Long noncoding RNA-MEG3 contributes to myocardial ischemia–reperfusion injury through suppression of miR-7-5p expression

Long noncoding RNA (lncRNA) maternally expressed gene 3 (MEG3) plays an important role in protection of ischemia–reperfusion (I/R) injury in brain and liver. However, role of MEG3 in myocardial I/R injury remains unclear. Here, the role of MEG3 in protection of myocardial I/R injury and its association with microRNA-7-5p (miR-7-5p) was investigated using rat cardiac I/R model and myocardial I/R cell model. Our results showed that MEG3 was significantly up-regulated and miR-7-5p was significantly down-regulated after I/R. Following I/R, the levels of intact PARP and intact caspase-3 were reduced, while the cleaved fragments of PARP and caspase-3 were increased. TUNEL assay showed an increase in cardiomyocyte apoptosis after I/R. The levels of I/R-induced creatine kinase (CK) and lactate dehydrogenase (LDH) were inhibited by knockdown of MEG3 (siMEG3). SiMEG3 increased cell proliferation and inhibited cell apoptosis after I/R. In contrast, overexpression of MEG3 increased the I/R-induced CK and LDH activities and cell apoptosis and decreased cell proliferation. The dual-luciferase reporter system showed a direct binding of MEG3 to miR-7-5p. The level of miR-7-5p was negatively associated with the change in levels of MEG3 in H9c2 cells. The levels of intact RARP1 and caspase-3 were significantly increased by knockdown of MEG3. Co-transfection of miR-7-5p inhibitor with siMEG3 activates CK and LDH, significantly decreased cell proliferation, increased cell apoptosis, and decreased intact poly(ADP-ribose) polymerase 1 (PARP1) and caspase-3. In summary, down-regulation of MEG3 protects myocardial cells against I/R-induced apoptosis through miR-7-5p/PARP1 pathway, which might provide a new therapeutic target for treatment of myocardial I/R injury.


Introduction
Ischemia-reperfusion (I/R) injury is a common pathological mechanism of ischemic cardiovascular disease in clinical anesthesia [1,2]. Timely reperfusion including primary percutaneous coronary intervention and thrombolytic treatment are the most efficient therapeutic strategies of ischemic heart disease, which results in cardiomyocyte damage and is commonly referred to as myocardial I/R injury [3]. Moreover, approaches for controlling and decreasing I/R injury in the perioperative period are always concerned by medical practitioners [4,5]. Although numerous drugs are utilized immediately after surgery to reduce myocardial necrosis and myocardial infarct area, the mechanism underlying cardio-protective effects, however, remains unclear. Increasing evidence suggests many cellular signaling pathways involving noncoding transcription regulation were triggered by I/R injury and responsible for cell survival or death [6][7][8].
Noncoding RNAs include microRNA (miRNA) and long noncoding RNAs (lncRNAs). MiRNAs contain 20-22 nt and lncRNAs contain >200 nt. Both miRNAs and lncRNAs are involved in myocardial I/R injury and cardiovascular diseases. MiR-103/107 modulates programmed necrosis and myocardial I/R injury by targeting FAS-associated protein, with death domain (FADD) [9]. LncRNA metastasis-associated lung adenocarcinoma transcript 1 (MALAT1) regulates miR-145/Bnip3 signaling in cardioprotective effects of fentanyl against myocardial I/R injury [10]. LncRNA necrosis-related factor (NRF) regulates miR-873 in protection of cardiomyocytes against necrosis and myocardial I/R injury [11]. LncRNA urothelial carcinoma-associated 1 (UCA1) plays a pro-apoptotic role in cardiomyocytes by suppressing p27 expression after cardiac I/R injury [12]. Although the roles of many miR-NAs have been well studied in myocardial I/R injury, the role of lncRNA and its association with miRNAs remains unclear. Recently, a novel rat lncRNA, human and mouse maternally expressed gene 3 (MEG3, also known as gene trap locus 2, GTL2) has been identified in many tissues and cells, such as brain and endothelial cells [13][14][15]. It was demonstrated that overexpression of MEG3 suppressed tumor cell growth and promote cell apoptosis in vitro [16,17]. A recent study suggested that MEG3 is involved in angiogenesis after ischemia brain injury by modulating the notch signaling [18]. MEG3 directly binds with the p53 DNA binding domain that stimulates p53-mediated transactivation and mediates ischemic neuronal death in ischemic neuronal damage [14]. MEG3 protects hepatocytes from hepatic ischemia-reperfusion through down-regulating miR-34a expression [19]. These findings suggest that MEG3 plays an important role in protection of IR injury. However, the role of MEG3 in myocardial I/R injury remains unclear. miR-7, a novel tumor suppressor, increases tumor cell apoptosis and reduced proliferation in malignant gastric cancer, breast cancer, nasopharyngeal carcinoma, and glioblastoma cells [20][21][22][23]. A recent study showed that miR-7 was up-regulated in murine heart after I/R [24]. miR-7 protects myocardial cells against I/R-induced apoptosis by targeting poly(ADP-ribose) polymerase 1 (PARP1) [25]. In the present study, we studied the role of lncRNA MEG3 in protection of myocardial I/R injury and its association with miR-7-5p in established rat cardiac I/R model and in vitro myocardial I/R cell model and its regulatory role in PARP in protecting cardiomyocytes from apoptosis.

