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miR‑320‑3p is involved in morphine pre‑conditioning to protect rat cardiomyocytes from ischemia/reperfusion injury through targeting Akt3

  • Authors:
    • Lan Cao
    • Shijun Chai
  • View Affiliations

  • Published online on: May 27, 2020     https://doi.org/10.3892/mmr.2020.11190
  • Pages: 1480-1488
  • Copyright: © Cao et al. This is an open access article distributed under the terms of Creative Commons Attribution License.

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Abstract

Morphine pre‑conditioning (MPC) can significantly reduce myocardial ischemic injury and inhibit cardiomyocyte apoptosis, but the underlying mechanism still remains unclear. The aim of the present study was to investigate the protective mechanism of MPC in myocardial hypoxia/reoxygenation (H/R) injury at the microRNA (miR) level. H9c2 cells were used as a model of H/R and subjected to morphine pre‑treatment. The protective effects of MPC on H/R injury in cardiomyocytes were evaluated using MTT and colorimetric assay, as well as flow cytometry. In addition, reverse transcription‑quantitative PCR, western blotting and dual‑luciferase reporter assay experiments were performed to determine the relationship between MPC, miR‑320‑3p and Akt3, and their effects on H/R injury. The present study demonstrated that MPC enhanced cell activity, decreased LDH content, and reduced apoptosis in rat cardiomyocytes, suggesting that MPC could protect these cells from H/R injury. Moreover, MPC partially reversed the increase in miR‑320‑3p expression and the decrease in Akt3 levels caused by H/R injury. Inhibition of miR‑320‑3p expression also attenuated the effects of H/R on cardiomyocyte activity, LDH content and apoptosis. Furthermore, Akt3 was predicted to be a target gene of miR‑320‑3p, and overexpression of miR‑320‑3p inhibited the expression of Akt3, blocking the protective effects of MPC on the cells. The current findings revealed that MPC could protect cardiomyocytes from H/R damage through targeting miR‑320‑3p to regulate the PI3K/Akt3 signaling pathway.

Introduction

Myocardial ischemia/reperfusion injury (MI/RI) refers to rapid tissue damage caused by the recovery of blood flow from myocardial cells following ischemia (1). MI/RI is a common complication during the perioperative period of cardiac and macrovascular surgeries that can lead to the aggravation of myocardial damage and seriously affect the recovery of postoperative cardiac function (2). Cardiomyocyte hypoxia/reoxygenation (H/R) injury can be used to mimic myocardial ischemia/reperfusion in vitro (3,4). Morphine is a widely used analgesic in cardiac and macrovascular anesthesia (5,6). Compared with ischemic pre-conditioning, morphine could be conveniently and easily administered and cause less trauma to patients (7). Previous studies demonstrated that morphine pre-conditioning (MPC) significantly reduced myocardial ischemic injury and inhibited cardiomyocyte apoptosis, although the mechanism remains unclear (8,9).

MicroRNAs (miRNAs/miRs) are a class of endogenous, single-stranded, non-coding RNAs that regulate multiple biological processes such as cell proliferation, differentiation and apoptosis (10). Cardiomyocyte apoptosis is an important characteristic of MI/RI (11). Accumulating evidence indicates that miRNAs can regulate apoptosis of cardiomyocytes through their target genes and downstream signaling pathways (12). For example, Dong et al (13) suggested that miR-21 could inhibit ischemia-induced apoptosis by studying acute myocardial infarction in rats. Cheng et al (14) demonstrated that miR-21 also protected cardiomyocytes from hydrogen peroxide-induced damage by regulating programmed cell death 4. Moreover, miR-320 was also implicated in the regulation of myocardial ischemia/reperfusion injury, and miR-320 upregulation could promote cardiomyocyte apoptosis (15). Additionally, miR-320 has multiple functions in different environments (16), and its expression level is closely related to tumor migration and invasion. Indeed, overexpression of miR-320 is associated with high risk of metastasis and poor prognosis (17).

Previous studies demonstrated that 5′-adenosine monophosphate-activated protein kinase (AMPK) could ameliorate myocardial ischemia through the regulation of oxidative stress (18,19), autophagy (20,21) and apoptosis (22) in cardiomyocytes. Moreover, AMPK exerts its protective effects with other molecules that may have crosstalk with each other against myocardial ischemia, for example, mesenchymal stem cell (MSC)-derived exosomes could reduce MI/RI by inducing cardiomyocyte autophagy via AMPK/mTOR and Akt/mTOR pathways (21,23,24). Sun et al (25) demonstrated that dexmedetomidine protected mice against MI/RI by activating the AMPK/phosphatidylinositol-4,5-bisphosphate 3-kinase (PI3K)/Akt/endothelial nitric oxide synthase pathway. PI3K/Akt signaling is an important signaling pathway associated with many diseases, including cancer and neurological diseases (26). Akt kinase consists of three subtypes, Aktl, Akt2 and Akt3, which are the key factors downstream of the PI3K signaling pathway that regulate cell proliferation, apoptosis and metastasis (27). Previous studies indicated that activation of the PI3K-Akt signaling pathway protected the heart from reperfusion injury (28). However, whether morphine pre-conditioning participates in myocardial H/R injury through the regulation of miR-320-3p and the PI3K/Akt signaling pathway is not fully understood. Therefore, the current study examined the myocardial protection mechanism of MPC following at the miRNA level, and aimed to elucidate the relationship between MPC, miR-320-3p and the PI3K/Akt signaling pathway in the regulation of H/R injury of cardiomyocytes.

