that targeting SIRT1 might help to establish effectively therapeutic approaches for Doxinduced cardiotoxicity. MicroRNAs (miRs) is reported to modify gene expression at the post-transcriptional level through binding to the 30 -untranslated regions (UTR) of target mRNAs [12,13]. Numerous microRNAs have been suggested to participate in the regulation of Dox-induced cardiotoxicity, and even could be used as early sensitive cardiotoxicity biomarkers [14]. Horie et al. found that miR-146a significantly inhibited neuregulin-1/ErbB signaling pathway and
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Please cite this article as: C. Xu et al., MicroRNA-22 inhibition prevents doxorubicin-induced cardiotoxicity via upregulating SIRT1, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/j.bbrc.2019.10.140
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exacerbated cardiomyocyte apoptosis in Dox-induced cardiotoxicity [15]. Recent studies showed that miR-140-5p overexpression promoted oxidative stress and Dox-induced cardiac injury [16]. MiR-22 was previously identified as a tumor suppressive microRNA that triggered cellular senescence in tumor cells [17]. Yet, emerging researches identified miR-22 as a cardiac- and skeletal muscleenriched microRNA that functioned as a key regulator in stressinduced cardiac injury. MiR-22 was upregulated during myocyte differentiation and was essential for hypertrophic cardiac growth in response to neurohumoral stress [18,19]. Moreover, Du et al. observed that miR-22 was upregulated in ischemic hearts and contributed to cardiomyocyte injury in mice [20]. Based on these findings, we supposed that miR-22 might be involved in the regulation of Dox-induced cardiotoxicity.
system [24]. AMCs were viewed by a computer monitor using an IonOptix MyoCam camera to record the amplitude and velocity of shortening or relengthening after superfused with a Tyrode buffer (1 ml/min) and stimulated by the STIM-AT stimulator/thermostat (0.5 Hz, 30 V).
2. Materials and methods
2.6. H9C2 cell culture and treatment
2.1. Reagents and antibodies
H9C2 cell line was purchased from the American Type Culture Collection (Manassas, USA) and cultured in Dulbecco’s modified Eagle’s medium (DMEM) with 10% fetal bovine serum (FBS). After resynchronization, cells were treated with miR-22 antagomir (50 nM) or the negative control for 24 h, followed by Dox (1 mM) for additional 24 h [13]. To inhibit SIRT1 activity, H9C2 cells were incubated with EX-527 (50 nM) at the meantime with Dox treatment [25].
Dox and EX-527 were purchased from Sigma (St. Louis, MO, USA). MiR-22 antagomir and the negative control were obtained from RiboBio Co. Ltd (Guangzhou, China). Superoxide dismutase (SOD), glutathione peroxidases (Gpx), NADPH oxidase (NOX) activity assay kits, glutathione (GSH), oxidized glutathione (GSSG) and malondialdehyde (MDA) assay kits were all purchased from Nanjing Jiancheng Bioengineering Institute (Nanjing, China). 4Hydroxynonenal (4-HNE) and 3-nitrotyrosine (3-NT) levels were detected using commercial kits from Abcam (Cambridge, UK). 20 ,70 dichlorodihydrofluorescein diacetate (DCFH-DA) and cell counting kit-8 (CCK-8) were obtained from Beyotime Biotechnology (Shanghai, China). TUNEL detection kit was obtained from Millipore (Billerica, MA, USA). Anti-SIRT1 was purchased from Abcam (Cambridge, UK), whereas anti-glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was from Cell Signaling Technology (Danvers, MA, USA). 2.2. Experimental procedures All animals received proper care in compliance with the Guide for the Care and Use of Laboratory Animals published by the United States National Institutes of Health (NIH Publication, revised 1996), and the experimental procedures were approved by the Animal Ethics Committee of the University of South China. Mice were randomly subjected to reduplicative injections of Dox (i.p., 4 mg/ kg) weekly for consecutive 4 weeks to generate Dox-induced cardiotoxicity or equal volume of saline as a control referring to previous studies [21]. At the meantime with Dox treatment, mice were intraperitoneally injected with miR-22 antagomir or the negative control (25 mg/kg/day) every other day to investigate the effect of miR-22 in vivo according to a previous study [12]. EX-527 (1 mg/kg, every other day) was used to inhibit SIRT1 activity in vivo [10]. 2.3. Cardiac function assessment Echocardiographic assessment was performed using a Vevo® 2100 Imaging System (Visual Sonics) with a 40 MHz MicroScan transducer (model MS-550D) [22]. SPR-839 microtip cardiac catheter (Millar Instruments, Houston, TX) was used for the collection of hemodynamic parameters [23].
2.5. Histological examination Murine hearts were fixed in formalin for 48 h and then were processed for picrosirius red (PSR) staining to detect cardiac fibrosis [22]. Fibrotic area was counted with a Zeiss Axioskop microscope equipped with Image-Pro Plus imaging software (Media Cybernetics) in a blinded manner.