Animals and cardiac I/R model
Male Sprague-Dawley (SD) rats (250-300 g) were obtained from the Animal Laboratory of Sun Yat-sen University for the present study. The rats were housed in cages and fed standard food and water in the Animal Laboratory of Sun Yat-sen University. The Sun Yat-sen University Animal Care and Use Committee, China, approved the animal experimental protocol and all animal experiments were carried out according to its guidance. The rats were randomly divided into sham and I/R groups (n=8). For I/R, the rats were anesthetized with pentobarbital sodium (3%, intraperitoneal injection). The left anterior descending branch (LAD) was occluded for 30 min using a 4-silk suture and reperfused for 2 h as previously described [26]. The sham control rats underwent surgery with the silk beneath the coronary artery, but the LAD was not ligated [25]. The cardiac function parameters including heart rate, maximum and minimum action potential, and peak-peak value were detected by BL-410 bio-functional experiment system (Sichuan Taimeng, China). To identify the infarct size, the harvested hearts were cut into slices (1-mm-thick) and rinsed with 1% 2,3,5-triphenyltetrazolium chloride (TTC, Sigma, U.S.A.) at 37 • C for 10 min. After fixing with 4% paraformaldehyde, the infarct size was not stained by TTC (white).

General histology and TUNEL assay
The heart of each rat was fixed in 10% formalin, embedded in paraffin to obtain 5-μm-thick sections, and viewed under an optical microscope after HE staining as previously described [27]. TUNEL was conducted to detect the DNA fragmentation using DeadEnd™ Fluorometric TUNEL system (Promega, U.S.A.) according to the manufacturer's manual. penicillin/streptomycin at 37 • C. The MEG3 expressing or shRNA plasmid and miR-7-5p inhibitor were purchased from Genepharma, China. For transfection, H9c2 cells were seeded in 100-mm plate at 1 × 10 6 until 50% confluence. Then, 0.5 nmol MEG3 expressing or shRNA vector and miR-7-5p mimics/inhibitor were transfected into cells using Lipofectamine 2000 (Invitrogen, U.S.A.) according to the manufacturer's manual. After 48 h, the expression of MEG3 or miR-7 was detected and the cells were used for further experiments. To simulate I/R, the cells were kept in a hypoxic chamber at 37 • C for 10 h with serum-and glucose-deficient DMEM and reoxygenated for 2 h with DMEM containing 10% FBS.