Materials and methods

Cell culture

H9c2 cells (cat. no. CRL-1446) were obtained from the American Type Culture Collection and divided into four groups: i) Control, ii) MPC, iii) H/R, and iv) MPC+H/R. Cells from the control group were cultured in DMEM/F-12 containing 10% FBS, and 1% penicillin/streptomycin at 37°C with 5% CO2 (all from Thermo Fisher Scientific, Inc.). After one day of culture, the original medium was discarded, and the cells were washed once or twice with PBS, then resuspended in 5 ml fresh DMEM/F-12 medium and returned to the CO2 incubator. The cells were passaged when grown to 80 to 90% confluence. In the MPC group, cells were cultured normally following a 10-min treatment with 1 µM morphine (cat. no. 121206-2; Northeast Pharmaceutical Group Co., Ltd.). In the H/R group, DMEM/F12 medium containing 10% serum was replaced by anoxic solution (i.e., glucose-free and serum-free medium containing 2.3 mM CaCl2, 5.6 mM KCl, 154 mM NaCl, 5 mM Hepes, and 3.6 mM NaHCO3; pH 7.4). The culture plate was maintained in anoxic chamber for 5 h at 37°C, and then reoxygenation for 1 h in DMEM/F12 medium containing 10% serum under a humidified atmosphere (95% air and 5% CO2) at 37°C. For the MPC + H/R group, the cells were treated with 1 µM morphine in serum-free DMEM/F12 for 10 min. Following the treatment, serum-free DMEM/F12 was replaced by DMEM/F12 medium containing 10% serum and the MPC pre-treatment was completed after 30 min of normal culture. Cells then received the same treatment as the H/R group.

Cell transfection

Cells were cultured for 12–16 h to 60–70% confluence, and then transfected with Lipofectamine® 3000 (Thermo Fisher Scientific, Inc.) according to the manufacturer's protocol. miR-320-3p mimic (miR10000903-1-5; 5′-AAAAGCUGGGUUGAGAGGGCGA-3′), mimic negative control (miRB160401025525-2-1; 5′-UUUGUACUACACAAAAGUACUG-3′), and miR-320-3p inhibitor (miR20000903-1-5; 5′-UCGCCCUCUCAACCCGCUUUU-3′), and inhibitor negative control (miR2N0000001-1-5; 5′-CAGUACUUUUGUGUAGUACAAA-3′) were purchased from Guangzhou RiboBio Co., Ltd. Briefly, when cells were at 60 to 70% confluence, the transfection was performed using Lipofectamine® 3000 (Thermo Fisher Scientific, Inc.). miR-320-3p mimic, inhibitor, corresponding control and Lipofectamine® RNAiMAX (Thermo Fisher Scientific, Inc.) were diluted in Opti-MEM medium (Thermo Fisher Scientific, Inc.). Then, the diluted RNAs were added into the diluted Lipofectamine® reagent and incubated for 10 min at room temperature. The mixture containing the RNA was added to the cells. Following an 8-h incubation in the presence of transfection reagent, the medium was replaced, and the cells incubated for 2–4 days at 37°C prior to subsequent experimentation.

Bioinformatics prediction

TargetScan7.2. (http://www.targetscan.org) was used for the biological prediction of miR-320-3p target genes.

Dual luciferase activity assay

The sequence of Akt3-3′-UTR was cloned into the pmirGLO luciferase vector (cat. no. E1330; Promega Corp.). H9c2 cells were transfected with miR-320-3p mimic to detect the binding of Akt3 and miR-320-3p. Cells transfected with miR-320-3p mimic negative control were used as the blank group. The cell transfection was performed by transfected with Lipofectamine® 3000 (Thermo Fisher Scientific, Inc.) according to the manufacturer's protocol. Luciferase activity was determined using the Double-Luciferase Reporter Assay kit (cat. no. FR201-01; TransGen Biotech) according to the manufacturer's protocol. Briefly, the cell culture medium was removed, and the cells were carefully washed twice in PBS, then added to 20 µl 1X Cell Lysis Buffer from the kit. The cells were lysed for 10 min at room temperature and centrifuged at 12,000 × g for 10 min at 4°C to collect supernatant for later use. After centrifugation, 100 µl Luciferase Reaction Reagent was added to the tube at room temperature, and 20 µl cell lysate was carefully pipetted into the tube and mixed gently to measure the activity of the firefly luciferase reporter gene in a luminometer (SpectraMaxL, Molecular Devices, LLC). Finally, 100 µl Luciferase Reaction Reagent II was added to the above reaction tube at room temperature, vortexed, and the activity of the Renilla luciferase reporter gene was detected using a SpectraMaxL luminometer (Molecular Devices, LLC).

