Long Noncoding RNA SNHG4 Attenuates the Injury of Myocardial Infarction via Regulating miR-148b-3p/DUSP1 Axis

Objective Long noncoding RNAs (lncRNAs), including some members of small nucleolar RNA host gene (SNHG), are important regulators in myocardial injury, while the role of SNHG4 in myocardial infarction (MI) is rarely known. This study is aimed at exploring the regulatory role and mechanisms of SNHG4 on MI. Methods Cellular and rat models of MI were established. The expression of relating genes was measured by qRT-PCR and/or western blot. In vitro, cell viability was detected by MTT assay, and cell apoptosis was assessed by caspase-3 level, Bax/Bcl-2 expression, and/or flow cytometry. The inflammation was evaluated by TNF-α, IL-1β, and IL-6 levels. The myocardial injury in MI rats was evaluated by echocardiography, TTC/HE/MASSON/TUNEL staining, and immunohistochemistry (Ki67). DLR assay was performed to confirm the target relationships. Results SNHG4 was downregulated in hypoxia-induced H9c2 cells and MI rats, and its overexpression enhanced cell viability and inhibited cell apoptosis and inflammation both in vitro and in vivo. SNHG4 overexpression also decreased infarct and fibrosis areas, relieved pathological changes, and improved heart function in MI rats. In addition, miR-148b-3p was an action target of SNHG4, and its silencing exhibited consistent results with SNHG4 overexpression in vitro. DUSP1 was a target of miR-148b-3p, which inhibited the apoptosis of hypoxia-induced H9c2 cells. Both miR-148b-3p overexpression and DUSP1 silencing weakened the effects of SNHG4 overexpression on protecting H9c2 cells against hypoxia. Conclusions Overexpression of SNHG4 relieved MI through regulating miR-148b-3p/DUSP1, providing potential therapeutic targets.


Introduction
Myocardial infarction (MI) is a fatal disorder with a high risk of mortality due to no obvious warning and late diagnosis [1,2]. There are approximately 350 million people suffer from MI each year [1]. In recent years, the main therapeutic strategy for MI is reperfusion for ischemic myocardial tissues in time. However, restoration of the blood flow can induce adverse microstructural destruction and inflammation [3]. The secondary injury of reperfusion directly contributes to the poor survival of patients with MI [4,5]. Therefore, exploring of potential therapeutic strategy with less adverse effects for MI is urgently needed.
With the development of molecular biology, more and more molecular targets exhibit therapeutic potential against MI [2]. Long noncoding RNAs (lncRNAs) are critical regulators in the pathogenesis of MI through posttranscription and chromatin modification [6]. For example, overexpression of CARL represses the apoptosis of cardiomyocytes in a rat model of MI [3]. The upregulation of Gm2691 and H19 improves heart function and relieves inflammation in MI rats [4,5]. Small nucleolar RNA host gene (SNHG) is a group of lncRNAs that also participates in myocardial injury. Lu et al. have shown that overexpression of SNHG12 weakened hypoxia-reoxygenation (H/R) injury via inhibiting RAGE expression in H9c2 cells [7]. Xiong et al. have revealed that SNHG15 may be a potential apoptotic biomarker in myocardial cells in response to ischemia/reperfusion (I/R) injury [8]. Notably, SNHG4 also exerts a critical role in many inflammatory disorders, including proteinuria [9], neuropathic pain [10], and cerebral I/R injury [11]. However, the regulatory role of SNHG4 in MI is relatively reported.
lncRNAs can block the functional interaction between miRNAs and related target mRNAs by acting as miRNA sponges [12]. Recently, massive miRNAs have been determined as the targets of SNHG4 in cancers, such as miR-138 [13], -590-3p [9], -377-3p [14], -204-5p [15], and -224-3p [16]. There are also many miRNAs involved in MI progression. For example, miR-375 acts a pathogenic miRNA in MI through regulating myocardial dysfunction [17]. Downregulation of miR-327 decreases the infarct area of myocardial tissues in MI mice [18]. Both miR-154 [17] and miR-21 [19] function in the acceleration of cardiac fibrosis. In addition, miR-148b is also an important regulator in myocardial injury. Sun et al. have found that the downregulation of miR-148b decreases the apoptosis of H/R-induced myocardial cells [18]. Yang et al. have revealed that silencing of miR-148b-3p relieves myocardial I/R injury through enhancing antioxidation and antiapoptosis [20]. However, the function of miR-148b-3p on MI and related regulatory relationship with SNHG4 remain unclear.
In this research, the function of SNHG4 was investigated in both hypoxia-induced H9c2 cells and a rat model of MI. The regulatory mechanisms of SNHG4/miR-148b-3p/ DUSP1 axis in MI were further evaluated in vitro. Our study is aimed at revealing potential therapeutic targets for MI.  [32]. Simply, rats were intraperitoneal injected with 50 mg/kg pentobarbital sodium for anaesthesia, and then the left anterior descending artery (LAD) was ligated for 30 min (n = 30) [32]. A same procedure without LAD ligation was performed in rats of the sham group (n = 8). Before suturing the chest, 10 μL pcDNA-SNHG4 and pcDNA-NC that packaged in adenovirus (5 × 10 10 pfu/mL) were injected into the apex of the left ventricle with a 30gauge needle (MI + Ad-SNHG4 and MI + Ad-NC groups; n = 10 each group). After the surgery for 4 weeks, the survival rate was 87.5%, 80%, 70%, and 80% in sham, MI, MI + Ad-SNHG4, and MI + Ad-NC groups, respectively. Six survival rats in each group were randomly selected for the following analyses. Transthoracic echocardiography was performed to detect the parameters of left ventricular fraction shortening (LVFS), left ventricular ejection fraction (LVEF), left ventricular end-diastolic diameter (LVEDd), and left ventricular end-systolic diameter (LVESd).

