LncRNA AK020546 protects against cardiac ischemia–reperfusion injury by sponging miR-350-3p

Long non-coding RNAs (lncRNAs) have been implicated in the development of cardiovascular diseases. We observed that lncRNA AK020546 was downregulated following ischemia/reperfusion injury to the myocardium and following H2O2 treatment in H9c2 cardiomyocytes. In vivo and in vitro studies showed that AK020546 overexpression attenuated the size of the ischemic area, reduced apoptosis among H9c2 cardiomyocytes, and suppressed the release of reactive oxygen species, lactic acid dehydrogenase, and malondialdehyde. AK020546 served as a competing endogenous RNA for miR-350-3p and activated the miR-350-3p target gene ErbB3. MiR-350-3p overexpression reversed the effects of AK020546 on oxidative stress injury and apoptosis in H9c2 cardiomyocytes. Moreover, ErbB3 knockdown alleviated the effects of AK020546 on the expression of ErbB3, Bcl-2, phosphorylated AKT, cleaved Caspase 3, and phosphorylated Bad. These findings suggest lncRNA AK020546 protects against ischemia/reperfusion and oxidative stress injury by sequestering miR-350-3p and activating ErbB3, which highlights its potential as a therapeutic target for ischemic heart diseases.


INTRODUCTION
Acute myocardial infarction (AMI), characterized by inadequate blood flow and oxygen supply to the heart due to blocked arteries, is the leading cause of morbidity and mortality worldwide [1]. The most effective therapeutic approach to protect the heart against AMI is to restore its blood supply [2]. However, reperfusion and reoxygenation exacerbate tissue injury as well as induce inflammatory response [3]. One of the major factors contributing to I/R injury is apoptosis induced by mitochondrial dysfunction and increased lipid peroxides [4].
LncRNAs participate in the progression of AMI by sponging microRNAs (miRNAs). The expression of several miRNAs is induced by oxidative stress. For AGING example, tomato supplementation protected against oxidative stress and left ventricular mass and restored heart function by downregulating miR-350 [14]. Moreover, overexpression of miR-350 has been reported to contribute to heart hypertrophy by inactivating JNK and p38 pathways [15].
In the present work, we studied the function of lncRNA AK020546 in cardiac ischemic injury. Functional studies revealed that AK020546 reduced the infarct size and myocardial cell apoptosis, and decreased H2O2induced apoptosis of H9c2 cardiomyocytes. In addition, it binds to and sequesters miR-350-3p to increase the expression of its downstream target gene ErbB3. These findings highlight the function of lncRNA AK020546 in maintaining cardiovascular health and provide potential therapeutic targets for cardiac ischemic injury.

LncRNA AK020546 was downregulated in I/R injury
To assess the function of AK020546 in I/R injury, we established an oxidative stress model using H9c2 cells and an I/R model in rats and studied its expression. The results indicated that AK020546 expression was notably reduced in H9c2 cells 1 h after H2O2 treatment and in the myocardium 1 h after ischemia ( Figure 1A, 1B).

Overexpression of lncRNA AK020546 attenuated H2O2-induced apoptosis and oxidative stress in H9c2 cardiomyocytes
Because AK020546 was downregulated in I/R injuryexposed myocardium and H2O2-treated H9c2 cardiomyocytes, we speculated that it participated in the progression of I/R injury. First, we established an in vitro model of oxidative stress injury using H2O2 in H9c2 cardiomyocytes. Quantitative realtime PCR (qPCR) analysis revealed a good efficiency of AK02054overexpressing adenovirus (Figure 2A). Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) results revealed that AK020546 reduced H2O2induced apoptosis of H9c2 cardiomyocytes ( Figure 2B). Flow cytometry revealed similar results ( Figure 2C, 2D). Because H9c2 cardiomyocyte apoptosis is linked to oxidative stress, we next evaluated the change in the levels of malondialdehyde (MDA), lactic acid dehydrogenase (LDH), and reactive oxygen species

LncRNA AK020546 overexpression inhibited cardiac I/R injury in vivo
To further confirm the functions of AK020546 in I/R injury, we constructed an I/R model in rats. Figure 3A shows increased expression of AK020546 in the myocardium following its overexpression using an adenovirus construct. We next conducted in vitro studies that showed that AK020546 overexpression reduced the elevated levels of MDA, LDH, and ROS ( Figure 3B-3D). Furthermore, compared with the Adcontrol (Ad-nc) group, 2,3,5-triphenyltetrazolium chloride (TTC) staining revealed that AK020546 overexpression reduced I/R injury-induced infarct area. The reduction in the infarct size was 28.45 ± 14.64% ( Figure 3E). TUNEL assay was conducted to evaluate apoptosis ( Figure 3F). We next investigated cardiac function, a parameter impaired by I/R injury. The results indicated that AK020546 elevated left ventricular systolic pressure (LVSP) and maximal LV contractility rate (±dp/dt); however, it decreased left ventricular end-systolic diameter (LVEDP). Moreover, AK020546 overexpression increased the heart rate during both I/R injury and reperfusion ( Figure 4A-4F).