2.7. Biochemical detection GSH/GSSG, MDA levels and SOD, Gpx, NOX activities were detected using a microplate reader (Synergy HT; Bio-Tek, USA) according to the instructions [26]. 4-HNE and 3-NT levels were quantified using commercial ELISA kits from Abcam (Cambridge, UK) [27]. Serum levels of creatine kinase (CK), cardiac isoform of tropnin T (cTnT) and lactate dehydrogenase (LDH) in mice as well as LDH release in cultured H9C2 cells were detected using the commercial kits according to the instructions [3]. 2.8. Western blot and quantitative real-time PCR detection Proteins extracted by RIPA lysis were transferred to PVDF membranes after SDS-PAGE separation, which were then incubated with 5% nonfat milk, primary antibodies and secondary antibodies, respectively. Membranes were visualized with ECL reagents and scanned by the Bio-Rad ChemiDoc Touch Imaging System (Hercules, CA, USA) [28,29]. Transcripts levels were analyzed by ABI realtime PCR system (7900HT FAST, Applied Biosystems, Foster City, CA) [30]. 2.9. DCFH-DA and TUNEL staining After treatment, cells were cultured in DMEM medium containing DCFH-DA (5 mmol/L) for 30 min and the images were captured by an Olympus IX53 fluorescence microscope [3]. TUNEL staining was performed as previously described [31]. Apoptotic index was quantified as the ratio of TUNEL þ nuclei to DAPI þ nuclei. 2.10. Caspase3 activity determination and cell viability
2.4. Adult mouse cardiomyocytes (AMCs) isolation and contractility measurement AMCs from indicating groups were isolated for mechanical property detection using a temperature-controlled Langendorff
Caspase3 activity was detected in the supernatant based on a fluorogenic peptide derived Ac-DEVD-pNA (acetyl-Asp-Glu-ValAsp p-nitroanilide) as previously described [32]. Cell viability was detected by the CCK-8 kit according to the instructions.
Please cite this article as: C. Xu et al., MicroRNA-22 inhibition prevents doxorubicin-induced cardiotoxicity via upregulating SIRT1, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/j.bbrc.2019.10.140
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2.11. Luciferase reporter assay Wild type (Wt) that carrying miR-22 binding sites or mutant (Mut) 30 -UTR of Sirt1 were synthesized by Aiyou Biological Technology Co., Ltd (Guangzhou, China) and cloned into the psi-CHECK2 luciferase reporter plasmid (Promega, USA) [33]. Cells were cotransfected with psiCHECK-2 plasmid containing Wt or Mut derivatives, along with the miR-22 mimic or its control. Lysates were collected 24 h after transfection and luciferase activity was measured by dual luciferase reporter system (Promega, USA). 2.12. Statistical analysis Data were expressed as mean ± standard deviation. The two tailed Student’s t-test was used to compare differences between two groups, whereas multi-group’s comparisons were performed by one-way ANOVA analysis (SPSS 23.0). Survival rate was evaluated by the Kaplan-Meier method and survival curves were compared using the Mantel-Cox log-rank test. Values of P < 0.05 were considered statistically significant. 3. Results 3.1. MiR-22 inhibition protected against Dox-induced cardiotoxicity in mice As shown in Fig. 1A, myocardial miR-22 level was markedly increased by Dox treatment. And miR-22 inhibition notably
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alleviated Dox-induced cardiac dysfunction in mice, as evidenced by the improved ejection fraction (EF), stroke volume (SV), stroke work (SW) and ±dP/dt (Fig. 1BeE). Dox treatment caused a decrease of heart weight/tibia length ratio (HW/TL), which was overtly restored after miR-22 inhibition (Fig. 1F). In addition, Doxinduced cardiac fibrosis and upregulation of fibrotic markers were also alleviated by miR-22 inhibition (Fig. 1GeH). However, miR-22 antagomir made no alleviation on Dox-triggered bradycardia and hypotension (Fig. 1I). The elevated levels of serum CK, cTnT and LDH in mice with Dox treatment were also decreased by miR-22 antagomir (Fig. 1J). Moreover, mortality rate in Dox-treated mice was largely prevented with miR-22 antagomir (Fig. 1K). 3.2. MiR-22 inhibition prevented Dox-induced cardiomyocyte contractile dysfunction As depicted in Fig. S1A, miR-22 was upregulated in AMCs isolated from Dox-treated murine hearts. Despite no difference in resting length of AMCs was observed, cardiomyocyte peaking shortening was increased by miR-22 inhibition (Fig. S1B). The maximal velocity of shortening/relengthening (±dL/dt) were also alleviated in AMCs isolated from miR-22 antagomir-treated hearts (Fig. S1C). 3.3. MiR-22 inhibition attenuated Dox-induced oxidative stress and cardiomyocyte apoptosis in vivo and in vitro Dox administration resulted in significantly increased oxidative
Fig. 1. MiR-22 inhibition protected against Dox-induced cardiotoxicity in mice. A. MiR-22 expression in doxorubicin (Dox)-treated murine hearts (n ¼ 6). B. The efficiency of miR-22 antagomir (n ¼ 6). C. Ejection fraction (EF) and stroke volume (SV) (n ¼ 8). D. Stroke work index (n ¼ 8). E. Hemodynamic parameters of murine hearts (n ¼ 8). F. Heart weight (HW) to tibia length (TL) ratio (n ¼ 8). G. Picrosirius red (PSR) staining results (n ¼ 6). H. Gene expressions in murine hearts (n ¼ 8). I. Heart rate and blood pressure (n ¼ 10). J. Serum levels of creatine kinase (CK), cardiac isoform of tropnin T (cTnT) and lactate dehydrogenase (LDH) (n ¼ 8). K. Survival rate data, black arrows indicate Dox injection (n ¼ 20). All data are expressed as the mean ± standard deviation (SD), *P < 0.05.
Please cite this article as: C. Xu et al., MicroRNA-22 inhibition prevents doxorubicin-induced cardiotoxicity via upregulating SIRT1, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/j.bbrc.2019.10.140
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stress, which was improved by miR-22 inhibition, as confirmed by the attenuation of SOD, Gpx, NOX activities and MDA, 4-HNE, GSH/ GSSG, 3-NT levels (Fig. 2AeE). Besides, Dox-triggered the decrease of Bcl2/Bax and the increase of caspase3 activity were also blunted in miR-22 inhibited hearts (Fig. 2FeG). TUNEL staining further implied that miR-22 inhibition attenuated Dox-induced cardiomyocyte apoptosis (Fig. 2H). We further investigated the beneficial effect of miR-22 antagomir in vitro. As shown in Fig. 2I, miR-22 inhibition suppressed ROS generation, further confirmed by the decreased levels of MDA and 3-NT (Fig. 2J). SOD, Gpx activities downregulation and NOX activity upregulation were also restored after miR-22 inhibition (Fig. 2KeL). In line with the in vivo data, Dox-induced H9C2 cardiomyocyte apoptosis was also ameliorated by miR-22 inhibition (Fig. 2MeP).
