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

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) miR-320-3p is involved in morphine pre-conditioning to protect rat cardiomyocytes from ischemia/reperfusion injury through targeting Akt3 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% co 2 (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 co 2 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 cacl 2 , 5.6 mM Kcl, 154 mM nacl, 5 mM Hepes, and 3.6 mM naHco 3 ; 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% co 2 ) 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.
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 x 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).

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 Figure 3. downregulation of mir-320-3p attenuates H/r damage to cells. (a) reverse transcription-quantitative Pcr was used to determine the effect of mir-320-3p inhibitor transfection. (B) H9c2 cell viability in the control, H/r, MPc+H/r, inhibitor control+H/r and inhibitor+H/r groups using an MTT assay. (c) ldH content was measured in H9c2 cells in the control, H/r, MPc+H/r, inhibitor control+H/r and inhibitor+H/r groups using a colorimetric assay. (D) Apoptosis of H9c2 cells in the control, H/R, MPC+H/R, inhibitor control+H/R and inhibitor+H/R groups was detected using flow cytometry. & P<0.05 vs. inhibitor control; * P<0.05 and ** P<0.001 vs. control; # P<0.05 and ## P<0.001 vs. H/r; ^P <0.05 and ^^P <0.001 vs. MPc+H/r; Δ P<0.05 and ΔΔ P<0.001 vs. inhibitor control+H/r. MPc, morphine pre-conditioning; H/r, hypoxia/reoxygenation; mir, microrna; ldH, lactate dehydrogenase; od, optical density; PI, propidium iodide; FITC, fluorescein isothiocyanate. 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  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 (50)(51)(52). 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.
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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.
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