LncRNA AK020546 increased the expression of ErbB3 and activated the downstream AKT pathway
ErbB3 is involved in cell apoptosis and oxidative stress during I/R injury. For instance, ErbB3 was reported to regulate the phosphorylation of AKT and Bad that are involved in cardiac I/R injury. We further evaluated the expression of apoptotic proteins such as Bcl-2, Bax, Bad, and cleaved Caspase 3 (c-Caspase 3). AK020546 elevated the expression of ErbB3, Bcl-2, pAKT, whereas it attenuated the expression of c-Caspase 3 and the phosphorylation of Bad ( Figure 8A, 8B). Figure 8C shows the proposed mechanism of the effect of lncRNA AK020546.

DISCUSSION
LncRNAs have been implicated in several physiological and pathological processes such as cell proliferation, differentiation, development, and apoptosis. Moreover, lncRNAs can function as competing endogenous RNAs (ceRNAs) that sequester miRNAs to regulate the expression of miRNA target genes. For instance, lncRNAs such as APF, MALAT1, and PFL participate in I/R injury by sponging miRNAs [22][23][24][25]. LncRNA AK020546, also known as CAIF, has been implicated in several cellular processes. For instance, AK020546 is downregulated in osteoarthritis and its overexpression improves the condition by sponging miR-1246 [26]. Zhou et al. reported that AK020546 participated in myocardial remodeling [27]. Similarly, Wu et al. reported MALAT1 to be a promising diagnostic and prognostic marker in end-stage cardiomyopathy. AK020546 has been reported to inhibit autophagy and AGING attenuate acute myocardial infarction via the p53myocardin axis [27]. We demonstrated that AK020546 was downregulated in AMI both in vivo and in vitro. Its overexpression reduced the infarct area and restored heart function. Moreover, AK020546 overexpression inhibited the release of ROS and apoptosis of H9c2 cardiomyocytes, implying its cardioprotective function in AMI.
Bioinformatic tools predicted miR-350-3p as a potential target of AK020546, which was experimentally confirmed by luciferase assay. MiR-350-3p promotes RAW264.7 cell apoptosis by repressing the PIK3R3/ MAPK signaling pathway [28]. In addition, a study reported miR-350 as a promising biomarker of smoking-related interstitial fibrosis (SRIF) [29]. Another study reported elevated expression of miR-350 in late-stage transverse aortic constriction (TAC) in rat heart. Moreover, miR-350-3p contributes to pathological cardiac hypertrophy and apoptosis by inactivating the p38/JNK pathway [14,15]. These findings demonstrated the function of miR-350-3p in AGING cardiovascular diseases. Considering the relationship between myocardial hypertrophy and myocardial ischemic injury, we hypothesized that miR-350-3p exerted a substantial effect during I/R injury. We confirmed miR-350-3p as a target of AK020546 and that AK020546 exerted its cardioprotective function via sponging miR-350-3p. Overexpression of miR-350-3p reversed the effects of AK020546 on I/R injury including ROS release and apoptosis.
ErbB3 belongs to the PA2G4 family and participates in multiple cellular processes including cell cycle, proliferation, and survival [30][31][32][33][34]. Several studies have reported the involvement of ErbB3 in cardiovascular diseases. For instance, methylation of the ErbB3 gene has been observed in human dilated cardiomyopathy. In addition, ErbB3 degradation by Nrdp1, an E3 ligase, following I/R injury in cardiomyocytes promotes apoptosis and alleviates I/R injury [21,35]. ErbB3 activates several protein kinases involved in cell apoptosis, including AKT, STAT3, and mitogen-activated protein kinases (MAPKs) [36][37][38]. The PI3K/AKT pathway is triggered in response to I/R injury and promotes cell survival by inhibiting caspase-induced apoptosis [39,40]. We found that AK020546 elevated the expression of Bcl-2 and phosphorylation of AKT, whereas decreased the protein levels of c-Caspase 3 and phosphorylation of Bad; these effects were reversed by ErbB3 knockdown. We speculated that AK020546 sponged miR-350-3p and blocked the inhibitory effect of miR-350-3p on ErbB3 expression to promote AKT phosphorylation and the expression of anti-apoptotic protein Bcl-2, consequently decreasing cell apoptosis in H9c2 cardiomyocytes.
Our study had certain limitations. Several studies have implicated altered mitochondrial functions, including increased mitochondrial membrane depolarization [41,42] and disrupted activity of enzymes of the electron AGING transport chain [43], in cardiac I/R injury. However, our study lacked data on the involvement of mitochondrial dysfunction. Furthermore, we did not study the function of gap junction protein Cx43, increased expression of which is associated with reduced reperfusion arrhythmia in cardiac IR injury [43][44][45].
In conclusion, lncRNA AK020546 was downregulated in AMI and its overexpression restored heart function, suppressed ROS release, and reduced the apoptosis of H9c2 cardiomyocytes. LncRNA AK020546 exerted these functions by activating ErbB3 via sponging miR-350-3p.