3.4. MiR-22 inhibition alleviated Dox-induced oxidative stress and cardiomyocyte apoptosis via upregulating SIRT1 We then explored the possible mechanism through which miR22 antagomir exerted the beneficial effects. Previous studies implied that miR-22 could target the 30 -UTR of Sirt1 and caused SIRT1 downregulation [18,20]. Surprisingly, we found that miR-22 inhibition increased SIRT1 protein and mRNA levels in the presence or absence of Dox (Fig. 3AeB). To notify whether Sirt1 is a direct target of miR-22, we performed luciferase reporter assay. Compared with the control group, miR-22 mimic significantly decreased the luciferase activity, however, with no alteration when the binding site was mutated (Fig. 3C). EX-527 was used for SIRT1 inhibition in
further studies (Fig. 3D). As shown in Fig. 3EeF, SIRT1 inhibitor blunted miR-22 antagomir-mediated the upregulation of SOD, Gpx activities and the downregulation of NOX activity. Consistently, MDA and 3-NT induction were prevented in miR-22-inhibited cells, but not in that with EX-527 incubation (Fig. 3G). The attenuation on cardiomyocyte apoptosis were also reversed by SIRT1 inhibition, as evidenced by the Bcl2/Bax ratio, caspse3 activity, cell viability and LDH leakage (Fig. 3HeK). Accordingly, the restoration of AMCs contractile capacity by miR-22 inhibition was completely blocked after SIRT1 suppression (Fig. 3L). These data collectively proved that the protective effects of miR-22 antagomir on oxidative stress and cardiomyocyte apoptosis was mediated via upregulating SIRT1.
3.5. MiR-22 inhibition-mediated beneficial effects were abolished by SIRT1 inhibitor in mice Consistent with the data in vitro, we observed that miR-22 antagomir-induced upregulation of SOD, Gpx activities and downregulation of NOX activity were reversed in murine hearts with EX-527 administration (Fig. 4AeB). Accordingly, the attenuation of MDA and 3-NT production was reduced in miR-22-deficient hearts, but not in that with SIRT1 inhibition (Fig. 4CeD). Caspase3 activity detection and TUNEL assay revealed the fact that SIRT1 inhibition abolished the beneficial effect of miR-22 antagomir on cardiomyocyte apoptosis (Fig. 4E). Improved cardiac function by miR-22 inhibition was also abolished by the SIRT1 inhibitor (Fig. 4FeH). Moreover, miR-22 inhibition-induced cTnT downregulation was also blunted by EX-527 (Fig. 4I).
Fig. 2. MiR-22 inhibition attenuated Dox-induced oxidative stress and cardiomyocyte apoptosis in vivo and in vitro. A-B. Superoxide dismutase (SOD), glutathione peroxidases (Gpx) and NADPH oxidase (NOX) activities in murine hearts (n ¼ 6). C-E. Myocardial malondialdehyde (MDA), 4-hydroxynonenal (4-HNE), glutathione to oxidized glutathione (GSH/ GSSG), 3-nitrotyrosine (3-NT) levels in mice (n ¼ 6). F. Statistical result of Bcl2/Bax in murine hearts (n ¼ 6). G-H. Caspase3 activity and TUNEL þ nuclei index (n ¼ 6). I. DCFH-DA staining in H9C2 cells (n ¼ 6). J. MDA and 3-NT production in H9C2 cells (n ¼ 6). K-L. SOD, Gpx and NOX activities in H9C2 cells (n ¼ 6). M. Data of Bcl2/Bax in H9C2 cells (n ¼ 6). NeO. Statistical results of caspase3 activity and cell viability (n ¼ 6). P. LDH level release to the medium (n ¼ 6). All data are expressed as the mean ± SD, *P < 0.05.
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Fig. 3. MiR-22 inhibition alleviated Dox-induced oxidative stress and cardiomyocyte apoptosis via upregulating SIRT1. A. Western blots and the statistical results in H9C2 cells (n ¼ 6). B. Sirt1 mRNA level in H9C2 cells (n ¼ 6). C. Statistical result of the dual luciferase assay (n ¼ 8). D. SIRT1 activity detection in H9C2 cells treated with or without EX-527 (n ¼ 6). E-F. SOD, Gpx and NOX activities in H9C2 cells (n ¼ 6). G. MDA and 3-NT production in H9C2 cells (n ¼ 6). H. The relative mRNA level of Bcl2/Bax (n ¼ 6). I. Statistical result of caspase3 activity in H9C2 cells (n ¼ 6). J. Cell viability detection using H9C2 cells (n ¼ 6). K. LDH level in the medium of H9C2 cells (n ¼ 6). L. Peaking shortening and þdL/dt in AMCs isolated from indicating murine hearts (n ¼ 6). All data are expressed as the mean ± SD, *P < 0.05.