Materials and Methods
2.3. Triphenyltetrazolium Chloride (TTC) Staining. Followed by hemodynamic analyses, rats were anaesthetized with intraperitoneal injection of pentobarbital sodium (50 mg/kg) and were sacrificed via cervical dislocation. The heart was immediately resected, and the ventricle samples at a same depth were sliced into sections at 2 mm or 5 μm. The sections were subsequently stained with 1% TTC for 15 min under darkness. After 6 h of fixing with 10% formaldehyde, the infarction area (white) was captured under a microscope (Olympus, Japan).

Cell Apoptosis
Assay. The caspase-3 in H9c2 cells was detected using a caspase-3 Assay Kit (QCbio, Shanghai, China) following the manufacturer's protocol. In addition, the apoptotic rate was measured using an annexin V-FITC Apoptosis Kit (Thermo Fisher Scientific). Briefly, 2 × 10 5 cells that resuspended in 500 μL binding buffer were incubated with 5 μL annexin V-EGFP and 5 μL PI for 15 min at 4°C under darkness. The apoptotic data was measured by a FACScan flow cytometer (BD, Franklin Lakes, NJ, USA).

Western
Blot. H9c2 cells were lysed in RIPA buffer to isolate total proteins. After quantification using a BCA Assay Kit (Thermo Fisher Scientific), 30 μg protein samples were run on 10% sodium dodecyl sulphate-polyacrylamide gel electrophoresis and transferred onto polyvinylidene fluoride membranes. The proteins in the membranes were then blocked with 5% skimmed milk and incubated with specific primary antibodies (anti-Bax, -Bcl-2, and -DUSP1; 1 : 1,500, Sigma) for 12 h at 4°C. Subsequently, the proteins were further incubated with HRP-conjugated secondary antibody (1 : 5,000, Sigma) for 1 h at 25°C. Using an ECL Kit (Thermo Fisher Scientific), the protein blots were visualized and captured under a Gel-Pro analyzer (Media Cybernetics, Silver Spring, MD, USA). β-Actin was an internal control for western blot (anti-β-actin, 1 : 2,000, Sigma).
2.9. Enzyme-Linked Immunosorbent Assay (ELISA). The TNF-α, IL-1β, and IL-6 in hypoxia-induced H9c2 cells and in serum samples were measured using specific ELISA kits (enzyme-linked Biotechnology, Shanghai, China). In addition, the serum level of lactate dehydrogenase (LDH) was detected using a LDH Assay Kit (Abcam). All the above assays were performed following the manufacturers' protocols.