Creatine kinase and lactate dehydrogenase release assays
At the end of reperfusion of rats and treatment of cells, plasma samples and culture media were collected form the coronary effluent and cell cultures, respectively. The activities of lactate dehydrogenase (LDH) and creatine kinase (CK) were assayed using LDH and CK kits (Nanjing Jiancheng Bioengineering, China) according to the manufacturer's instructions.

Flow cytometry
After I/R, cell apoptosis was assessed by Annexin V-FITC and propiduim iodide (BD Biosciences, U.S.A.) according to the manufacturer's instructions. The stained cells were analyzed by flow cytometry (BD, U.S.A.).

Western blot analysis
Proteins were isolated from homogenized hearts and cultured with H9c2 cells, and quantified by BCA assay  In other assays, more than three mice in each group were assayed. Experiments were done thrice. P-values were determined using Student's t tests.

Statistical analysis
All the data were expressed as means + − SD from three or more independent experiments. Statistical analysis was carried out using two-tailed Student's t test or the ANOVA test using SPSS 15.0. Statistical significance was considered at P<0.05.

Expression of MEG3 and miR-7-5p in established rat I/R model
The cardiac function of the two groups in response to reperfusion is shown in Figure 1. The hearts of the rats were subjected to myocardial ischemia for 30 min followed by 2 h of reperfusion. The I/R model group showed a decrease in value of ST segments' elevations with increased heart rate, increased maximum action potential and minimum action potential, and decreased peak-peak value, compared with the sham group ( Figure 1A).
To detect the infarct size in the heart, TTC staining was performed after reperfusion ( Figure 1B). The I/R model group showed an increase in the size of infarct.
The myocardial damage was evaluated by measuring the activity of CK and LDH released into the coronary effluent ( Figure 1C,D). The activities of CK and LDH were significantly increased in the I/R group compared with the sham group.
To detect the infarct size in the heart, TTC staining was performed after reperfusion ( Figure 1B). The I/R model group showed the presence of an infarct in hearts. Further, transthoracic echocardiography and M-mode tracings were used to evaluate LVIDd, LVIDs, LVFS% and LVEF%. As expected, I/R injury significantly increased the LVIDd and LVIDs (Supplementary Figure S1A,B), while reduced LVFS% and LVEF% (Supplementary Figure S1C,D) relative to sham group. The myocardial damage was evaluated by activity of CK and LDH released into the coronary effluent ( Figure 1C,D). The activities of CK and LDH were significantly increased in the I/R group in comparison with the sham group.
The myocardial damage was evaluated by activity of CK and LDH released into the coronary effluent ( Figure 1C,D). The activities of CK and LDH were significantly increased in the I/R group in comparison with the sham group.
The alterations in the morphology of myocardial tissues were evaluated by HE staining ( Figure 1E). Myocardial tissues in I/R model group showed loosely and irregularly arranged muscle fibers and necrosis of muscle fibers, while normal morphology with tightly and orderly arranged cardiomyocytes, and no necrosis of muscle fibers were observed in the sham group ( Figure 1E).
As I/R-induced cell apoptosis contributes significantly to impairment of cardiac function, TUNEL assay was performed to observe the cardiomyocyte apoptosis ( Figure 1F). The apoptosis rate in I/R model group was significant higher compared with sham group.
It was reported that miR-7 was up-regulated in H9c2 cells after simulating I/R by negatively regulating PARP [25]. To detect the levels of MEG3 and miR-7-5p and their roles in I/R injury, the expression of PARP1 and caspase 3 were detected by Western blot ( Figure 1G) and the levels of MEG3 and miR-7-5p were detected by qRT-PCR ( Figure  1H,I). After I/R, the levels of intact PARP and intact caspase-3 were reduced and the cleaved fragments of PARP and caspase-3 were increased ( Figure 1G), which could be attributed to the cardiomyocyte apoptosis. MEG3 was markedly up-regulated and miR-7-5p was significantly down-regulated ( Figure 1H,I), thereby indicating an important role of MEG3 and miR-7-5p in I/R-induced cardiomyocyte apoptosis.