Reverse transcription-quantitative PCR (RT-qPCR)

Total RNA extraction from lysed H9c2 cells was carried out at 4°C using TRIzol® (Invitrogen; Thermo Fisher Scientific, Inc.), and all the consumables and reagents used in the extraction process were subjected to DEPC treatment. The RT of miRNA and mRNA was conducted using TaqMan MicroRNA Reverse Transcription kit (cat. no. 4366597; Thermo Fisher Scientific, Inc.) and TaqMan Reverse Transcription Reagents (cat. no. N8080234; Thermo Fisher Scientific, Inc.). GAPDH served as internal control for mRNA, while U6 served as internal control for miRNA quantification. The primer sequences were as follows: miR-320-3p-forward (F), 5′-TAAGTGCTTCCATGTTTTGGTGA-3′; miR-320-3p-reverse (R), 5′-GAACATGTCTGCGTATCTCAGACTTC-3′; Akt3-F, 5′-TCCCCCGAACACTCTCTTCA-3′; Akt3-R, 5′-CCCTCCACCAAGGCGTTTAT-3′; GAPDH-F, 5′-ATGACTCTACCCACGGCAAG-3′; GAPDH-R, 5′-GGAAGATGGTGATGGGTTTC-3′; U6-F, 5′-CTCGCTTCGGCAGCACA-3′; U6-R, 5′-AACGCTTCACGAATTTGCGT-3′. RT-qPCR was performed using the ABI7500 Real-Time PCR System (Thermo Fisher Scientific, Inc.) according to the manufacturer's instructions. The thermocycling conditions were set as follows: Initial denaturation at 95°C for 10 min, followed by 45 cycles at 95°C for 30 sec, 59°C for 1 min, and finally at 60°C for 1 min, and preservation at 4°C. The relative expression levels were calculated using the 2−ΔΔCq method (29).

MTT assay

Cell viability was detected using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (cat. no. M6494; Thermo Fisher Scientific, Inc.). H9c2 cells were seeded in 96-well plates at a density of 5×104/ml and incubated for 24, 48 and 72 h at 37°C. A volume of 10 µl MTT reagent was added into each well and cells were then cultured for an additional 4 h at 37°C. After removing the MTT, 100 µl DMSO was added. Finally, the optical density was measured at 490 nm using a microplate reader (cat. no. 24072800; Thermo Fisher Scientific, Inc.). Cells were seeded in triplicate wells and the experiment was repeated three times.

Lactate dehydrogenase (LDH) content determination

LDH content was measured using the Cytotoxicity LDH Assay Kit-WST (cat. no. CK12; Dojindo Molecular Technologies, Inc.), according to the manufacturer's protocol. Briefly, the cell suspension was diluted in 50 µl medium, added to the 96-well plate with 50 µl medium and incubated in a CO2 incubator at 37°C for 24 h. A volume of 10 µl Lysis Buffer was added into the control well and the reaction was incubated at 37°C with 5% CO2 for 30 min. After adding 100 µl Working Solution into each well, the cells were incubated in the dark at room temperature for 30 min. Next, 50 µl Stop Solution was added to terminate the reaction, and the absorbance at 490 nm was determined immediately using a microplate reader (cat. no. 24072800; Thermo Fisher Scientific, Inc.).

Flow cytometry

A total of 1×106 H9c2 cells were harvested then centrifuged at 1,200 × g for 1 min at 4°C, then resuspended in 300 µl pre-chilled PBS containing 10% calf serum. Cells were then fixed in 700 µl absolute ethanol at −20°C for >24 h, and centrifuged at 140 × g for 30 sec at 4°C. Fixed cells were resuspended in 500 µl cold PBS, and centrifuged at 1,200 × g for 1 min at 4°C. After discarding the supernatant, the pelleted cells were suspended in 480 µl cold PBS and 10 µl of 0.1 mg/ml RNase A, and allowed to stand at 37°C for 30 min. Annexin V-FITC and propidium iodide (PI) staining solution (5 µl each; cat. no. APOAF; Sigma-Aldrich; Merck KGaA) were added to the cell suspension for 15 min at room temperature in the dark. After the incubation, cells were filtered once through a 400-mesh sieve, and apoptosis was detected using a BD FACS Canto™ flow cytometer. Analysis was carried out using FlowJo version 10.0 (FlowJo, LLC). The lower left quadrant (FITC-H/PI) represents healthy living cells, while the lower right quadrant (FITC-H+/PI) represents early apoptotic cells. Late apoptotic cells are gated in the upper right quadrant (FITC-H+/PI+) and necrotic cells are found in the upper left quadrant (FITC-H/PI+).

Western blot analysis

Total protein was extracted from H9c2 cells using RIPA buffer (cat. no. 89901; Thermo Fisher Scientific, Inc.) containing protease inhibitor and phosphatase inhibitor. The protein concentrations were measured using Pierce Rapid Gold BCA Protein Assay kit (cat. no. A53227; Thermo Fisher Scientific, Inc.). 30 µg protein was separated by SDS-PAGE on 10% gels, then transferred to PVDF membranes (cat. no. HVLP04700; EMD Millipore). The films were fully rinsed in TBS, and then transferred to a dish containing 5% skim milk solution and blocked at 37°C for 1 h, then shaken for 2 h at room temperature on a decolorizing shaker. The membranes were then incubated with rabbit primary antibody against Akt3 (72 kDa; 1:2,000; cat. no. ab152157; Abcam) or mouse anti-GAPDH (36 kDa; 1:20,000; cat. no. ab8245; Abcam) overnight at 4°C. The membrane was then incubated with an HRP-conjugated secondary antibody (1:5,000; cat. nos. ab205718 and ab205719; Abcam) for 2 h at room temperature. Finally, SignalFire electrochemiluminescence reagent (cat. no. 6883; Cell Signaling Technology, Inc.) was used for visualization. The ImageJ version 5.0 software (Bio-Rad Laboratories, Inc.) for quantification. GAPDH was used as the reference protein.