Animals
Wistar rats were purchased from Charles River Laboratories (Beijing, China). These rats were housed in a environment of specific pathogen-free (SPF) and are with ad lib access to water and food. All in vivo studies were approved by the Animal Studies Committee of Zhejiang Renmin Hospital and were performed following the institutional guidelines.

Plasmid construction and transfection
MiR-350-3p mimic, siRNA against ErbB3, adenovirus overexpressing AK020546 along with their controls were purchased from GenePharma (Shanghai, China). The wild type and mutant type of binding cites among AK020546 and ErbB3 sequence were synthesized by GenePharma followed by cloning into the pGL3 reporter vector (Promega, CA, USA).

I/R model establishment
All rats were kept on a 12-h light/dark cycle and in an environment of 25 and 60% humidity with ad lib access to water and food. 32 rats were randomly divided into 4 different groups: sham group, I/R group, IR + Ad-nc group, and IR + Ad-AK020546 group. Afterward, the rats were administered of Ad-AK020546 or Ad-nc at the dose of 1 × 109 plaqueforming units (PFUs) for 3 consecutive days via tail vein, whereas the rat in sham control group was injected with saline. Four days later, the I/R injury was induced in rats as per the method described previously [46]. Briefly, rats were intraperitoneally injected with 1% pentobarbital sodium (50 mg/kg; Sigma-Aldrich, P3761, USA). After thoracotomy, the intercostal muscle was separated in the 3rd and 4th intercostal space to expose the heart. The middle of the left anterior descending coronary artery was ligated using a 6-0 surgical suture. The sham rats underwent surgery but without ligation of left anterior descending (LAD) coronary artery ligated using a 6-0 silk suture. The I/R procedure included a 30 min of ischemia and 2 h of reperfusion. All rats were sacrificed immediately after anesthetization with 3% pentobarbital sodium (160 mg/kg). Subsequently, heart tissue and blood samples were collected for further experiments following I/R injury.

Echocardiography
Transthoracic echocardiography detection was carried out with a VisualSonics Vevo 2100 (VisualSonics) ultrasound system and a 40-MHz transducer. A 1.4-F Millar catheter (Millar Instruments, UT, USA) was inserted into the carotid artery of mice, and then left ventricular end-diastolic pressure (LVEDP) left ventricular systolic pressure (LVSP), the maximal rate of LV contractility (+dp/dt), and the maximal rate of LV relaxation (−dp/dt) were obtained using the ultrasound device as described previously [47].

TTC staining
For TTC staining, 2% Evans blue was injected into the heart from the femoral vein after the ligation of LAD. Heart tissues were collected immediately after the injection and rinsed with ice-cold normal saline (NS). The samples were frozen at -20° C for 30 min and subsequently transversely cut into 1 mm-thick slices. These tissues were incubated with TTC at 37° C for 15 min. After fixation with formaldehyde for 24 h, the samples were visualized and photographed.

Cell culture
H9c2 cardiomyocytes were provided by the Cell Bank of Chinese Academy of Sciences. Cells were maintained in DMEM (Gibco, USA) containing 10% fetal bovine serum (FBS, Gibco, USA) and supplemented with 100 U/mL penicillin, 100 μg/mL streptomycin, and 110 mg/mL sodium pyruvate at 37° C under a humidified conditions of 95% air and 5% CO2. For the establishment of oxidative injury model, H9c2 cells were cultured with 600 µM of H2O2.