4. Discussion MicroRNAs are emerging as important regulators in Doxinduced cardiotoxicity through binding to the 30 -UTR of target genes, and even could be used as early sensitive cardiotoxicity biomarkers [14]. MiR-22 was previously shown to be ubiquitously expressed in multiple tissues that was recently identified as a cardiac- and skeletal muscle-enriched microRNA [18]. Gurha et al. proved that miR-22 played critical roles in regulating myocardial calcium homeostasis and myofibrillar protein content, and was required for the heart to adapt to pressure overload-induced cardiac hypertrophy [19]. Conversely, cardiomyocyte-specific deletion of miR-22 repressed the progression of cardiac hypertrophy and impaired cardiac response to stress [18]. In addition, miR-22 was also involved in adjusting mitochondrial function, and miR-22 upregulation contributed to cardiac mitochondrial damage and the progression of ischemia reperfusion injury [20]. In the current study, we showed that miR-22 was upregulated in Dox-treated murine hearts and cardiomyocytes, and miR-22 inhibition prevented Dox-induced cardiac dysfunction. Oxidative stress and cardiomyocyte apoptosis were proposed as the key mechanisms responsible for Dox-triggered cardiotoxicity [2,34]. Previous studies indicated that the Dox showed a specific affinity to the heart, and could be accumulated in cardiac mitochondria. In the mitochondrial inner membrane, Dox formed a nearly-irreversible complex with cardiolipin and then disrupted its normal physiological function, which subsequently promoted massive ROS generation and cell death [35]. Previous studies
showed that oxidative stress could be observed in heart samples within 3 h after Dox treatment, and interference on ROS overproduction significantly improved Dox-induced cardiac dysfunction [3,36]. MiR-22 level was previously suggested to be positively related to MDA, whereas negatively correlated to GSH/GSSG ratio in the myocardium, implying the possible role of miR-22 in regulating myocardial oxidative stress [37]. Herein, we found that miR-22 antagomir enhanced the total anti-oxidant capacity of the heart and markedly reduced Dox-induced oxidative stress and cardiomyocyte apoptosis. SIRT1 is known to exert various bioactivities via suppressing oxidative stress in cardiovascular diseases, and SIRT1 activation was linked to the attenuation on Dox-induced oxidative stress and cardiomyocyte apoptosis [10,11]. In the present study, we found that miR-22 could target the 30 -UTR of Sirt1 and thereby leading to SIRT1 downregulation. MiR-22 inhibition partially restored the SIRT1 level after Dox treatment, and SIRT1 suppression abolished the beneficial effects of miR-22 antagomir on Dox-induced oxidative stress and cardiomyocyte apoptosis in vivo and in vitro. Beyond the role in controlling cardiac pathophysiology, miR-22 was also indicated to be involved in regulating tumorigenesis. Numerous studies found that miR-22 expression was downregulated in several tumor cell lines and clinical samples, whereas miR-22 overexpression caused cellular senescence and inhibited tumor progression [17]. However, Palacios et al. demonstrated that miR-22 was upregulated in proliferative B cells in chronic lymphocytic leukemia, which then induced PTEN downregulation, PI3K/AKT activation and excessive cell proliferation [38]. Hence the efficiency
Please cite this article as: C. Xu et al., MicroRNA-22 inhibition prevents doxorubicin-induced cardiotoxicity via upregulating SIRT1, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/j.bbrc.2019.10.140
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Fig. 4. MiR-22 inhibition-mediated beneficial effects were abolished by SIRT1 inhibitor in mice. A-B. SOD, Gpx and NOX activities in the indicating murine hearts (n ¼ 6). C-D. Myocardial MDA and 3-NT production in mice (n ¼ 6). E. Statistical result of caspase3 and TUNEL þ nuclei in murine hearts (n ¼ 6). FeH. Statistical results of EF, SV and þdP/dt (n ¼ 8). I. Serum level of cTnT in mice (n ¼ 8). All data are expressed as the mean ± SD, *P < 0.05.
and safety of miR-22 inhibition on Dox-induced cardiotoxicity need to be further detected. Taken together, we found that miR-22 inhibition prevented oxidative stress and cardiomyocyte apoptosis via upregulating SIRT1 and miR-22 might be a new target for treating Dox-induced cardiotoxicity. Declaration of competing interest None. Acknowledgements None. Appendix A. Supplementary data Supplementary data to this article can be found online at https://doi.org/10.1016/j.bbrc.2019.10.140. References [1] M. Li, V. Sala, M.C. De Santis, et al., Phosphoinositide 3-kinase gamma inhibition protects from anthracycline cardiotoxicity and reduces tumor growth, Circulation 138 (2018) 696e711, https://doi.org/10.1161/ CIRCULATIONAHA.117.030352. [2] S. Zhang, X. Liu, T. Bawa-Khalfe, et al., Identification of the molecular basis of doxorubicin-induced cardiotoxicity, Nat. Med. 18 (2012) 1639e1642, https:// doi.org/10.1038/nm.2919. [3] X. Zhang, C. Hu, C.Y. Kong, et al., FNDC5 alleviates oxidative stress and cardiomyocyte apoptosis in doxorubicin-induced cardiotoxicity via activating AKT, Cell Death Differ. (2019), https://doi.org/10.1038/s41418-019-0372-z. [4] H. Zhou, P. Zhu, J. Wang, et al., Pathogenesis of cardiac ischemia reperfusion injury is associated with CK2alpha-disturbed mitochondrial homeostasis via suppression of FUNDC1-related mitophagy, Cell Death Differ. 25 (2018) 1080e1093, https://doi.org/10.1038/s41418-018-0086-7. [5] A. Prola, D.S.J. Pires, A. Guilbert, et al., SIRT1 protects the heart from ER stress-
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MicroRNA-22 inhibition prevents doxorubicin-induced cardiotoxicity via upregulating SIRT1 Can Xu a, Chang-Hui Liu a, Da-Li Zhang b, * a b
Department of Cardiology, The First Affiliated Hospital of University of South China, Hengyang, Hunan, 421001, PR China Department of Emergency, The First Affiliated Hospital of University of South China, Hengyang, Hunan, 421001, PR China
a r t i c l e i n f o
a b s t r a c t
Article history: Received 30 September 2019 Accepted 19 October 2019 Available online xxx
Oxidative stress and cardiomyocyte apoptosis contributed to the progression of doxorubicin (Dox)induced cardiotoxicity. Recent studies identified microRNA-22 (miR-22) as a cardiac- and skeletal muscle-enriched microRNA that functioned as a key regulator in stress-induced cardiac injury. The present study aimed to investigate the role and possible mechanism of miR-22 on Dox-induced oxidative stress and cardiomyocyte apoptosis. Mice were exposed to reduplicative injections of Dox (i.p., 4 mg/kg) weekly for consecutive 4 weeks to generate Dox-induced cardiotoxicity. Herein, we found that miR-22 level was significantly increased in murine hearts subjected to chronic Dox treatment. MiR-22 inhibition attenuated oxidative stress and cardiomyocyte apoptosis in vivo and in vitro, thereby preventing Doxinduced cardiac dysfunction. Mechanistically, we observed that miR-22 directly bound to the 30 -UTR of Sirt1 and caused SIRT1 downregulation. Conversely, miR-22 antagomir upregulated SIRT1 expression and SIRT1 inhibitor abolished the beneficial effects of miR-22 antagomir. In conclusion, miR-22 inhibition prevented oxidative stress and cardiomyocyte apoptosis via upregulating SIRT1 and miR-22 might be a new target for treating Dox-induced cardiotoxicity. © 2019 Elsevier Inc. All rights reserved.