Statistical analysis

Data are presented as the mean ± SD. Multi-group comparisons were conducted using one-way ANOVA, followed by Tukey's post hoc test. Statistical analysis was carried out using GraphPad Prism 7.0 (GraphPad Software, Inc.). P<0.05 was considered to indicate a statistically significant difference. All experiments were performed in triplicate.

Results

MPC protects rat cardiomyocytes from H/R damage

To investigate the effects of MPC on reperfusion injury in rat cardiomyocytes, H9c2 cells were divided into Control, MPC, H/R, MPC+H/R groups. An MTT assay demonstrated that the viability of cells in the H/R group was reduced, compared with the control. However, cell viability in the MPC+H/R group was significantly higher than that of the H/R group (P<0.05; Fig. 1A).

LDH levels indicate the number of dead and damaged cells. In the H/R group, the LDH content was increased, compared with the control. However, the LDH content in the MPC+H/R group was significantly lower than that in the H/R group (P<0.05; Fig. 1B). In addition, the levels of apoptosis were also examined in each of the four groups of cells. The frequency of apoptotic cells in the H/R group was increased, compared with control, but significantly decreased in the MPC+H/R group, compared with the H/R group (P<0.05; Fig. 1C).

MPC regulates the expression of miR-320-3p and Akt3

To further investigate the molecular mechanism of MPC in rat cardiomyocytes, RT-qPCR was performed to detect the expression of miR-320-3p. MPC treatment alone did not affect the expression of miR-320-3p. The expression of miR-320-3p in the MPC+H/R group was significantly lower than that in the H/R group (P<0.05; Fig. 2A). Moreover, the mRNA and protein expression levels of Akt3 were also examined. MPC treatment alone did not affect the expression of Akt3. However, the expression of Akt3 in the H/R group was reduced, compared with the control. Additionally, expression of Akt3 in the MPC+H/R group was significantly higher than that in the H/R group (P<0.05; Fig. 2B and C).

Downregulation of miR-320-3p reduces H/R damage to H9c2 cells

To further understand the biological role of miR-320-3p in reperfusion injury of rat cardiomyocytes, H9c2 cells were transfected with an miR-320-3p inhibitor (Fig. 3A). In the H/R group, an MTT assay demonstrated that the viability of cells transfected with the inhibitor (Inhibitor+H/R) was significantly higher than untransfected cells and was similar to the MPC+H/R group (P<0.05; Fig. 3B). Furthermore, the LDH content in the Inhibitor+H/R group was lower than that in the unstranfected H/R group and was similar to the MPC+H/R group (P<0.05; Fig. 3C). As indicated by flow cytometry, the rates of apoptosis of in the Inhibitor+H/R group was similar to that of the MPC+H/R group. However, this was significantly lower than that of the H/R group (P<0.001; Fig. 3D).

Upregulation of miR-320-3p targeting Akt3 inhibits the protective effect of MPC on H9c2 cells

Using TargetScan v7.2 bioinformatics analysis and luciferase assay, Akt3 was identified as the direct target of miR-320-3p (Fig. 4A and B). Both at the mRNA and protein levels, the expression of Akt3 was significantly reduced in the untransfected cells of the H/R group, compared with control. However, Akt3 expression in the H/R-treated cells transfected with the inhibitor was significantly higher, compared to the untransfected cells (P<0.001; Fig. 4C and D).

To better understand the effects of MPC, miR-320-3p and Akt3 on reperfusion injury of rat cardiomyocytes, H9c2 cells were transfected with an miR-320-3p mimic (Fig. 5A). The expression of Akt3 in transfected cells of the MPC+H/R group (Mimic+MPC+H/R group) was significantly lower than that in the untransfected MPC+H/R group (P<0.05; Fig. 5B and C). Moreover, an MTT assay suggested that cell viability in the Mimic+MPC+H/R group was significantly lower than that in the MPC+H/R group (P<0.05; Fig. 5D). To further assess the biological function of miR-320-3p in H9c2 cells, LDH content was measured by colorimetry in each group. The LDH content of the Mimic+MPC+H/R group was significantly higher than that of the MPC+H/R group (P<0.05; Fig. 5E). In addition, the rates of apoptosis in the Mimic+MPC+H/R group was significantly increased, compared with the MPC+H/R group (P<0.05; Fig. 5F).

Discussion

Myocardial ischemia/reperfusion injury (MI/RI) is a pathophysiological process that occurs after revascularization in patients with ischemic heart disease, such as myocardial infarction (30,31). Morphine is a non-selective opioid receptor agonist widely used as an anesthetic and in the treatment and myocardial infarction (32). Previous research has demonstrated that morphine has an early pre-conditioning effect on cardiomyocytes that is triggered by activation of opioid receptors (33). In the present study, the H9c2 cardiomyocyte hypoxia/reoxygenation injury model was established as previously described (34) but modified to incorporate 1 µM morphine as pre-treatment (35). In the present study, MPC significantly enhanced the viability of cardiomyocytes, reduced the levels of LDH resulting from H/R injury, and inhibited the apoptosis of cardiomyocytes, indicating that MPC alleviates H/R injury and plays a role in myocardial protection.

miRNAs are abundantly expressed in cardiomyocytes and participate in pathophysiological processes, such as cardiomyocyte apoptosis, myocardial remodeling and heart failure (10). Several previous studies suggest that MI/RI could be alleviated through the upregulation or downregulation of the expression of target miRNAs, pointing to a new therapeutic target for clinical myocardial protection (36,37). Yang et al (38) demonstrated that miRNA-22 can reduce apoptosis in MI/RI in a rat model by inhibiting CREB-binding protein expression and its downstream signaling pathway. Li et al (39) suggested that miRNA-145 expression was downregulated in an MI/RI model, whereas upregulation of miRNA-145 inhibited hydrogen peroxide-induced apoptosis, reactive oxygen species production, and activation of key signaling proteins in the mitochondrial apoptotic pathway. In addition, it has been reported that morphine can upregulate the expression of miR-133b-5p and protect cardiomyocytes from H/R injury, indicating that the biological function of morphine may be achieved by regulating miRNA expression (9).

