Silencing of ATP2B1-AS1 contributes to protection against myocardial infarction in mouse via blocking NFKBIA-mediated NF-κB signalling pathway.

Abstract Myocardial infarction (MI) is an acute coronary syndrome that refers to tissue infarction of the myocardium. This study aimed to investigate the effect of long intergenic non‐protein‐coding RNA (lincRNA) ATPase plasma membrane Ca2+ transporting 1 antisense RNA 1 (ATP2B1‐AS1) against MI by targeting nuclear factor‐kappa‐B inhibitor alpha (NFKBIA) and mediating the nuclear factor‐kappa‐B (NF‐κB) signalling pathway. An MI mouse model was established and idenepsied by cardiac function evaluation. It was determined that ATP2B1‐AS1 was highly expressed, while NFKBIA was poorly expressed and NF‐κB signalling pathway was activated in MI mice. Cardiomyocytes were extracted from mice and introduced with a series of mouse ATP2B1‐AS1 vector, NFKBIA vector, siRNA‐mouse ATP2B1‐AS1 and siRNA‐NFKBIA. The expression of NF‐κBp50, NF‐κBp65 and IKKβ was determined to idenepsy whether ATP2B1‐AS1 and NFKBIA affect the NF‐κB signalling pathway, the results of which suggested that ATP2B1‐AS1 down‐regulated the expression of NFKBIA and activated the NF‐κB signalling pathway in MI mice. Based on the data from assessment of cell viability, cell cycle, apoptosis and levels of inflammatory cytokines, either silencing of mouse ATP2B1‐AS1 or overexpression of NFKBIA was suggested to result in reduced cardiomyocyte apoptosis and expression of inflammatory cytokines, as well as enhanced cardiomyocyte viability. Our study provided evidence that mouse ATP2B1‐AS1 silencing may have the potency to protect against MI in mice through inhibiting cardiomyocyte apoptosis and inflammation, highlighting a great promise as a novel therapeutic target for MI.


| In silico analysis
The microarray data and annotated probe files of microarray data sets (GSE66360, GSE48060, GSE65705 and GSE97320) were downloaded from the Gene Expression Omnibus (GEO) database (http://www.ncbi.nlm.nih.gov/geo/), which were normalized and corrected using the Affy package of the R software. 16 Then, linear models with empirical Bayes moderation (Limma) combined with t-statistics were performed for the microarray analysis in order to select the differentially expressed long non-coding RNAs (lncRNAs). 17 Prediction of the differentially expressed lncRNA expression was performed using Multi-Experiment Matrix (MEM) database (http://biit.cs.ut.ee/mem/). 18 Based on the WebGestalt (http://www.webge stalt.org) database, a Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis of the candidates was proceeded to determine their involvement in major physiological metabolic processes and signalling pathways. 19 The lncRNA targets were predicted on a website available at http:// www.herbb ol.org:8001/lrt/index.php. 20

| Sequence analysis
The conserved sequences of homologous genes among different species shared some similarities. The nucleotide sequence of the full-length LINC00936 gene for human (available at https://www. ncbi.nlm.nih.gov/nucco re/NR_028138.1) was used as the query sequence to search the sequence of mice from genomic database (GRCm38/mm10) in UCSU (http://genome.ucsc.edu/) using basic local alignment search tool (BLAST), and the homologous sequences of LINC00936 gene in human and mouse were compared. The conserved nature of the lncRNA gene between two species was analysed.

| Establishment of a mouse model of MI
Forty-five adult male C57BL/6 mice (aged 12 weeks, weighing 25-30 g) were provided by the Animal Lab Center of Capital Medical University (Beijing, China). All mice were acclimatized for 1 week in 22-26°C under a 12/12-hours light/dark cycle with free access to food and water. Thirty mice were randomly selected for the establishment of the MI model. 21 The mice were anaesthetized by intraperitoneal administration of 1% pentobarbital sodium (P3761, 50 mg/kg; Sigma-Aldrich Chemical Company) and placed in the supine position on a heating pad. The mice were intubated and connected to a rodent ventilator (SAR-830/P; CWE Inc) at a respiratory rate of 30 breaths/min and tidal volume of 7 mL/kg. Following a left thoracotomy at the fourth intercostal space, the left thoracic cavity was exposed, and ligation of the left anterior descending (LAD) coronary artery was performed 2-3 mm around the lower edge of the left atrial appendage using an 8-0 atraumatic suture. The immediate colour change in the heart surface, which turned dark red, and the ST-segment elevation in leads II were signs of a successful coronary ligation. The chest was then closed and sutured. Mice received an intramuscular injection of penicillin (130 437; BIOBW Biological Technology Co., Ltd.) at a dosage of 10 000 U/d for three consecutive days. During the recovery period, the mice were allowed to breath spontaneously without tracheal intubation and placed on a heating pad maintained at 39°C. The remaining fifteen mice were sham-operated with the same procedures without LAD ligation. The infarct area was pale in the visual inspection during the surgery, and electrocardiogram (ECG) was recorded before and after the surgery.