Statistical Analyses.
The experiments in vitro were performed in triplicate, and those in vivo were performed in six rats of each group, respectively. Data that expressed as mean ± standard deviation (SD) were statistically analyzed using SPSS 23.0. The comparison between two groups was analyzed by Student's t-test and that among multiple groups was analyzed by one-way ANOVA with Tukey's test. Pvalue < 0:05 presented significantly different.

SNHG4 Overexpression Relieves the Myocardial Injury in MI Rats.
The effects of SNHG4 overexpression on the myocardial injury of MI rats were evaluated. TTC staining showed an obvious reduction of infarct area in rats of Ad-SNHG4 + MI group than in the MI group (P < 0:01; Figures 7(a)-7(b)). Overexpression of SNHG4 also significantly reduced fibrosis area in MI rats (Ad-SNHG4 + MI vs. MI, P < 0:01; Figure 7(c)). HE staining revealed swelling cardiomyocytes, irregular arranged nucleus, interstitial enlargement, and neutrophil infiltration in MI rats. These pathological changes were partially relieved in MI rats injected with ad-SNHG4 (Figure 7(d)). Furthermore, hemodynamic analyses uncovered lower LVEF and LVFS and higher LVESd and LVEDd in the MI group than in the sham group (P < 0:01), and these changes were all weakened by the injection of ad-SNHG4 (MI + Ad-SNHG4 vs. MI, P < 0:01; Figure 7(e)). However, no significantly differences on the heart rate were revealed among different groups (Figure 7(f)).

SNHG4 Overexpression Inhibits Cell Apoptosis and
Inflammation in MI Rats. In MI rats, cell apoptosis and inflammation in myocardial tissues were further explored. TUNEL assay showed that there are more apoptotic cells in the MI group than in the sham group and less apoptotic cells in the MI + Ad-SNHG4 group than in the MI group (Figure 8(a)). On the contrary, IHC determined that there are less Ki67-positive cells in the MI group than in the sham group and more Ki-67-positive cells in the MI + Ad-SNHG4 group than in the MI group (Figure 8(b)). In addition, the serum TNF-α, IL-6, IL-1β, and LDH were higher in MI rats than in the sham rats (P < 0:01). The injection of ad-SNHG4 inhibited the increasing of the above parameters in MI rats (MI + Ad-SNHG4 vs. MI, P < 0:01; Figures 8(c) and 8(d)).