Keywords: MicroRNA-22 Doxorubicin Oxidative stress Cardiomyocyte apoptosis SIRT1
1. Introduction Doxorubicin (Dox) is regarded as the first-line drug in human cancer chemotherapy, however, its clinical use is hampered by the cumulative cardiotoxicity [1]. Previous studies prove that Dox targets topoisomerase-II alpha (Top2a) and therefore forms the ternary Top2a-Dox-DNA cleavage complex to kill tumor cells. Top2b, which is extensively expressed in mammalian cardiomyocytes, is also a target for Dox. The formation of Top2b-DoxDNA ternary cleavage complex is essential for Dox-induced structural and functional damage in cardiomyocyte mitochondria, which ultimately leads to reactive oxygen species (ROS) overproduction, cardiomyocyte apoptosis and the occurrence of Dox-induced cardiotoxicity [2e4]. Therefore, a better understanding about the pathogenesis of Dox-induced oxidative stress and cardiomyocyte apoptosis and the identification of novel therapeutic targets are of great significance. Silent information regulator 1 (SIRT1) is implicated in kinds of
* Corresponding author. Department of Emergency, The First Affiliated Hospital of University of South China, Chuanshan road 69, Hengyang, 421001, PR China.. E-mail address: [email protected] (D.-L. Zhang).
pathophysiological processes, including oxidative stress and cell apoptosis [5,6]. Growing interests have been laid on SIRT1 for its protective roles in cardiovascular diseases [7]. In the myocardium, SIRT1 is essential for normal growth of cardiomyocytes, and conversely, Sirt1 knockout mice exhibited cardiac malformation [8]. In addition, SIRT1 activation exerted beneficial effects on cardiac ischemia, remodeling and electrical disorder in mice [9]. Recent studies identified SIRT1 as the potential node in regulating Doxinduced cardiotoxicity. Zhang et al. determined that SIRT1 activation by resveratrol suppressed p53 acetylation and p53-dependent transcription of Bax in Dox-treated murine hearts, and Yuan et al. found that SIRT1 upregulation prevented Dox-induced cell loss and cardiac dysfunction [10,11]. These data implied that targeting SIRT1 might help to establish effectively therapeutic approaches for Doxinduced cardiotoxicity. MicroRNAs (miRs) is reported to modify gene expression at the post-transcriptional level through binding to the 30 -untranslated regions (UTR) of target mRNAs [12,13]. Numerous microRNAs have been suggested to participate in the regulation of Dox-induced cardiotoxicity, and even could be used as early sensitive cardiotoxicity biomarkers [14]. Horie et al. found that miR-146a significantly inhibited neuregulin-1/ErbB signaling pathway and
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Please cite this article as: C. Xu et al., MicroRNA-22 inhibition prevents doxorubicin-induced cardiotoxicity via upregulating SIRT1, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/j.bbrc.2019.10.140
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exacerbated cardiomyocyte apoptosis in Dox-induced cardiotoxicity [15]. Recent studies showed that miR-140-5p overexpression promoted oxidative stress and Dox-induced cardiac injury [16]. MiR-22 was previously identified as a tumor suppressive microRNA that triggered cellular senescence in tumor cells [17]. Yet, emerging researches identified miR-22 as a cardiac- and skeletal muscleenriched microRNA that functioned as a key regulator in stressinduced cardiac injury. MiR-22 was upregulated during myocyte differentiation and was essential for hypertrophic cardiac growth in response to neurohumoral stress [18,19]. Moreover, Du et al. observed that miR-22 was upregulated in ischemic hearts and contributed to cardiomyocyte injury in mice [20]. Based on these findings, we supposed that miR-22 might be involved in the regulation of Dox-induced cardiotoxicity.
system [24]. AMCs were viewed by a computer monitor using an IonOptix MyoCam camera to record the amplitude and velocity of shortening or relengthening after superfused with a Tyrode buffer (1 ml/min) and stimulated by the STIM-AT stimulator/thermostat (0.5 Hz, 30 V).
2. Materials and methods
2.6. H9C2 cell culture and treatment
2.1. Reagents and antibodies
H9C2 cell line was purchased from the American Type Culture Collection (Manassas, USA) and cultured in Dulbecco’s modified Eagle’s medium (DMEM) with 10% fetal bovine serum (FBS). After resynchronization, cells were treated with miR-22 antagomir (50 nM) or the negative control for 24 h, followed by Dox (1 mM) for additional 24 h [13]. To inhibit SIRT1 activity, H9C2 cells were incubated with EX-527 (50 nM) at the meantime with Dox treatment [25].