Previous studies indicate that the regulation of multiple signaling pathways could improve myocardial injury. For example, melatonin protects the diabetic heart against ischemia-reperfusion injury through membrane receptor-dependent activation of cyclic GMP-dependent guanosine monophosphate protein kinase (40). The inhibition of miR-101a-3p alleviates H/R injury in H9c2 cells by regulating the JAK2/STAT3 pathway (41). Downregulation of miR-134 protects against myocardial H/R injury through targeting nitric oxide synthase 3 and regulating the PI3K/Akt pathway (42). However, whether myocardial protection by MPC involves the regulation of miR-320-3p and PI3K/Akt signaling pathways is still unclear.

The present findings demonstrated that miR-320-3p expression was upregulated and Akt3 expression was downregulated in cardiomyocytes after H/R injury, while MPC partially reversed the changes in miR-320-3p and Akt3 expression induced by the injury. This indicated that MPC plays an important role in the protection of H9c2 cardiomyocytes by regulating miR-320-3p expression and the PI3K/Akt signaling pathway. Previous studies also demonstrated that miR-320 serves critical functions in many diseases such as glioma, ovarian cancer and osteosarcoma (16,4346). Another study reported that miR-320 targeted A-kinase interacting protein 1 to induce mitochondrial apoptosis, and contributed to MI/RI (47). In the present study, the role of miR-320-3p in myocardial ischemia/reperfusion was further investigated using transfection of an miR-320-3p inhibitor, which indicated that inhibition of miR-320-3p expression enhanced cell viability, reduced LDH content and inhibited apoptosis. Moreover, these results were consistent with a previous study that suggested that miR-320-3p is involved in MI/RI (47).

In line with previous studies, it was hypothesized that downregulation of miR-320-3p may be involved in mediating MPC to protect myocardial cells from H/R injury through the regulation of apoptosis. Through bioinformatics analysis and experimental validation, it was determined that miR-320-3p could directly interact with Akt3. The Akt3 gene encodes protein kinase B and plays an important role in the regulation of several physiological processes (48,49). Transgenic mice display significant cardiac hypertrophy, suggesting that one of the functions of Akt3 is to promote cell growth (28). The PI3K/Akt signaling pathway is also involved in cardiac hypertrophy, myocardial cell remodeling and the prevention of inflammation, thereby alleviating myocardial ischemia-reperfusion injury (5052). The present study suggested that overexpression of miR-320-3p inhibited the expression Akt3 following MPC, and reduced the protective effect of MPC on H/R injury in cardiomyocytes. This indicated that MPC may protect cardiomyocytes from H/R injury through inhibiting miR-320-3p expression and the PI3K/Akt signaling pathway.

In conclusion, the present study demonstrated that MPC could significantly reduce H/R injury of H9c2 cardiomyocytes, and its mechanism of action may be related to miR-320-3p and the PI3K/Akt3 signaling pathway. The present findings provide a basis for further research on miRNA regulation mechanism of morphine pre-treatment.

Acknowledgements

Not applicable.

Funding

No funding was received.

Availability of data and materials

The analyzed data sets used and/or analyzed during the current study are available from the corresponding author on reasonable request.

Authors' contributions

LC and SC made substantial contributions to conception and design. LC and SC acquired, analyzed and interpreted data. LC and SC drafted the article and critically revised the draft for important intellectual content. LC and SC agree to be accountable for all aspects of the work in ensuring that questions related to the accuracy or integrity of the work are appropriately investigated and resolved. All authors approved the final version of the manuscript.

Ethics approval and consent to participate

Not applicable.

Patient consent for publication

Not applicable.

Competing interests

The authors declare that they have no competing interests.

References

1 

Frank A, Bonney M, Bonney S, Weitzel L, Koeppen M and Eckle T: Myocardial ischemia reperfusion injury: From basic science to clinical bedside. Semin Cardiothorac Vasc Anesth. 16:123–132. 2012. View Article : Google Scholar : PubMed/NCBI

2 

Konstantinidis K, Whelan RS and Kitsis RN: Mechanisms of cell death in heart disease. Arterioscler Thromb Vasc Biol. 32:1552–1562. 2012. View Article : Google Scholar : PubMed/NCBI

3 

Chen C, Jia KY, Zhang HL and Fu J: MiR-195 enhances cardiomyocyte apoptosis induced by hypoxia/reoxygenation injury via downregulating c-myb. Eur Rev Med Pharmacol Sci. 20:3410–3416. 2016.PubMed/NCBI

4 

Cao H, Xu H, Zhu G and Liu S: Isoquercetin ameliorated hypoxia/reoxygenation-induced H9C2 cardiomyocyte apoptosis via a mitochondrial-dependent pathway. Biomed Pharmacother. 95:938–943. 2017. View Article : Google Scholar : PubMed/NCBI

