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Long Non-Coding RNA SNHG8 Plays a Key Role in Myocardial Infarction Through Affecting Hypoxia-Induced Cardiomyocyte Injury

Background The objective of the study was to explore the role of long non-coding RNA SNHG8 (lncRNA SNHG8) in myocardial infarction (MI) and the related mechanism of action. Material/Methods In vitro model of MI was established by hypoxia induction in cardiomyocyte line H9c2 cells. H9c2 cells were transfected with control-plasmid, SNHG8-plasmid, control-shRNA and SNHG8-shRNA. Quantitative real-time polymerase chain reaction (qRT-PCR) assay was performed to measure transfection efficiency. Creatine kinase-muscle/brain (CK-MB) release, cardiac troponin 1 (cTnI) release and mitochondria viability were detected by using related detection kits. MTT (3-(45)-dimethylthiahiazo (-z-y 1)-35-diphenytetrazoliumromide) assay was used to detect cell viability and flow cytometry analysis was used to detect cell apoptosis. Western blot assay was performed to measure protein expression of cleaved-Caspase3, p-p65 and p65. Enzyme-linked immunosorbent assay (ELISA) and qRT-PCR assay were performed to detect expression of interleukin (IL)-1β, tumor necrosis factor (TNF)-α and IL-6. Results LncRNA SNHG8 was overexpressed in hypoxia-induced cardiomyocytes. SNHG8-plasmid increased lncRNA SNHG8 expression, CK-MB release, cTnI release, and mitochondria viability in hypoxia-induced H9c2 cells. In addition, SNHG8-plasmid reduced cell viability, induced cell apoptosis, and increased expression of cleaved-caspase3, IL-1β, TNF-α, IL-6, and p-p65 in hypoxia-induced H9c2 cells, while the effects of SNHG8-shRNA were opposite. Conclusions We demonstrated that lncRNA SNHG8 affected myocardial infarction by affecting hypoxia-induced cardiomyocyte injury via regulation of the NF-κB pathway.


Background
Myocardial infarction (MI) refers to ischemic necrosis of the myocardium. Clinically, there are severe and long-lasting sternum pains, accompanied by increased serum myocardial enzyme activity, and progressive electrocardiogram changes, which can be complicated by arrhythmia, shock, or heart failure and ultimately threaten life [1][2][3]. At present, MI pathogenesis is not fully understood, but aging, overwork, overeating, and smoking are major pathogenic factors [4]. MI is most common in Europe and the United States, with about 1.5 million people suffering from it each year in the United States. China has shown a clear upward trend in recent years, with at least 500,000 new issues each year and a current incidence of at least 2 million [5,6]. The primary objective of treatment of MI is clearance of the blocked coronary artery and restoration of function of ischemic and hypoxic myocardium [7]. Treatment is usually done by dilating the coronary arteries and removing the pathogenic blockages. In addition, reduction of cardiac load, such as oxygen absorption, absolute bedrest, and avoidance of emotional fluctuations is also conducive to recovery of patients. MI is known to be harmful, but there is no effective treatment for it so far. Therefore, new, safe, and more effective treatments for MI are desperately needed. Previous reports have used an in vitro model of MI created by inducting hypoxia in cardiomyocytes to study the related mechanism of MI, and we used hypoxia-induced a similar model to further study MI [8,9].
Long non-coding RNAs (lncRNAs) are usually defined as RNA molecules longer than 200 nucleotides and that lack biological functions [10,11]. To date, lncRNAs have been shown to play an extensive regulatory role in life activities and have been implicated in various diseases, such as cervical, gastric, and esophageal cancers; Parkinson's disease; and myocarditis and MI [12][13][14]. LncRNA SNHG8, known as a novel small nucleolar guide RNA, is located on 4q26 [15]. LncRNA SNHG8 has been well studied in various types of cancers, including cervical, gastric and colorectal cancers and esophageal squamous cell carcinomas [16][17][18][19][20]. A recent study indicated that lncRNA SNHG8 was significantly overexpressed in patients with MIMI, and found that lncRNA SNHG8 was a risk factor for acute MI, but the specific mechanism of action still needed further study [21].
In this study, we explored whether lncRNA SNHG8 played an important role in hypoxia-induced myocardial cell injury in vitro, and further studied its molecular mechanism. Our results provide more theoretical basis and new strategies for treatment of MI.