Dox and EX-527 were purchased from Sigma (St. Louis, MO, USA). MiR-22 antagomir and the negative control were obtained from RiboBio Co. Ltd (Guangzhou, China). Superoxide dismutase (SOD), glutathione peroxidases (Gpx), NADPH oxidase (NOX) activity assay kits, glutathione (GSH), oxidized glutathione (GSSG) and malondialdehyde (MDA) assay kits were all purchased from Nanjing Jiancheng Bioengineering Institute (Nanjing, China). 4Hydroxynonenal (4-HNE) and 3-nitrotyrosine (3-NT) levels were detected using commercial kits from Abcam (Cambridge, UK). 20 ,70 dichlorodihydrofluorescein diacetate (DCFH-DA) and cell counting kit-8 (CCK-8) were obtained from Beyotime Biotechnology (Shanghai, China). TUNEL detection kit was obtained from Millipore (Billerica, MA, USA). Anti-SIRT1 was purchased from Abcam (Cambridge, UK), whereas anti-glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was from Cell Signaling Technology (Danvers, MA, USA). 2.2. Experimental procedures All animals received proper care in compliance with the Guide for the Care and Use of Laboratory Animals published by the United States National Institutes of Health (NIH Publication, revised 1996), and the experimental procedures were approved by the Animal Ethics Committee of the University of South China. Mice were randomly subjected to reduplicative injections of Dox (i.p., 4 mg/ kg) weekly for consecutive 4 weeks to generate Dox-induced cardiotoxicity or equal volume of saline as a control referring to previous studies [21]. At the meantime with Dox treatment, mice were intraperitoneally injected with miR-22 antagomir or the negative control (25 mg/kg/day) every other day to investigate the effect of miR-22 in vivo according to a previous study [12]. EX-527 (1 mg/kg, every other day) was used to inhibit SIRT1 activity in vivo [10]. 2.3. Cardiac function assessment Echocardiographic assessment was performed using a Vevo® 2100 Imaging System (Visual Sonics) with a 40 MHz MicroScan transducer (model MS-550D) [22]. SPR-839 microtip cardiac catheter (Millar Instruments, Houston, TX) was used for the collection of hemodynamic parameters [23].
2.5. Histological examination Murine hearts were fixed in formalin for 48 h and then were processed for picrosirius red (PSR) staining to detect cardiac fibrosis [22]. Fibrotic area was counted with a Zeiss Axioskop microscope equipped with Image-Pro Plus imaging software (Media Cybernetics) in a blinded manner.
2.7. Biochemical detection GSH/GSSG, MDA levels and SOD, Gpx, NOX activities were detected using a microplate reader (Synergy HT; Bio-Tek, USA) according to the instructions [26]. 4-HNE and 3-NT levels were quantified using commercial ELISA kits from Abcam (Cambridge, UK) [27]. Serum levels of creatine kinase (CK), cardiac isoform of tropnin T (cTnT) and lactate dehydrogenase (LDH) in mice as well as LDH release in cultured H9C2 cells were detected using the commercial kits according to the instructions [3]. 2.8. Western blot and quantitative real-time PCR detection Proteins extracted by RIPA lysis were transferred to PVDF membranes after SDS-PAGE separation, which were then incubated with 5% nonfat milk, primary antibodies and secondary antibodies, respectively. Membranes were visualized with ECL reagents and scanned by the Bio-Rad ChemiDoc Touch Imaging System (Hercules, CA, USA) [28,29]. Transcripts levels were analyzed by ABI realtime PCR system (7900HT FAST, Applied Biosystems, Foster City, CA) [30]. 2.9. DCFH-DA and TUNEL staining After treatment, cells were cultured in DMEM medium containing DCFH-DA (5 mmol/L) for 30 min and the images were captured by an Olympus IX53 fluorescence microscope [3]. TUNEL staining was performed as previously described [31]. Apoptotic index was quantified as the ratio of TUNEL þ nuclei to DAPI þ nuclei. 2.10. Caspase3 activity determination and cell viability
2.4. Adult mouse cardiomyocytes (AMCs) isolation and contractility measurement AMCs from indicating groups were isolated for mechanical property detection using a temperature-controlled Langendorff
Caspase3 activity was detected in the supernatant based on a fluorogenic peptide derived Ac-DEVD-pNA (acetyl-Asp-Glu-ValAsp p-nitroanilide) as previously described [32]. Cell viability was detected by the CCK-8 kit according to the instructions.