Down-regulation of MEG3 inhibited I/R-induced levels of CK and LDH and myocardial cell apoptosis
To detect the role of MEG3 in I/R-induced cardiac injury, down-or over-expression of MEG3 in H9c2 cells was determined ( Figure 2). The level of I/R-induced CK and LDH were inhibited by siMEG3, and increased by overexpression of MEG3 (Figure 2A,B), thereby suggesting that MEG3 promoted myocardial damage.
The cell proliferation and apoptosis were also detected. SiMEG3 increased cell proliferation in I/R H9c2 cells; however, cell proliferation was significantly decreased by overexpression of MEG3 ( Figure 2C). Similarly, siMEG3 inhibited I/R induced cell apoptosis in H9c2 cells; however, overexpression of MEG3 significantly promoted cell apoptosis in I/R H9c2 ( Figure 2D). Thus, MEG3 promoted cell apoptosis, inhibited cell proliferation, and promoted myocardial damage.

MEG3 regulated cardiomyocyte by directly binding to miR-5-7p
To examine whether MEG3 promoted myocardial damage through miR-7-5p, the lentivirus with miR-7-5p mimics and GV126-MEG3-3 UTR-wide type (WT) or its mutant type (MUT) vectors were transfected into 293 cells ( Figure  3A). The luciferase activity was significantly decreased in the miR-7-5p mimics and WT group, but no change in the activity was observed in the MUT group ( Figure 3B), thereby suggesting a direct binding of MEG3 to miR-7-5p. The role of MEG3 in regulating miR-7-5p was evaluated by qRT-PCR ( Figure 3C,D). Negatively associated with the change in levels of MEG3 in H9c2 cells that overexpressed or knocked down MEG3 (Figure 3C), the level of miR-7-5p was significantly down-or up-regulated ( Figure 3D). The intact RARP1 was significantly increased by knockdown of MEG3, while significant overexpression of MEG3 was observed in the I/R-induced cleaved PARP1 levels ( Figure 3E). Similarly, intact caspase-3 was significantly increased by knockdown of MEG3. These results indicated that MEG3 regulated cardiomyocyte apoptosis by directly binding to miR-5-7p.

Co-transfection of siMEG3 and miR-7-5p inhibitor attenuated the inhibition of siMEG3 on I/R-inhibited cell proliferation and cell apoptosis
To detect the role of miR-5-7p in the protection of MEG3 against I/R injury, both siMEG3 and miR-7-5p inhibitor were transfected into H9c2 cells. Knockdown of MEG3 and miR-7-5p was observed in the co-transfected cells with I/R ( Figure 4A,B). The activities of CK and LDH were significantly increased in co-transfection group compared with the siMEG3 transfected group after I/R ( Figure 4C,D); thereby indicating that miR-7-5p inhibitor attenuated the role of siMEG3 in protection against the myocardial damage.
The up-regulation of target gene PARP1 of miR-7-5p by siMEG3 was significantly attenuated by miR-7-5p in I/R cells ( Figure 4G). The level of intact PARP1 was decreased and cleaved PARP1 was increased in co-transfection group in I/R condition. Similarly, the level of intact caspase-3 was decreased and the cleaved caspase-3 was increased in co-transfection group in I/R condition.
Thus, MEG3 protected cardiomyocyte against I/R injury by directly regulating miR-5-7p and its downstream target gene PARP1 and the caspase 3 signaling pathway.