Cell culture
Rat cardiomyocyte line H9c2 cells were purchased from the American Type Culture Collection (ATCC, Manassas, Virginia, United States). The cells were cultured in DMEM medium (Gibco, United States) containing 10% fetal bovine serum (FBS, Gibco), 1% penicillin/streptomycin and incubated in 5% CO 2 at 37°C.

Establishment of in vitro MI model
We established an in vitro MI model by inducting hypoxia in cardiomyocytes. Briefly, rat cardiomyocyte line H9c2 cells were cultured under hypoxic conditions (94% N 2 , 5% CO 2 , and 1% O 2 ) for 48 h [22].

Detection of CK-MB release, cTnI release and mitochondria viability
To explore the role of lncRNA SNHG8 in cellular damage of hypoxia-induced cardiomyocytes, CK-MB release, cTnI release, and mitochondria viability were detected by the CK-MB Detection Kit, cTnI Detection Kit (ReLIA, California, United States) and mitochondria activity assay kit (Jiancheng Bioengineering Institute, China), respectively, after the cells were transfected for 48 h.

3-(4,5-dimethylthiazol-2-yl)-2,5-dipheyltetrazolium bromide (MTT) assay
After specific treatment, the cells were incubated with 10 μL MTT solution (Beyotime, Shanghai, China) for 4 h. Subsequently, 100 μL dimethyl sulfoxide was added into each well to solubilize the formazan product after the solution was removed. Finally, absorbance at 490 nm was recorded with a microplate reader (Bio-Rad, Hercules, California, United States). Relative cell viability was normalized with the control group using optical density values.

Apoptosis analysis
Detection of cell apoptosis was performed by flow cytometric (FCM) analysis. The cells were transferred into six-well plates for 48 h, and collected by trypsinization after transfection, washed once with phosphate-buffered saline (PBS) and then resuspended in 1×binding buffer. A 100-µL cell suspension was transferred to a 5-mL tube, and then the cells were incubated with 5 μL Annexin V PE and 7-Aminoactinomycin D (7-AAD) (BD Biosciences, San Diego, California, United States), respectively, according to specifications. The stained cells were analyzed by with a FACSCalibur flow cytometer (BD Biosciences, United States) within 1 hr. FlowJo software (version 7.2.4; FlowJo LLC) was used to analyze the data and calculate early and late stage apoptosis rates.

Western blot analysis
Protein expression levels of cleaved-Caspase3, p-p65 and p65 in cells were investigated with Western blot analysis. Briefly, we extracted the protein using a total protein extraction kit (Beyotime, Shanghai, China), and determined protein concentrations using a BCA protein kit (Pierce, United States). The total protein extracted was resolved with SDS-PAGE using a 10% polyacrylamide gel under reduced conditions, and then transferred to polyvinylidene difluoride membranes. The membranes were blocked with 5% nonfat milk PBS Tween (PBST) solution at room temperature for 1 h. Afterwards, we incubated cleaved-Caspase3 (cat. no. ab2302; 1: 1,000; Abcam), p-p65 (cat no. 3033; 1: 1,000; Cell Signaling Technology, Inc.) and p65 (cat no. 8242; 1: 1,000; Cell Signaling Technology, Inc.) or GAPDH (cat. no. 5174; 1: 1,000; Cell Signaling Technology, Inc.) at 4°C overnight. Afterwards, the membranes were washed three times with PBST and incubated with horseradish peroxidaseconjugated secondary antibody (cat. no. 7074; 1: 2,000; Cell Signaling Technology, Inc.) at room temperature for 1 h. Protein bands were detected by enhanced chemiluminescent reagent (ECL Advance Western Blotting Detection Kit; GE Healthcare Life Sciences) according to the protocol and protein levels were quantified with Image J software. In addition, we calculated the value of Pp65/p65.