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2.11. Luciferase reporter assay Wild type (Wt) that carrying miR-22 binding sites or mutant (Mut) 30 -UTR of Sirt1 were synthesized by Aiyou Biological Technology Co., Ltd (Guangzhou, China) and cloned into the psi-CHECK2 luciferase reporter plasmid (Promega, USA) [33]. Cells were cotransfected with psiCHECK-2 plasmid containing Wt or Mut derivatives, along with the miR-22 mimic or its control. Lysates were collected 24 h after transfection and luciferase activity was measured by dual luciferase reporter system (Promega, USA). 2.12. Statistical analysis Data were expressed as mean ± standard deviation. The two tailed Student’s t-test was used to compare differences between two groups, whereas multi-group’s comparisons were performed by one-way ANOVA analysis (SPSS 23.0). Survival rate was evaluated by the Kaplan-Meier method and survival curves were compared using the Mantel-Cox log-rank test. Values of P < 0.05 were considered statistically significant. 3. Results 3.1. MiR-22 inhibition protected against Dox-induced cardiotoxicity in mice As shown in Fig. 1A, myocardial miR-22 level was markedly increased by Dox treatment. And miR-22 inhibition notably
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alleviated Dox-induced cardiac dysfunction in mice, as evidenced by the improved ejection fraction (EF), stroke volume (SV), stroke work (SW) and ±dP/dt (Fig. 1BeE). Dox treatment caused a decrease of heart weight/tibia length ratio (HW/TL), which was overtly restored after miR-22 inhibition (Fig. 1F). In addition, Doxinduced cardiac fibrosis and upregulation of fibrotic markers were also alleviated by miR-22 inhibition (Fig. 1GeH). However, miR-22 antagomir made no alleviation on Dox-triggered bradycardia and hypotension (Fig. 1I). The elevated levels of serum CK, cTnT and LDH in mice with Dox treatment were also decreased by miR-22 antagomir (Fig. 1J). Moreover, mortality rate in Dox-treated mice was largely prevented with miR-22 antagomir (Fig. 1K). 3.2. MiR-22 inhibition prevented Dox-induced cardiomyocyte contractile dysfunction As depicted in Fig. S1A, miR-22 was upregulated in AMCs isolated from Dox-treated murine hearts. Despite no difference in resting length of AMCs was observed, cardiomyocyte peaking shortening was increased by miR-22 inhibition (Fig. S1B). The maximal velocity of shortening/relengthening (±dL/dt) were also alleviated in AMCs isolated from miR-22 antagomir-treated hearts (Fig. S1C). 3.3. MiR-22 inhibition attenuated Dox-induced oxidative stress and cardiomyocyte apoptosis in vivo and in vitro Dox administration resulted in significantly increased oxidative
Fig. 1. MiR-22 inhibition protected against Dox-induced cardiotoxicity in mice. A. MiR-22 expression in doxorubicin (Dox)-treated murine hearts (n ¼ 6). B. The efficiency of miR-22 antagomir (n ¼ 6). C. Ejection fraction (EF) and stroke volume (SV) (n ¼ 8). D. Stroke work index (n ¼ 8). E. Hemodynamic parameters of murine hearts (n ¼ 8). F. Heart weight (HW) to tibia length (TL) ratio (n ¼ 8). G. Picrosirius red (PSR) staining results (n ¼ 6). H. Gene expressions in murine hearts (n ¼ 8). I. Heart rate and blood pressure (n ¼ 10). J. Serum levels of creatine kinase (CK), cardiac isoform of tropnin T (cTnT) and lactate dehydrogenase (LDH) (n ¼ 8). K. Survival rate data, black arrows indicate Dox injection (n ¼ 20). All data are expressed as the mean ± standard deviation (SD), *P < 0.05.
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stress, which was improved by miR-22 inhibition, as confirmed by the attenuation of SOD, Gpx, NOX activities and MDA, 4-HNE, GSH/ GSSG, 3-NT levels (Fig. 2AeE). Besides, Dox-triggered the decrease of Bcl2/Bax and the increase of caspase3 activity were also blunted in miR-22 inhibited hearts (Fig. 2FeG). TUNEL staining further implied that miR-22 inhibition attenuated Dox-induced cardiomyocyte apoptosis (Fig. 2H). We further investigated the beneficial effect of miR-22 antagomir in vitro. As shown in Fig. 2I, miR-22 inhibition suppressed ROS generation, further confirmed by the decreased levels of MDA and 3-NT (Fig. 2J). SOD, Gpx activities downregulation and NOX activity upregulation were also restored after miR-22 inhibition (Fig. 2KeL). In line with the in vivo data, Dox-induced H9C2 cardiomyocyte apoptosis was also ameliorated by miR-22 inhibition (Fig. 2MeP).
3.4. MiR-22 inhibition alleviated Dox-induced oxidative stress and cardiomyocyte apoptosis via upregulating SIRT1 We then explored the possible mechanism through which miR22 antagomir exerted the beneficial effects. Previous studies implied that miR-22 could target the 30 -UTR of Sirt1 and caused SIRT1 downregulation [18,20]. Surprisingly, we found that miR-22 inhibition increased SIRT1 protein and mRNA levels in the presence or absence of Dox (Fig. 3AeB). To notify whether Sirt1 is a direct target of miR-22, we performed luciferase reporter assay. Compared with the control group, miR-22 mimic significantly decreased the luciferase activity, however, with no alteration when the binding site was mutated (Fig. 3C). EX-527 was used for SIRT1 inhibition in
further studies (Fig. 3D). As shown in Fig. 3EeF, SIRT1 inhibitor blunted miR-22 antagomir-mediated the upregulation of SOD, Gpx activities and the downregulation of NOX activity. Consistently, MDA and 3-NT induction were prevented in miR-22-inhibited cells, but not in that with EX-527 incubation (Fig. 3G). The attenuation on cardiomyocyte apoptosis were also reversed by SIRT1 inhibition, as evidenced by the Bcl2/Bax ratio, caspse3 activity, cell viability and LDH leakage (Fig. 3HeK). Accordingly, the restoration of AMCs contractile capacity by miR-22 inhibition was completely blocked after SIRT1 suppression (Fig. 3L). These data collectively proved that the protective effects of miR-22 antagomir on oxidative stress and cardiomyocyte apoptosis was mediated via upregulating SIRT1.
3.5. MiR-22 inhibition-mediated beneficial effects were abolished by SIRT1 inhibitor in mice Consistent with the data in vitro, we observed that miR-22 antagomir-induced upregulation of SOD, Gpx activities and downregulation of NOX activity were reversed in murine hearts with EX-527 administration (Fig. 4AeB). Accordingly, the attenuation of MDA and 3-NT production was reduced in miR-22-deficient hearts, but not in that with SIRT1 inhibition (Fig. 4CeD). Caspase3 activity detection and TUNEL assay revealed the fact that SIRT1 inhibition abolished the beneficial effect of miR-22 antagomir on cardiomyocyte apoptosis (Fig. 4E). Improved cardiac function by miR-22 inhibition was also abolished by the SIRT1 inhibitor (Fig. 4FeH). Moreover, miR-22 inhibition-induced cTnT downregulation was also blunted by EX-527 (Fig. 4I).
