lncRNA DLX6-AS1 Promotes Myocardial Ischemia-Reperfusion Injury through Mediating the miR-204-5p/FBXW7 Axis

Myocardial ischemia-reperfusion (IR) injury is the restoration of blood flow post ischemia, which threatens the human life. Long noncoding RNA distal-less homeobox 6 antisense 1 (DLX6-AS1) has been found to take part in the IR-induced cerebral injury. Here, we determined the functional role of DLX6-AS1 in IR-induced myocardial injury. We ligated the left anterior descending coronary artery of rats to induce IR injury. IR injury rats exhibited severe tissue damage and increase of infraction size. The levels of lactate dehydrogenase (LDH), creatine kinase (CK), proinflammatory factors including MCP-1, IL-6, and IL-1β, and cell apoptosis were also enhanced in IR rats, indicating that IR induced significant myocardial injury in rats. DLX6-AS1 expression was elevated in the myocardial tissues of IR injury rats, while DLX6-AS1 deficiency alleviated IR-induced myocardial injury in rats by reducing inflammatory response and cell apoptosis. Moreover, rat embryonic cardiomyocyte cell line H9c2 was subjected to hypoxia reoxygenation (HR). DLX6-AS1 was upregulated in the HR-treated H9c2 cells, and DLX6-AS1 enhanced the expression of F-box and WD40 repeat domain-containing 7 (FBXW7) by sponging miR-204-5p. Inhibition of DLX6-AS1 inhibited inflammatory response and cell apoptosis in H9c2 cells via miR-204-5p/FBXW7 axis. In conclusion, this work demonstrates that DLX6-AS1 accelerates myocardial IR injury through regulating miR-204-5p/FBXW7 axis. Thus, this work provides a novel ceRNA DLX6-AS1/miR-204-5p/FBXW7 axis in myocardial IR injury, and DLX6-AS1 may be a potential target for the treatment of myocardial IR injury.


Introduction
Acute myocardial infarction (AMI) is a common and frequently occurring disease in the cardiovascular system [1]. Timely reperfusion therapy is the most effective treatment for AMI patients, which can save more cardiomyocytes that are on the verge of necrosis [2,3]. However, a lot of cardiomyocytes died owing to reperfusion injury after revascularization. Although AMI patients survived form myocardial ischemic necrosis, they still have severe cardiac dysfunction, which affects the long-term prognosis of AMI patients [4]. This kind of reperfusion therapy after ischemia restores the blood supply but aggravates the tissues injury, which is called ischemiareperfusion (IR) injury [5]. The main feature of IR is the severe mucosal and submucosal osmotic inflammation, which leads to the necrosis of endothelial cells [6].
lncRNA and microRNA (miRNA) are widely involved in competitive endogenous RNA (ceRNA) model. lncRNA relieves the inhibitory effect of miRNA on the downstream target mRNA by sponging miRNA [14]. Many miRNAs have become important regulators in the physiological and pathological processes of cardiovascular diseases and participate in regulating cardiac functions, such as contraction, cardiac growth, and morphogenesis [15,16]. Previous study has revealed that the expression of miR-204-5p is lower in the myocardial tissue of the myocardial IR injury mouse model. KCNQ1OT1 binds to miR-204-5p and aggravates myocardial IR injury in mice by upregulating LGALS3, which provides a new idea for the treatment of myocardial IR injury [17]. Whether miR-204-5p has a functional role in IR-induced myocardial injury remains unclear. Additionally, Bioinformatical analysis (Starbase) revealed that miR-204-5p may be a downstream target of DLX6-AS1, and there may be binding sites between miR-204-5p and oxidized lowdensity lipoprotein receptor-1 (FBXW7). A previous study has uncovered that FBXW7 expression was elevated in myocardial IR injury. Inhibition of FBXW7 significantly alleviated inflammatory response, cell apoptosis, and IR-induced myocardial injury in mice [18,19]. Thus, we speculated that DLX6-AS1 may alleviate myocardial IR injury by regulating miR-204-5p/FBXW7 axis.
In this work, we constructed a rat model of IR injury and determined the impact of DLX6-AS1 inhibition on IR injury in rats. Next, we further verified the mechanism of action of DLX6-AS1/miR-204-5p/FBXW7 axis in myocardial IR injury in vitro.

Materials and Methods
2.1. IR Injury Rat Model. Wistar male rats with 14 weeks were purchased from the Shanghai SLAC Laboratory Animal Co., Ltd (China). All protocols were carried out under the authorization of the Ethics Committee of Zhongshan Hospital of Fudan University. IR injury rat model was established by ligation of the left anterior descending (LAD) coronary artery as previously described [20]. Briefly, rats were anesthetized with intraperitoneal injection of 45 mg/kg pentobarbitone. Subsequently, the trachea of rats was carefully exposed. Then, a rodent ventilator TOPO Dual Mode Ventilator (Kent Scientific Corporation, Torrington, CT, USA) was connected to the trachea for establishment of mechanical ventilation. The left ventricle was exposed from the chest under the rats with steady breathing. The LAD coronary artery was ligated with a 6-0 suture to induce ischemia for 30 min. Subsequently, the ligature was cut to mimic reperfusion. After that, the chest was sutured utilizing 6-0 Prolene sutures. In sham-operated rats, the procedure was the same except for artery ligation. The rats were sacrificed 6 h after the LAD ligation-reperfusion intervention. After the rats were sacrificed, tissues about 5 mm around the infarct area were selected for subsequent experiments.