Statistical analysis
Data were displayed as the mean±standard deviation (SD) of a least three independent experiments. Statistical analyses between groups were estimated by Student's t-test or one-way ANOVA followed by Tukey's post-hoc test by using SPSS 18.0 software package (SPSS Inc, United States). P<0.05 was considered a statistically significant difference.

Hypoxia induced lncRNA SNHG8 accumulation in H9c2 cells
For preliminary assessment of the role of lncRNA SNHG8 in MI, we detected expression of lncRNA SNHG8 in H9c2 cells. As shown in Figure 1, expression of lncRNA SNHG8 was upregulated in hypoxia-induced H9c2 cells significantly compared with the control group.

Effects of high expression of lncRNA SNHG8 on cell injury in hypoxia-induced cardiomyocytes
To study the effect of high expression of lncRNA SNHG8 on cell injury, control-plasmid or SNHG8-plasmid was transfected into hypoxia-induced cardiomyocytes. After transfection, e924016-3

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the cells were separated into four groups: control, hypoxia, hypoxia+control-plasmid and hypoxia+SNHG8-plasmid group. Transfection efficiency was evaluated by qRT-PCR assay. Compared with the control-plasmid group, expression of lncRNA SNHG8 was dramatically increased after cells were transfected with SNHG8-plasmid ( Figure 2A). In comparison with the control group, hypoxia significantly induced release of creatine kinase isoenzyme-MB (CK-MB) and cardiac troponin I (cTnI), while SNHG8-plasmid further promoted release of CK-MB and cTnI ( Figures 2B, 2C). Meanwhile, we detected viability of mitochondria in H9c2 cells. The results showed that hypoxia reduced mitochondrial viability remarkably compared to the control group, and SNHG8-plasmid further reduced mitochondrial viability ( Figure 2D).

Effects of high expression of lncRNA SNHG8 on cell viability and apoptosis of hypoxia-induced cardiomyocytes
To assess whether lncRNA SNHG8 interfered with cell viability and apoptosis of hypoxia-induced cardiomyocytes, cell viability, apoptosis, and cleaved-Caspase3 protein expression were measured following transfection. Compared with the control group, hypoxia markedly reduced the cell viability of cardiomyocytes ( Figure 3A), induced cell apoptosis ( Figure 3B, 3C), and promoted cleaved-Caspase3 protein expression ( Figure 3D, 3E), while SNHG8-plasmid deepened these effects.

Effect of high expression of lncRNA SNHG8 on secretion of inflammatory factors in hypoxia-induced cardiomyocytes
To study the mechanism of lncRNA SNHG8 in cardiomyocytes, expression of related inflammatory factors was detected. As shown in Figure 4A-4C, in contrast with the control group, hypoxia markedly increased secretion of TNF-a, L-1b, and IL-6, while SNHG8-plasmid further induced secretion of TNF-a, L-1b and IL-6. Similarly, hypoxia increased mRNA expression of TNF-a, L-1b and IL-6 remarkably compared with the control group, and all these effects were further improved by SNHG8plasmid ( Figure 4D-4F).

Effects of knockdown of lncRNA SNHG8 on cell damage in hypoxia-induced cardiomyocytes
Rat cardiomyocyte line H9c2 cells were transfected with control-shRNA or SNHG8-shRNA in anoxic condition and the cells were

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separated into four groups: control, hypoxia, hypoxia+control-shRNA group, and hypoxia+SNHG8-shRNA group. Results of transfection efficiency detection showed that the expression of lncRNA SNHG8 was lower in the SNHG8-shRNA group than that in the control-shRNA group ( Figure 5A). In contrast with the control group, hypoxia significantly increased release of CK-MB and cTnI, while SNHG8-shRNA significantly reduced release of CK-MB and cTnI ( Figure 5B, 5C). As shown in Figure 5D, hypoxia remarkably reduced mitochondrial viability compared with the control group, while SNHG8-shRNA improved mitochondrial viability.