Fig. 2. MiR-22 inhibition attenuated Dox-induced oxidative stress and cardiomyocyte apoptosis in vivo and in vitro. A-B. Superoxide dismutase (SOD), glutathione peroxidases (Gpx) and NADPH oxidase (NOX) activities in murine hearts (n ¼ 6). C-E. Myocardial malondialdehyde (MDA), 4-hydroxynonenal (4-HNE), glutathione to oxidized glutathione (GSH/ GSSG), 3-nitrotyrosine (3-NT) levels in mice (n ¼ 6). F. Statistical result of Bcl2/Bax in murine hearts (n ¼ 6). G-H. Caspase3 activity and TUNEL þ nuclei index (n ¼ 6). I. DCFH-DA staining in H9C2 cells (n ¼ 6). J. MDA and 3-NT production in H9C2 cells (n ¼ 6). K-L. SOD, Gpx and NOX activities in H9C2 cells (n ¼ 6). M. Data of Bcl2/Bax in H9C2 cells (n ¼ 6). NeO. Statistical results of caspase3 activity and cell viability (n ¼ 6). P. LDH level release to the medium (n ¼ 6). All data are expressed as the mean ± SD, *P < 0.05.
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Fig. 3. MiR-22 inhibition alleviated Dox-induced oxidative stress and cardiomyocyte apoptosis via upregulating SIRT1. A. Western blots and the statistical results in H9C2 cells (n ¼ 6). B. Sirt1 mRNA level in H9C2 cells (n ¼ 6). C. Statistical result of the dual luciferase assay (n ¼ 8). D. SIRT1 activity detection in H9C2 cells treated with or without EX-527 (n ¼ 6). E-F. SOD, Gpx and NOX activities in H9C2 cells (n ¼ 6). G. MDA and 3-NT production in H9C2 cells (n ¼ 6). H. The relative mRNA level of Bcl2/Bax (n ¼ 6). I. Statistical result of caspase3 activity in H9C2 cells (n ¼ 6). J. Cell viability detection using H9C2 cells (n ¼ 6). K. LDH level in the medium of H9C2 cells (n ¼ 6). L. Peaking shortening and þdL/dt in AMCs isolated from indicating murine hearts (n ¼ 6). All data are expressed as the mean ± SD, *P < 0.05.
4. Discussion MicroRNAs are emerging as important regulators in Doxinduced cardiotoxicity through binding to the 30 -UTR of target genes, and even could be used as early sensitive cardiotoxicity biomarkers [14]. MiR-22 was previously shown to be ubiquitously expressed in multiple tissues that was recently identified as a cardiac- and skeletal muscle-enriched microRNA [18]. Gurha et al. proved that miR-22 played critical roles in regulating myocardial calcium homeostasis and myofibrillar protein content, and was required for the heart to adapt to pressure overload-induced cardiac hypertrophy [19]. Conversely, cardiomyocyte-specific deletion of miR-22 repressed the progression of cardiac hypertrophy and impaired cardiac response to stress [18]. In addition, miR-22 was also involved in adjusting mitochondrial function, and miR-22 upregulation contributed to cardiac mitochondrial damage and the progression of ischemia reperfusion injury [20]. In the current study, we showed that miR-22 was upregulated in Dox-treated murine hearts and cardiomyocytes, and miR-22 inhibition prevented Dox-induced cardiac dysfunction. Oxidative stress and cardiomyocyte apoptosis were proposed as the key mechanisms responsible for Dox-triggered cardiotoxicity [2,34]. Previous studies indicated that the Dox showed a specific affinity to the heart, and could be accumulated in cardiac mitochondria. In the mitochondrial inner membrane, Dox formed a nearly-irreversible complex with cardiolipin and then disrupted its normal physiological function, which subsequently promoted massive ROS generation and cell death [35]. Previous studies
showed that oxidative stress could be observed in heart samples within 3 h after Dox treatment, and interference on ROS overproduction significantly improved Dox-induced cardiac dysfunction [3,36]. MiR-22 level was previously suggested to be positively related to MDA, whereas negatively correlated to GSH/GSSG ratio in the myocardium, implying the possible role of miR-22 in regulating myocardial oxidative stress [37]. Herein, we found that miR-22 antagomir enhanced the total anti-oxidant capacity of the heart and markedly reduced Dox-induced oxidative stress and cardiomyocyte apoptosis. SIRT1 is known to exert various bioactivities via suppressing oxidative stress in cardiovascular diseases, and SIRT1 activation was linked to the attenuation on Dox-induced oxidative stress and cardiomyocyte apoptosis [10,11]. In the present study, we found that miR-22 could target the 30 -UTR of Sirt1 and thereby leading to SIRT1 downregulation. MiR-22 inhibition partially restored the SIRT1 level after Dox treatment, and SIRT1 suppression abolished the beneficial effects of miR-22 antagomir on Dox-induced oxidative stress and cardiomyocyte apoptosis in vivo and in vitro. Beyond the role in controlling cardiac pathophysiology, miR-22 was also indicated to be involved in regulating tumorigenesis. Numerous studies found that miR-22 expression was downregulated in several tumor cell lines and clinical samples, whereas miR-22 overexpression caused cellular senescence and inhibited tumor progression [17]. However, Palacios et al. demonstrated that miR-22 was upregulated in proliferative B cells in chronic lymphocytic leukemia, which then induced PTEN downregulation, PI3K/AKT activation and excessive cell proliferation [38]. Hence the efficiency
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Fig. 4. MiR-22 inhibition-mediated beneficial effects were abolished by SIRT1 inhibitor in mice. A-B. SOD, Gpx and NOX activities in the indicating murine hearts (n ¼ 6). C-D. Myocardial MDA and 3-NT production in mice (n ¼ 6). E. Statistical result of caspase3 and TUNEL þ nuclei in murine hearts (n ¼ 6). FeH. Statistical results of EF, SV and þdP/dt (n ¼ 8). I. Serum level of cTnT in mice (n ¼ 8). All data are expressed as the mean ± SD, *P < 0.05.
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Please cite this article as: C. Xu et al., MicroRNA-22 inhibition prevents doxorubicin-induced cardiotoxicity via upregulating SIRT1, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/j.bbrc.2019.10.140