TUNEL staining
The TUNEL staining was carried out using an in situ Cell Death Detection Kit (Roche, Mannheim, Germany). After fixation and dehydration, the heart tissues were embedded in paraffin followed by cutting into 4 μm sections. The experimental procedures were performed as per the manufacturer's instructions. Slices were mounted and imaged under a fluorescence microscope (Nikon, Japan). Subsequently, the ratio of apoptotic cells to total cardiomyocytes was calculated.

Flow cytometry
Approximately 1× 106 /mL H9c2 cells were collected, centrifuged at 500-1000 r/min for 5 min. After washing with 3 ml PBS and another centrifugation (500-1000 r/min for 5 min), the cells were fixed with precooled 70% ethanol for 2 h at 4° C. Then, the cells were incubated with FITC-AnnexinV (300ng/mL, 4° C) for 10 min to label apoptotic cells. The samples were further incubated with propidium iodide (PI) for 5 min. The apoptotic cells were then detected with a Fortessa flow-cytometer system (Becton Dickinson, NJ, USA).

JC-1 staining
JC-1 (10 mM, Beyotime, China) was applied to incubate with H9c2 cells at 37° C for 30 min in dark condition. Then, the H9c2 cells were rinsed and resuspended with PBS and imaged under a fluorescence microscope (Nikon, Japan). When the level of JC-1 is reduced, green fluorescence could be observed. Green fluorescence indicates the mitochondrial inner membrane potential is disrupted.

Malondialdehyde (MDA) and lactate dehydrogenase (LDH) evaluation
The heart tissues were grind with lysis buffer and centrifuged at 1,500 g for 15 min at 4° C. After the supernatant was extracted, the amount of LDH and MDA in the supernatant was detected using commercial kits following the manufacture's protocols at -20° C (Beyotime).

ROS detection
2',7'-dichlorofluorescin diacetate (DCFH) (Jiancheng, Nanjing, China) was applied to assess the level of ROS. Briefly, after H/R or I/R treatment, cells or myocardial samples were rinsed with PBS (4° C). Then, the samples were incubated with DCFH (10 μM) at 37° C for 20 min in dark incubator. The stained samples were photographed under a fluorescence microscope (Nikon, Japan) at 488 nm excitation and 590 nm emission, respectively. The mean fluorescence intensity (MFI) was analyzed using ImageQuant version 5.2.

Mitochondrial ROS detection
Mitochondrial ROS was assessed using a MitoSOX kit (Thermo Fisher Scientific, M36008, USA) following manufacturer's protocols. After transfection and treatment, MitoSOX was added to the H9c2 cells and incubated at 37° C for 20 min. The relative fluorescence intensity was calculated in different groups.

Pull-down assay
The biotin-labeled miR-350-3p probe and the control were purchased from Sangon Biotech (Shanghai, China). These probes were incubated with the streptavidin-coated beads (Invitrogen) at 25° C for 2 h. Thereafter, H9c2 cells were lysed, and the biotin labeled probes were added into the lysate. The incubation last overnight at 4° C. Subsequently, the bound RNAs were eluted from the beads and purified using with TRIzol reagent (Takara, China). Finally, the enrichment of AK020546 or ErbB3 in the complex was evaluated by qPCR as mentioned above.

Luciferase assay
The wild or mutant type of binding sites among AK020546 or ErbB3 sequence were cloned into pGL3 luciferase reporter vector. H9c2 cells were co-transfected with miR-350-3p mimic or mimic control along with the reporters carrying those wild type or mutant binding citesTRL-SV40 vector served as internal control (Promega, USA) was transfected. 48 h later, the H9c2 cells were collected followed by detecting the luciferase activity with a dual-luciferase assay kit (Promega, USA).

Statistical analysis
All data was presented as the means ± standard deviations (SDs) of at least three independent experiments. Statistical analysis was performed with the SPSS 17.0 (SPSS, Inc., IL, USA). One-way analysis of variance (ANOVA) followed by Tukey's post-hoc test was performed to evaluate the difference between multiple groups. p< 0.05 was considered as statistical significant.

AUTHOR CONTRIBUTIONS
Meiqi Zhang established the I/R model and performed the in vivo experiments; Kang Cheng contributed to the cell culture and in vitro experiments; Huan Chen contributed to the luciferase assay; Jianfeng Tu collected the data; Ye Shen performed the statistical analysis; Lingxiao Pang contributed to the bioinformatic analysis; Weihua Wu raised the animal; Zhenfei Yu designed the study and wrote the manuscript.