5 

McGilliard KL and Takemori AE: Alterations in the antagonism by naloxone of morphine-induced respiratory depression and analgesia after morphine pretreatment. J Pharmacol Exp Ther. 207:884–891. 1978.PubMed/NCBI

6 

Swartjes M, Mooren RA, Waxman AR, Arout C, van de Wetering K, den Hartigh J, Beijnen JH, Kest B and Dahan A: Morphine induces hyperalgesia without involvement of µ-opioid receptor or morphine-3-glucuronide. Mol Med. 18:1320–1326. 2012. View Article : Google Scholar : PubMed/NCBI

7 

Okubo S, Tanabe Y, Takeda K, Kitayama M, Kanemitsu S, Kukreja RC and Takekoshi N: Ischemic preconditioning and morphine attenuate myocardial apoptosis and infarction after ischemia-reperfusion in rabbits: Role of delta-opioid receptor. Am J Physiol Heart Circ Physiol. 287:H1786–H1791. 2004. View Article : Google Scholar : PubMed/NCBI

8 

Wang Y, Wang L, Li JH, Zhao HW and Zhang FZ: Morphine alleviates myocardial ischemia/reperfusion injury in rats by inhibiting TLR4/NF-κB signaling pathway. Eur Rev Med Pharmacol Sci. 23:8616–8624. 2019.PubMed/NCBI

9 

He SF, Zhu HJ, Han ZY, Wu H, Jin SY, Irwin MG and Zhang Y: MicroRNA-133b-5p is involved in cardioprotection of morphine preconditioning in rat cardiomyocytes by targeting fas. Can J Cardiol. 32:996–1007. 2016. View Article : Google Scholar : PubMed/NCBI

10 

Bartel DP: MicroRNAs: Genomics, biogenesis, mechanism, and function. Cell. 116:281–297. 2004. View Article : Google Scholar : PubMed/NCBI

11 

Whelan RS, Kaplinskiy V and Kitsis RN: Cell death in the pathogenesis of heart disease: Mechanisms and significance. Annu Rev Physiol. 72:19–44. 2010. View Article : Google Scholar : PubMed/NCBI

12 

Hwang CK, Wagley Y, Law PY, Wei LN and Loh HH: MicroRNAs in opioid pharmacology. J Neuroimmune Pharmacol. 7:808–819. 2012. View Article : Google Scholar : PubMed/NCBI

13 

Dong S, Cheng Y, Yang J, Li J, Liu X, Wang X, Wang D, Krall TJ, Delphin ES and Zhang C: MicroRNA expression signature and the role of microRNA-21 in the early phase of acute myocardial infarction. J Biol Chem. 284:29514–29525. 2009. View Article : Google Scholar : PubMed/NCBI

14 

Cheng Y, Liu X, Zhang S, Lin Y, Yang J and Zhang C: MicroRNA-21 protects against the H(2)O(2)-induced injury on cardiac myocytes via its target gene PDCD4. J Mol Cell Cardiol. 47:5–14. 2009. View Article : Google Scholar : PubMed/NCBI

15 

Ren XP, Wu J, Wang X, Sartor MA, Jones K, Qian J, Nicolaou P, Pritchard TJ and Fan GC: MicroRNA-320 is involved in the regulation of cardiac ischemia/reperfusion injury by targeting heat-shock protein 20. Circulation. 119:2357–2366. 2009. View Article : Google Scholar : PubMed/NCBI

16 

Wu H, Li W, Zhang M, Zhu S, Zhang D and Wang X: Inhibitory roles of miR-320 in osteosarcoma via regulating E2F1. J Cancer Res Ther. 12:S68–S71. 2016. View Article : Google Scholar

17 

Wang W, Yang J, Xiang YY, Pi J and Bian J: Overexpression of hsa-miR-320 is associated with invasion and metastasis of ovarian cancer. J Cell Biochem. 118:3654–3661. 2017. View Article : Google Scholar : PubMed/NCBI

18 

Chen Q and Lesnefsky EJ: A new strategy to decrease cardiac injury in aged heart following ischaemia-reperfusion: Enhancement of the interaction between AMPK and SIRT1. Cardiovasc Res. 114:771–772. 2018. View Article : Google Scholar : PubMed/NCBI

19 

Tian L, Cao W, Yue R, Yuan Y, Guo X, Qin D, Xing J and Wang X: Pretreatment with Tilianin improves mitochondrial energy metabolism and oxidative stress in rats with myocardial ischemia/reperfusion injury via AMPK/SIRT1/PGC-1 alpha signaling pathway. J Pharmacol Sci. 139:352–360. 2019. View Article : Google Scholar : PubMed/NCBI

20 

Chen WR, Liu HB, Chen YD, Sha Y, Ma Q, Zhu PJ and Mu Y: Melatonin attenuates myocardial ischemia/reperfusion injury by inhibiting autophagy via an AMPK/mTOR signaling pathway. Cell Physiol Biochem. 47:2067–2076. 2018. View Article : Google Scholar : PubMed/NCBI

21 

Liu L, Jin X, Hu CF, Li R, Zhou Z and Shen CX: Exosomes derived from mesenchymal stem cells rescue myocardial ischaemia/reperfusion injury by inducing cardiomyocyte autophagy Via AMPK and Akt pathways. Cell Physiol Biochem. 43:52–68. 2017. View Article : Google Scholar : PubMed/NCBI