Discussion
Various studies have showed that myocardial ischemia, hypoxia, and myocardial tissues damage induce release of enzymes in blood, including the significant CK and LDH [28]. The elevation of CK and LDH is proportional to the intensity of myocardial infarction [29,30]. In the present study, an increase in CK and LDH activity, enhanced size of infarction with a significantly high apoptosis rate, and decrease in values of ST segments' elevations were observed model group compared with the sham control group. These findings suggested the establishment of rat I/R injury model. In the I/R model, MEG3 was significantly up-regulated and miR-7-5p was significantly down-regulated. The levels of intact target gene PARP of miR-7-5p and caspase-3 were reduced. It was reported that miR-7-5p targeting on PARP protects cardiomyocyte against I/R-induced cell apoptosis and reduces the intact caspase-3 [25]. This is consistent with our results. In the present study, our results demonstrated a role of MEG3 in I/R-induced myocardial injury through miR-7-5p/PARP pathway.
There are at least 18 members of PARP family, of which PARP-1 is the most abundant and well studied [31]. PARP inhibitors can protect myocardial I/R injury against cell apoptosis. During the process of apoptosis, PARP was cleaved into two fragments of 89 and 24 kDa by caspase-3, thereby avoiding unnecessary DNA repair in cells and the excessive consumption of NAD + and ATP in cells to ensure an adequate supply of energy during apoptosis [32,33]. Moreover, the activation of caspase-3 plays an important role in the process of cardiomyocyte apoptosis. Caspase-3 activity was increased in traumatic plasma-induced rat cardiomyocytes apoptosis [34]. In infarcted and ischemic myocardium of myocardial infarction mice, caspase-3 expression in heart tissues was up-regulated [35]. Hypoxia also induced caspase-3 and apoptosis in cardiomyocyte [36]. The activity of caspase-3 in cardiomyocytes is increased after I/R injury with the increase in apoptosis [37]. The levels of intact RARP1 and caspase-3 were significantly increased by knockdown of MEG3, while overexpression of MEG3 significantly increased I/R-induced cleaved PARP1 and caspase-3 levels. The levels of intact PARP1 and caspase-3 were decreased and the cleaved PARP1 and caspase-3 were increased in co-transfection group in I/R condition. Thus, MEG3 regulated PARP1 and caspase-3 levels.
H9c2 is a subclone of the original clonal cell line derived from embryonic BD1X rat heart tissues that exhibits many properties of skeletal muscle. Previous evidence showed similar hypertrophic responses between H9C2 cell line and primary neonatal cardiomyocyte cells in vitro [38]. H9c2 cell line has also proved to be a valuable in vitro model to study the drug metabolizing enzymes in the heart [39]. Therefore, we chose H9C2 cell line for our in vitro study in order to exclude disturbing factors caused by diverse batches of primary cardiac myocytes. Meanwhile, male SD rats were also included for the in vivo investigation. Of note, we found that down-regulation of MEG3 protected H9c2 cells from hypoxia-induced injury and prevented cardiac fibrosis and diastolic dysfunction [40]. MEG3 modulated cardiac remodeling in cardiac hypertrophy [41]. Consistently, MEG3 promoted cell apoptosis, inhibited cell proliferation, and promoted myocardial damage. The level of miR-7-5p was negatively associated with the change in levels of MEG3 in H9c2 cells. Down-regulation of MEG3 could reduce I/R-induced apoptosis of myocardial cells, while miR-7-5p inhibitor rescued cell apoptosis. SiMEG3 increased cell proliferation and inhibited cell apoptosis in I/R H9c2 cells; however, the cell proliferation was significantly increased and apoptosis was significantly decreased by overexpression of MEG3, respectively. Other miRNAs such as miR-183 involved in hypoxia-induced injury [42].
In conclusion, we have showed that lncRNA MEG3 plays a key role in the heart ischemia reperfusion injury via miR-7-5p/PARP1 pathway through in vivo and in vitro experiments. The mechanism of myocardial I/R injury is very complex and requires further investigation.

Ethics approval and consent to participate
The present study was conducted after obtaining approval from the The Third Affiliated Hospital of Sun Yat-sen University.