Effects of lncRNA SNHG8 on NF-kB signal pathway in hypoxia-induced cardiomyocytes
To investigate the signal pathway underlying the role of lncRNA SNHG8 in hypoxia-induced cardiomyocytes, we detected expression of p-p65 and p65 in H9c2 cells using Western blot assay. The results showed that hypoxia increased p-p65 protein expression and p-p65/p65 ratio remarkably in comparison with

Discussion
Coronary heart disease (CHD) is a cardiovascular disease with high incidence. Data indicate that the rate of CHD in China continues to increase every year, and it is being seen in younger ande younger patients [24]. MI is a type of CHD that involves myocardial necrosis and often results in malignant arrhythmia, heart failure, other serious complications, or even sudden death [25,26]. In previous studies, we developed an in vitro MI model by inducing hypoxia in cardiomyocytes. Studies show that lncRNA SNHG8 is highly expressed in patients with MI. Rat cardiomyocyte line H9c2 cells were cultured under oxygen-deficit conditions for 48 h in one study. We found that expression of lncRNA SNHG8 was obviously higher in hypoxiainduced H9c2 cells which is consistent with previous research.
To further study mechanism of action of lncRNA SNHG8, we upregulated and down-regulated lncRNA SNHG8 by transfection.  Creatine kinase, also known as phosphocreatine or creatine phosphokinase, is an important kinase found in a variety of tissues and cells. It consists of three isozyme forms: CK-BB, CK-MM, and CK-MB. CK-MB accounts for 20% of the total creatine kinase in the myocardial tissues. In addition, CK-MB is specific for myocardial tissues, and CK-MB level can be considered an important clinical indicator for diagnosis of MI [27,28]. Cardiac troponin (cTn) is mainly present in myocardial myofibroblasts, including three subunits: troponin I, troponin T, and troponin C. It is an important functional protein that maintains myocardial fiber contraction and relaxation. Cardiac troponin I (cTnI) has myocardial specificity and is diagnostic for myocardium. Combined with CK-MB, it can improve specificity of diagnosis of MI. Mitochondrial dysfunction and resulting oxidative stress also are closely related to myocardial ischemia-reperfusion injury [29][30][31].
In this study, we found that overexpression of lncRNA SNHG8 increased CCK-MB release, cTnI release, and mitochondria viability. Upregulation of lncRNA SNHG8 also reduced cell viability, induced cell apoptosis, and increased the expression of cleaved-Caspase3, while downregulation of lncRNA SNHG8 had the opposite effect. In addition, we found that lncRNA SNHG8
Several protein kinase pathways were activated in myocardial ischemia and hypoxia [32]. Some studies indicate that proinflammatory cytokine levels are elevated in an NF-kB activation-dependent manner during myocardial damage [33]. During treatment of ischemic myocardium, the NF-kB pathway is crucial to preventing myocardial cell death [34]. But the relationship between lncRNA SNHG8 and the NF-kB pathway remains unclear. Therefore, we analyzed whether the NF-kB pathway was involved in regulation of lncRNA SNHG8 in hypoxia-induced myocardial cell injury. Results indicated that lncRNA SNHG8 upregulation further enhanced the NF-kB signal pathway activation in hypoxia-induced cardiomyocytes, while lncRNA SNHG8 downregulation inhibited NF-kB signal pathway activation.  In other words, SNHG8 played a significant role in myocardial cell injury caused by hypoxia, knockdown of SNHG8 played a protective role in the myocardial cell injury caused by hypoxia, and overexpression of SNHG8 promoted myocardial cell injury caused by hypoxia.

Conclusions
We found that lncRNA SNHG8 affected MI by promoting hypoxia-induced cardiomyocyte injury via regulation of the NF-kB signaling pathway. These findings suggest that SNHG8 may be an effective target for treatment of MI, which could transltate into new opportunities for clinical therapies for MI.