22 

Kosuru R, Cai Y, Kandula V, Yan D, Wang C, Zheng H, Li Y, Irwin MG, Singh S and Xia Z: AMPK contributes to cardioprotective effects of pterostilbene against myocardial ischemia-reperfusion injury in diabetic rats by suppressing cardiac oxidative stress and apoptosis. Cell Physiol Biochem. 46:1381–1397. 2018. View Article : Google Scholar : PubMed/NCBI

23 

Gu C, Li T, Jiang S, Yang Z, Lv J, Yi W, Yang Y and Fang M: AMP-activated protein kinase sparks the fire of cardioprotection against myocardial ischemia and cardiac ageing. Ageing Res Rev. 47:168–175. 2018. View Article : Google Scholar : PubMed/NCBI

24 

Chen K, Li G, Geng F, Zhang Z, Li J, Yang M, Dong L and Gao F: Berberine reduces ischemia/reperfusion-induced myocardial apoptosis via activating AMPK and PI3K-Akt signaling in diabetic rats. Apoptosis. 19:946–957. 2014. View Article : Google Scholar : PubMed/NCBI

25 

Sun Y, Jiang C, Jiang J and Qiu L: Dexmedetomidine protects mice against myocardium ischaemic/reperfusion injury by activating an AMPK/PI3K/Akt/eNOS pathway. Clin Exp Pharmacol Physiol. 44:946–953. 2017. View Article : Google Scholar : PubMed/NCBI

26 

Liu J, Huang W, Ren C, Wen Q, Liu W, Yang X, Wang L, Zhu B, Zeng L, Feng X, et al: Flotillin-2 promotes metastasis of nasopharyngeal carcinoma by activating NF-κB and PI3K/Akt3 signaling pathways. Sci Rep. 5:116142015. View Article : Google Scholar : PubMed/NCBI

27 

Zhou K, Fan YD, Wu PF, Duysenbi S, Feng ZH, Du GJ and Zhang TR: MicroRNA-145 inhibits the activation of the mTOR signaling pathway to suppress the proliferation and invasion of invasive pituitary adenoma cells by targeting AKT3 in vivo and in vitro. Onco Targets Ther. 10:1625–1635. 2017. View Article : Google Scholar : PubMed/NCBI

28 

Khedr RM, Ahmed AAE, Kamel R and Raafat EM: Sitagliptin attenuates intestinal ischemia/reperfusion injury via cAMP/PKA, PI3K/Akt pathway in a glucagon-like peptide 1 receptor-dependent manner. Life Sci. 211:31–39. 2018. View Article : Google Scholar : PubMed/NCBI

29 

Livak KJ and Schmittgen TD: Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) method. Methods. 25:402–408. 2001. View Article : Google Scholar : PubMed/NCBI

30 

Tang H, Song X, Ling Y, Wang X, Yang P, Luo T and Chen A: Puerarin attenuates myocardial hypoxia/reoxygenation injury by inhibiting autophagy via the Akt signaling pathway. Mol Med Rep. 15:3747–3754. 2017. View Article : Google Scholar : PubMed/NCBI

31 

Xiao R, Xiang AL, Pang HB and Liu KQ: Hyperoside protects against hypoxia/reoxygenation induced injury in cardiomyocytes by suppressing the Bnip3 expression. Gene. 629:86–91. 2017. View Article : Google Scholar : PubMed/NCBI

32 

Coles JA Jr, Sigg DC and Iaizzo PA: Role of kappa-opioid receptor activation in pharmacological preconditioning of swine. Am J Physiol Heart Circ Physiol. 284:H2091–H2099. 2003. View Article : Google Scholar : PubMed/NCBI

33 

Peart JN, Patel HH and Gross GJ: Delta-opioid receptor activation mimics ischemic preconditioning in the canine heart. J Cardiovasc Pharmacol. 42:78–81. 2003. View Article : Google Scholar : PubMed/NCBI

34 

Sun L, Zhao M, Yu XJ, Wang H, He X, Liu JK and Zang WJ: Cardioprotection by acetylcholine: A novel mechanism via mitochondrial biogenesis and function involving the PGC-1α pathway. J Cell Physiol. 228:1238–1248. 2013. View Article : Google Scholar : PubMed/NCBI

35 

Liang BT and Gross GJ: Direct preconditioning of cardiac myocytes via opioid receptors and KATP channels. Circ Res. 84:1396–1400. 1999. View Article : Google Scholar : PubMed/NCBI

36 

Pan Z, Sun X, Ren J, Li X, Gao X, Lu C, Zhang Y, Sun H, Wang Y, Wang H, et al: miR-1 exacerbates cardiac ischemia-reperfusion injury in mouse models. PLoS One. 7:e505152012. View Article : Google Scholar : PubMed/NCBI

37 

Yang Y, Del Re DP, Nakano N, Sciarretta S, Zhai P, Park J, Sayed D, Shirakabe A, Matsushima S, Park Y, et al: miR-206 mediates YAP-induced cardiac hypertrophy and survival. Circ Res. 117:891–904. 2015. View Article : Google Scholar : PubMed/NCBI

38 

Yang J, Chen L, Yang J, Ding J, Li S, Wu H, Zhang J, Fan Z, Dong W and Li X: MicroRNA-22 targeting CBP protects against myocardial ischemia-reperfusion injury through anti-apoptosis in rats. Mol Biol Rep. 41:555–561. 2014. View Article : Google Scholar : PubMed/NCBI