Discussion
MI is a life-threatening cardiovascular disorder that characterized by myocardial necrosis on account of acute ischemia and hypoxia [26]. With the increasing of healthcare costs in patients with MI [35], exploring effective molecular targets is urgently helpful for the treatment MI. Until now, the diverse lncRNAs are abnormally expressed in MI. For examples, ANRIL [36], GAS5 [23], and KCNQ1OT1 [37] are upregulated; whereas, SLC8A1-AS1 [38], H19 [5], and Gm2619 [4] are downregulated in cell or animal models of MI. In our study, SNHG4 was significantly downregulated in 8 Cardiovascular Therapeutics hypoxia-treated H9c2 cells than in the controls and also in MI rats than in the sham rats. This result heralds a potential role of SNHG4 in the pathogenesis of MI. lncRNAs exert critical roles in MI through regulating various cellular processes, mainly including cell viability, apoptosis, and inflammation [8,10,11]. For example, overexpression of H19 represses the development of MI via inhibiting inflammatory reaction and apoptosis in myocardial cells [5]. The upregulation of Gm2691 ameliorates cardiac function through suppressing myocardial fibrosis, apoptosis, and inflammation [4]. The upregulation of CARL increases myocardial cell viability and decreases cell apoptosis in MI rats [3]. Here, researches on the regulatory role of SNHG4 in MI showed that overexpression of SNHG4 inhibited cell apoptosis and inflammation and enhanced cell viability in both hypoxia-treated H9c2 cells and MI rats (hypoxia + pcDNA-SNHG4 vs. hypoxia, MI + Ad-SNHG4 vs. MI). These results indicate a protective role of SNHG4 in myocardial cells under MI, which are similar with those reported lncRNAs mentioned above. In addition, SNHG4 overexpression also decreased infarct and fibrosis areas, relieved histopathological damages, and improved heart function in MI rats (MI + Ad-SNHG4 vs. MI). These findings further confirm the therapeutic potential of SNHG4 against MI. To lncRNAs are widely known as sponges for miRNAs, and this interaction has been studied throughout physiological and disease states [39]. In this study, the potential miRNA target of SNHG4 was explored to determine the action mechanisms of SNHG4 in MI. A target relation between SNHG4 and miR-148b-3p was predicted, which was subsequently confirmed by DLR assay. With the ability to regulate cell proliferation, apoptosis, migration, and invasion, miR-148b-3p acts an antioncogene in different human cancers, such as gastric cancer [40], gastrointestinal stromal tumor [41], hepatocellular carcinoma [42], and renal carcinoma [43]. miR-148b-3p also participates in the pathogenesis of cardiovascular disorders. Chen et al. have found that the miR-148b-3p in the blood is a diagnostic marker for ischemic stroke [44]. Wang et al. have revealed that miR-148b-3p is involved in inhibiting neural stem cell proliferation and differentiation, and its silencing relieves ischemic lesion in a rat model of ischemic stroke [45]. Notably, the downregulation of miR-148b ameliorates I/R-induced cardiac dysfunction and myocardial infarction and increases the viability of myocardial cells [20]. In this research, miR-148b-3p was upregulated in hypoxia-treated H9c2 cells compared with controls. Silencing of miR-148b-3p in hypoxia-treated H9c2 cells inhibited cell apoptosis and inflammation and promoted cell viability (hypoxia + miR-148b-3p inhibitor vs. hypoxia). These findings are similar with previous studies and indicate that miR-148b-3p inhibitor may protect myocardial cells against hypoxia-induced injury. To combine with the negative regulatory relation between SNHG4 and miR-148b-3p, we suspect that miR-148b-3p downregulation may contribute to the relieving role of SNHG4 on MI. Furthermore, our following feedback assays determined that miR-148b-3p overexpression eliminated the protective effects of SNHG4 overexpression on hypoxia-treated H9c2 cells, evidenced by weakened cell viability, as well as enhanced cell apoptosis and inflammation (hypoxia + pcDNA-SNHG4 vs. hypoxia + pcDNA-SNHG4 + miR-148b-3p mimics). Therefore, we believe that SNHG4 may attenuate the myocardial injury of MI by targeting miR-148b-3p.
Since lncRNAs and miRNAs are both noncoding RNAs, their functions rely on the translation or degradation of target mRNAs [46]. To further reveal a complete regulatory axis of lncRNA/miRNA/mRNA in MI, the mRNA target of miR-148b-3p was investigated. The results determined that DUSP1 was a downstream target of miR-148b-3p. As an antiapoptotic phosphatase, DUSP1 can act as an oncogene in gastric cancer [47], breast cancer [23], and osteosarcoma [48]. DUSP1 also exerts a critical regulatory role in myocardial I/R injury. He et al. have shown that the downregulation of TRIM11 inhibits the apoptosis of cardiomyocytes following I/R injury through regulating DUSP1-JNK1/2 pathway [49]. Ren et al. have revealed that DUSP1 enhances the antiapoptotic effect of NaHS in rats with myocardial I/R injury [50]. Jin et al. have revealed that DUSP1 alleviates myocardial I/R injury through inhibiting mitochondrial fission and mitophagy [51]. Therefore, the upregulation of DUSP1 may also be a benefit for the remission of MI via similar mechanisms mentioned above. Since DUSP1 can be

Conclusions
In conclusion, SNHG4 and DUSP1 were downregulated, and miR-148b-3p was upregulated in both cellular and rat models of MI. Overexpression of SNHG4 promoted the viability and repressed the apoptosis and inflammation of hypoxia-induced H9c2 cells through regulating miR-148b-3p/DUSP1. In addition, SNHG4 overexpression also relieved myocardial injury in MI rats. Our findings illustrate an important regulatory axis of SNHG4/miR-148b-3p/DUSP1 in MI, providing potential therapeutic targets.

Data Availability
All data in the manuscript are available through the responsible corresponding author.

Disclosure
The funder was not involved in the manuscript writing editing approval and decision to publish.