Histological
Analysis. Paraffin sections (5 μm) of myocardial tissues were subjected to dewaxing and hydration. Then, the sections were stained with hematoxylin and eosin sequentially using Hematoxylin-Eosin (HE) Staining Kit (Beyotime, Shanghai, China). For detection of apoptotic cells in myocardial tissues, the paraffin sections were stained with TUNEL Apoptosis Assay Kit (Beyotime, Shanghai, China). The nucleus was stained with DAPI (Beyotime, Shanghai, China). Finally, the pathological changes and apoptotic cells in myocardial tissues were observed under optical or fluorescence microscope.
2.3. 2,3,5-Triphenyte-Trazoliumchloride (TTC) Staining. The infarction size of myocardial tissues was determined as previously reported [20]. The hearts were separated form rats, and the left ventricle was sliced into sections with 2 mm perpendicular to the long axis of heart. The sections were incubated with 1% TTC (Solarbio, Beijing, China) at 37°C for 60 min. The sections were sealed in closed freezer bag containing PBS to avoid oxidation of samples. The red part with TTC staining was ischemic but viable tissue, and the unstained area indicated the infarction area of myocardial tissues. The infarction size of myocardial tissues was analyzed using Image Pro Plus 6.0.

Cell
Culture. The rat embryonic cardiomyocyte cell line H9c2 (CCTCC, Wuhan, China) was cultured in Dulbecco's Modified Eagle Medium (DMEM; Gibco BRL, Grand Island, NY, USA) containing 10% fetal bovine serum (FBS; Gibco) and 100 μg/mL penicillin/streptomycin at 37°C and 5% CO 2 . For inducing IR through hypoxia reoxygenation (HR) treatment, H9c2 cells were cultured in serum-and glucosefree DMEM in a hypoxic chamber for 6 h, followed by incubation in DMEM containing 10% FBS in normal oxygen environments.  The relative expression of lncRNA and mRNA was normalized to GAPDH, and miRNA expression was normalized to U6. The results were analyzed using 2 -ΔΔCT method for quantification.
2.9. Luciferase Reporter Assay. Starbase online tool uncovered that there may be binding sites between DLX6-AS1 and miR-204-5p or betweenmiR-204-5p and FBXW7. To verify this speculation, the luciferase reporter vectors pmir-GLO containing wild type (WT) or mutant type (Mut) of DLX6-AS1 and FBXW7 were constructed by GenePharma. H9c2 cells were transfected with WT/Mut of luciferase reporter vectors and miR-204-5p mimic or NC-mimic using Lipofectamine 2000 Transfection Reagent. Finally, Luciferase Assay System (Ambion, Austin, TX, USA) was used to assess the relative luciferase activity of H9c2 cells.
2.10. RNA-Binding Protein Immunoprecipitation (RIP). RIP assay was carried out to determine whether DLX6-AS1 and miR-204-5p interacted with Ago-2 to form a RNA-induced silencing complex (RISC) according to reported work [21].   2.12. Statistical Analysis. Each assay was performed for 3 times. The representative data were presented and expressed as mean ± standard deviation. Statistical analysis was carried out utilizing SPSS 22.0 statistical software (IBM, Armonk, NY, USA). Two-tailed Student's t-test (between two groups) and one-way ANOVA (among multiple groups) were utilized to analyze the statistical differences. P < 0:05 indicates significant differences.

The Expression of DLX6-AS1 Was Decreased in IR Injury
Rats. In order to determine the function of DLX6-AS1 in IR injury, a rat model of IR injury was constructed by LAD ligation. HE and TTC staining revealed that IR rats exhibited a severe damage and an increase of infarction size in the myocardial tissues compared with sham-operated rats (Figures 1(a) and 1(b)). Moreover, we detected the level of myocardial enzyme spectrum in the serum by ELISA, showing that LDH and CK were increased in IR rats (Figure 1(c)).
The proinflammatory factors such as MCP-1, IL-6, and IL-  Mediators of Inflammation 1β were also enhanced in the serum of IR rats as compared with sham-operated rats (Figure 1(d)). Furthermore, cell apoptosis in the myocardial tissues of rats was measured by TUNEL staining, revealing that the apoptotic cells were increased in IR rats (Figure 1(e)). Compared with that in sham-operated rats, Bax protein was upregulated, while Bcl-2 protein was downregulated in myocardial tissues of IR rats (Figure 1(f)). Interestingly, DLX6-AS1 expression was upregulated in the myocardial tissues of IR rats (Figure 1(g)). Thus, these data indicated that the upregulation of DLX6-AS1 may be associated with myocardial IR injury.