39 

Li R, Yan G, Li Q, Sun H, Hu Y, Sun J and Xu B: MicroRNA-145 protects cardiomyocytes against hydrogen peroxide (H2O2)-induced apoptosis through targeting the mitochondria apoptotic pathway. PLoS One. 7:e449072012. View Article : Google Scholar : PubMed/NCBI

40 

Yu LM, Di WC, Dong X, Li Z, Zhang Y, Xue XD, Xu YL, Zhang J, Xiao X, Han JS, et al: Melatonin protects diabetic heart against ischemia-reperfusion injury, role of membrane receptor-dependent cGMP-PKG activation. Biochim Biophys Acta Mol Basis Dis. 1864:563–578. 2018. View Article : Google Scholar : PubMed/NCBI

41 

Liu J, Wang J, Ning Y and Chen F: The inhibition of miR-101a-3p alleviates H/R injury in H9C2 cells by regulating the JAK2/STAT3 pathway. Mol Med Rep. 21:89–96. 2020.PubMed/NCBI

42 

Xiao JM, Wang JJ and Sun LL: Effect of miR-134 against myocardial hypoxia/reoxygenation injury by directly targeting NOS3 and regulating PI3K/Akt pathway. Acta Cir Bras. 34:e2019008022019. View Article : Google Scholar : PubMed/NCBI

43 

Luo L, Yang R, Zhao S, Chen Y, Hong S, Wang K, Wang T, Cheng J, Zhang T and Chen D: Decreased miR-320 expression is associated with breast cancer progression, cell migration, and invasiveness via targeting Aquaporin 1. Acta Biochim Biophys Sin (Shanghai). 50:473–480. 2018. View Article : Google Scholar : PubMed/NCBI

44 

Pan C, Gao H, Zheng N, Gao Q, Si Y and Zhao Y: miR-320 inhibits the growth of glioma cells through downregulating PBX3. Biol Res. 50:312017. View Article : Google Scholar : PubMed/NCBI

45 

Zhang T, Zou P, Wang T, Xiang J, Cheng J, Chen D and Zhou J: Down-regulation of miR-320 associated with cancer progression and cell apoptosis via targeting Mcl-1 in cervical cancer. Tumour Biol. 37:8931–8940. 2016. View Article : Google Scholar : PubMed/NCBI

46 

Zhu H, Jiang X, Zhou X, Dong X, Xie K, Yang C, Jiang H, Sun X and Lu J: Neuropilin-1 regulated by miR-320 contributes to the growth and metastasis of cholangiocarcinoma cells. Liver Int. 38:125–135. 2018. View Article : Google Scholar : PubMed/NCBI

47 

Tian ZQ, Jiang H and Lu ZB: miR-320 regulates cardiomyocyte apoptosis induced by ischemia-reperfusion injury by targeting AKIP1. Cell Mol Biol Lett. 23:412018. View Article : Google Scholar : PubMed/NCBI

48 

Alcantara D, Timms AE, Gripp K, Baker L, Park K, Collins S, Cheng C, Stewart F, Mehta SG, Saggar A, et al: Mutations of AKT3 are associated with a wide spectrum of developmental disorders including extreme megalencephaly. Brain. 140:2610–2622. 2017. View Article : Google Scholar : PubMed/NCBI

49 

Chin YR, Yoshida T, Marusyk A, Beck AH, Polyak K and Toker A: Targeting Akt3 signaling in triple-negative breast cancer. Cancer Res. 74:964–973. 2014. View Article : Google Scholar : PubMed/NCBI

50 

Carloni S, Girelli S, Buonocore G, Longini M and Balduini W: Simvastatin acutely reduces ischemic brain damage in the immature rat via Akt and CREB activation. Exp Neurol. 220:82–89. 2009. View Article : Google Scholar : PubMed/NCBI

51 

Yao H and Han X and Han X: The cardioprotection of the insulin-mediated PI3K/Akt/mTOR signaling pathway. Am J Cardiovasc Drugs. 14:433–442. 2014. View Article : Google Scholar : PubMed/NCBI

52 

Song HP, Chu ZG, Zhang DX, Dang YM and Zhang Q: PI3K-AKT pathway protects cardiomyocytes against hypoxia-induced apoptosis by MitoKATP-mediated mitochondrial translocation of pAKT. Cell Physiol Biochem. 49:717–727. 2018. View Article : Google Scholar : PubMed/NCBI

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August-2020
Volume 22 Issue 2

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Spandidos Publications style
Cao L and Cao L: miR‑320‑3p is involved in morphine pre‑conditioning to protect rat cardiomyocytes from ischemia/reperfusion injury through targeting Akt3. Mol Med Rep 22: 1480-1488, 2020
APA
Cao, L., & Cao, L. (2020). miR‑320‑3p is involved in morphine pre‑conditioning to protect rat cardiomyocytes from ischemia/reperfusion injury through targeting Akt3. Molecular Medicine Reports, 22, 1480-1488. https://doi.org/10.3892/mmr.2020.11190
MLA
Cao, L., Chai, S."miR‑320‑3p is involved in morphine pre‑conditioning to protect rat cardiomyocytes from ischemia/reperfusion injury through targeting Akt3". Molecular Medicine Reports 22.2 (2020): 1480-1488.
Chicago
Cao, L., Chai, S."miR‑320‑3p is involved in morphine pre‑conditioning to protect rat cardiomyocytes from ischemia/reperfusion injury through targeting Akt3". Molecular Medicine Reports 22, no. 2 (2020): 1480-1488. https://doi.org/10.3892/mmr.2020.11190