Inhibition of DLX6-AS1 Attenuated IR-Induced
Myocardial Injury. Next, we investigated whether inhibition of DLX6-AS1 can attenuate IR injury in rats. DLX6-AS1 was silenced in IR rats, and the results showed that DLX6-AS1 was severely downregulated in the myocardial tissues of IR rats following injection of sh-DLX6-AS1 (Figure 2(a)). Then, the myocardial infarction of IR rats was examined by TTC staining. As shown in Figures 2(b) and 2(c), the infarction size in myocardial tissues of IR rats was severely decreased. We also found that silencing DLX6-AS1 caused a decrease in the levels of LDH and CK in the serum of IR rats (Figure 2(d)).
Additionally, ELISA results revealed that the inflammatory cytokines including MCP-1, IL-6, and IL-1β in the serum of IR rats were decreased by DLX6-AS1 deficiency (Figure 3(a)). TUNEL staining and WB assays were carried out to assess apoptotic cells and the expression of apoptosis-related proteins in myocardial tissues of IR rats. Silence of DLX6-AS1 reduced the number of apoptotic cells through downregulating Bax and upregulating Bcl-2 in IR rats (Figures 3(b) and 3(c)).
Taken together, DLX6-AS1 deficiency reduced myocardial infarction, inflammatory response, and cell apoptosis in IR rats.

Mediators of Inflammation
In a word, DLX6-AS1 functioned as a ceRNA to interact with miR-204-5p, which contributed to regulate the expression of its down-stream target FBXW7.
It is also important that we discovered that the infraction size was reduced after silencing DLX6-AS1, but this effect could be reversed by overexpressing FBXW7 (Figure Supplementary A). Additionally, the decreased LDH and CK levels of mediated DLX6-AS1 knockdown were rescued by FBXW7 overexpression (Figure Supplementary B).

Discussion
Myocardial IR injury is a complex process, and many factors are involved in the damage of myocardial tissues [22]. In the past few decades, scholars attempt to clarify the molecular mechanism of myocardial IR injury and find the effective treatment to reduce the scope and extent of myocardial injury caused by IR [22].   [23]. DLX6-AS1 functions as ceRNA to regulate miR-149-5p/ERP44 axis, which contributes to aggravate preeclampsia progression. DLX6-AS1 is rich in the serum exosomes of cervical cancer patients, whose upregulation is positively related to the poor prognosis of cervical cancer [24]. DLX6-AS1 inhibits ANXA10 expression through ubiquitination-mediated degradation to accelerate hepatocellular carcinoma development, which can be attributed to the regulation on miR-513c/Cul4A axis [25]. Hu et al. have confirmed that DLX6-AS1 promoted I/R-induced cerebral neuron impairments by regulating miR-149-3p/BOK axis [13]. In the present work, the IR injury rats displayed a severe tissue damage and significant increase of the myocardial infarction size and cell apoptosis. The levels of serum myocardial enzyme spectrum LDH and CK and proinflammatory factors MCP-1, IL-6, and IL-1β were significantly increased in the myocardial tissues of IR injury rats. These data showed that IR injury rats had a significant myocardial damage, which was rescued by inhibition of DLX6-AS1 expression. Thus, DLX6-AS1 played a crucial role in the development of myocardial IR injury.
The miR-204-5p expression is lower in the myocardial tissue, and KCNQ1OT1/miR-204-5p/LGALS3 axis was involved in the myocardial IR injury in mice [17]. In this work, it was verified that DLX6-AS1 repressed miR-204-5p expression by targeting miR-204-5p. The expression of miR-204-5p was decreased in IR rats and HR-treated H9c2 cells. Both DLX6-AS1 and miR-204-5p interacted with Ago-2 to form a RISC. The assembly of RISC is the central link of RNAi and miRNA pathways, which has the activity of silencing the target mRNA [26]. Ago-2 is the core protein in RISC and has the ability to shear mRNA [27]. Moreover, miR-204-5p interacted with FBXW7, and inhibited FBXW7 expression. DLX6-AS1 rescued the miR-204-5p-mediated inhibition on FBXW7 expression.

Conclusion
This work demonstrated that lncRNA DLX6-AS1 promoted FBXW7 expression by sponging miR-204-5p, which contributed to accelerating myocardial IR injury. Thus, this work provides a novel ceRNA DLX6-AS1/miR-204-5p/FBXW7 axis in myocardial IR injury, and DLX6-AS1 may be a potential target for myocardial IR injury treatment.

Data Availability
All data generated or analyzed during this study are included in this published article.

Ethical Approval
Ethical approval was obtained from the Ethics Committee of Zhongshan Hospital of Fudan University.