| Ultrasonic cardiogram (UCG) test
After anaesthesia with pentobarbital sodium, the mice were fixed in the supine position before surgery and 2 weeks after surgery, respectively. An ultrasonic instrument (ACUSON, Siemens) was

| Haematoxylin-eosin (HE) staining
HE staining was performed to observe the MI-induced histological changes. 23 Two weeks after surgery, the mice from the MI and sham groups were anaesthetized with pentobarbital sodium.
Then, heart obtained by thoracotomy was rinsed with phosphate buffered saline (PBS), fixed with 10% formalin for 12 hours and dissected along the ligature to excise myocardial tissues. The paraffin-embedded tissues were sliced into 4-μm sections. After being heated at 60°C, the sections were dewaxed with xylene for 20 minutes, dehydrated using gradient ethanol and soaked in distilled water for 5 minutes. Afterwards, the sections were stained with haematoxylin (H8070; Solarbio) for 4 minutes and counterstained with eosin (E8090; Solarbio) for 3 minutes. Finally, the sections were observed under an optical microscope (DSX100, Olympus) and photographed.

| 2, 3, 5-triphenyltetrazolium chloride (TTC) staining
The infarct size (IS) was measured by conducting TTC staining. 23,24 The heart was isolated and washed with PBS, and the atrium and right ventricle were removed. The left ventricle was weighed and stored at −20°C for 20 minutes. Subsequently, the left ventricle was sliced into 2-mm sections and stained with 1% TTC solution (MHB0268a; Yuanye Biotechnology Co., Ltd.) at 37°C for 10 minutes. Then, the sections were dried, weighed and fixed with 4% paraformaldehyde (P1110, Solarbio) for 30 minutes. The IS was analysed using Image-Pro Plus software (IPP 7.0, Media Cybernetics) and calculated as follows: IS (%) = the weight of the infracted mycardium (IMW)∕ the weight of the left ventricular (LVW) × 100%. .

| Terminal deoxynucleotidyl transferasemediated dUTP nick-end labelling (TUNEL) staining
In compliance with the specifications of TUNEL Kit (C1086; Beyotime Biotechnology Co.), cell apoptosis was observed. Paraffin sections were dewaxed, rehydrated and immersed in 3% H 2 O 2 for 10 minutes at ambient temperature. The sections were hydrolysed with 50 μL of 20 μg/mL proteinase K (P6556; Sigma-Aldrich) for 20 minutes. The citrate was added for 30 minutes of antigen retrieval. Subsequently, the sections reacted with 50 μL deoxynucleotide transferase (TdT) in a wet box in the dark at 37°C for 1 hour. The TdT-free TUNEL reaction solution was used as a negative control (NC). Then, the sections were reacted with 50 μL peroxidase-labelled anti-digoxigenin in a wet box at 37°C in the dark for 30 minutes. The sections were stained with 4',6-diamidine-2-phenylindole (DAPI) solution (C1002; Beyotime Biotechnology Co.) for 10 minutes. Each step was followed by PBS washing. Finally, the sections were sealed using neutral balsam and observed under a fluorescence microscope (BX53; Olympus). In ten randomly selected visual fields, positive cells and the total cells were counted. The ratio (positive cells/total cells) represented the apoptosis index (AI).

| Immunohistochemical staining
After dewaxing and hydration of paraffin-embedded sections, the antigen retrieval was conducted for 10 minutes using 0.01 M sodium citrate buffer. The sections were subsequently immersed in 0.3% H 2 O 2 -methanol solution for 20 minutes. After PBS rinsing, the sections were blocked with 10% goat serum (36119ES03; Yeasen Biotechnology Co., Ltd.) for 10 minutes. The sections were then incubated with a rabbit antimouse antibody to NFKBIA (1:100; ab32518; Abcam Inc) or PBS as the NC, at 4°C overnight. The sections were incubated with horseradish peroxidase (HRP)-labelled goat anti-rabbit to IgG (1:1000; ab6721; Abcam) for 30 minutes. Development with 3, 3′-diaminobenzidine (DAB; P0203; Beyotime) was performed for 5 minutes. After counterstaining with haematoxylin for 3 minutes, the sealed sections were observed under a fluorescence microscope.
Five randomly selected fields of each tissue were observed to calculate the mean optical density (OD) using Image-Pro 7.0 software.

| Reverse transcription quantitative polymerase chain reaction (RT-qPCR)
The total RNA was extracted from the myocardial tissues and cells using a TRIzol Kit (15596-026; Invitrogen). The concentration and purity of extracted RNA were detected using a nucleic acid protein determinator (BioPhotometer D30, Eppendorf). The RNAs were reversely transcribed into cDNA by a Reverse Transcription Kit (K1621; Fermentas). The primers of the mouse ATP2B1-AS1, NFKBIA, NF-κBp50, NF-κBp65, IKKβ and β-actin (Table 1) were designed and synthesized by Shanghai Genechem Co., Ltd. The mRNA levels of the genes were determined by a fluorescent quantitative PCR kit (Takara). The qPCR was performed using an ABI 7500 PCR instrument (ABI). Using β-actin as an internal reference, 2 -ΔΔCT was used to calculate the relative target gene expression as follows:

| Western blot analysis
An amount of 50 mg myocardial tissue samples were extracted and lysed in protein lysis buffer (R0010; Solarbio), and centrifuged at 3000 g. After an ice bath for 30 minutes, the supernatant obtained by centrifugation at 5000 g at 4°C for 15 minutes were harvested for further use. The protein concentration was determined using a bicinchoninic acid protein assay kit (23225; Pierce) and adjusted to 1 μg/ μL. The proteins were separated with 10% sodium dodecyl sulphatepolyacrylamide gel electrophoresis (P1200; Solarbio). Subsequently, the proteins were transferred onto the polyvinylidene fluoride membranes (HVLP04700; Millipore). The membranes were blocked with 5% skim milk powder at ambient temperature for 2 hours. The membranes were incubated with primary antibodies, including rabbit antibodies to NFKBIA (1:1000, ab32518), NF-κBp50 (1:1000, ab32360), NF-κBp65 (1:50 000, ab32536), IKKβ (1:1000, ab32135) and β-actin (1:1000, ab8227) at 4°C overnight. HRP-labelled goat anti-rabbit IgG (1:2000, ab6721) served as secondary antibody for a 2-hours incubation. Each step was followed with Tris-buffered saline with Tween 20 (TBST) washing. All antibodies were purchased from Abcam Inc. After the DAB staining, the proteins were photographed using a gel imager (Gel Doc XR, Bio-Rad). β-actin was utilized as internal control. The experiment procedures were also suitable for the measurement of cellular proteins.

| Isolation of adult mouse cardiomyocytes
The adult mouse cardiomyocytes were isolated using the method outlined in a previous study. 25 In details, the mice were intraperitoneally injected with heparin and killed by cervical dislocation, followed by heart extraction. The heart was rinsed with Joklik's modified minimal essential medium, and tubes were inserted into the aorta. After perfusion at 37°C, the aorta was rinsed for 5 minutes, followed by circulatory perfusion with 40 mL digestive enzyme solution. After centrifugation at 1000 g for 10 minutes, the precipitation (cells) was resuspended with M199 culture medium containing 10% foetal bovine serum (FBS) and cultured in a 5% CO 2 incubator.

| Isolation of neonatal mouse cardiomyocytes
The neonatal mouse cardiomyocytes were isolated as previously described. 26 In brief, hearts of 1-day-old neonatal mice (aged 1 day) were detached, and the myocardial tissues were extracted. The tissues were minced into 1 mm 3 tissue blocks and detached with 0.1% collagenase I and 0.01% trypsin in a 37°C water bath for 80 minutes.

| Isolation of myocardial fibroblasts
The mouse hearts were extracted, and the ventricle was minced into CO 2 . Then, the cells were incubated with 10 mL DMEM/F12 containing 100 mL/L FBS, and the medium was renewed every 3 days.

| Isolation of endothelial cells
The mice were killed by cervical dislocation, immersed in 75% ethanol for disinfection and fixed on an operation table. The pleuroperitoneal cavity was exposed through thoracolaparotomy, and the aorta

| Isolation of peripheral blood mononuclear cells (PBMCs)
The cardiac valves of the mice were punctured to collect 0.5 ~ 1 mL blood. Anticoagulant blood was uniformly mixed with the same volume of Royal Park Memorial Institute 1640. The sample was added with lymphocyte separation medium and centrifuged at 600 g for 30 minutes. The monocyte layer was removed. After cell suspension, the cells were seeded in a 24-well plate at 2 × 10 6 cells/well and cultured at 37°C with 5% CO 2 for 2 ~ 4 hours. The non-adherent cells were discarded. Finally, the purified PBMCs were obtained through the adherent method. 27

| Cardiac troponin T (cTNT) fluorescent staining
Cardiomyocytes were seeded in a 24-well plate at the density of 1 × 10 5 cells/well. The cells were incubated at 37°C for 12 hours.

| Construction of the overexpression and interference plasmids
According to the known transcription sequences in the GenBank database, the full-length sequences of mouse ATP2B1-AS1 and NFKBIA, small interfering RNA (siRNA) sequences

| Cell transfection and grouping
The cardiomyocytes at passage 3 were grouped as follows:

| Cell transfection efficiency determination
Cardiomyocytes were isolated from the mice and cultured in vitro. The cells were seeded in a 6-well plate with M199 medium containing 10% ( Figure S1) showed that the primary cardiomyocytes from the mice were well-transfected, and the positive rate was 68.3%.

| Dual-luciferase reporter gene assay
Human

| Flow cytometry
At 48 hours after transfection, the cells detached with 0.25% trypsin were harvested and centrifuged at 1000 g for 5 minutes.
Subsequently, the cells were centrifuged again at 1000 g for 5 minutes and fixed with pre-cooled 70% ethanol at 4°C overnight, and centrifuged at 1000 g for 5 minutes. Then, the cells were incubated with 10 µL RNase at 37°C for 5 minutes and

| Enzyme-linked immunosorbent assay (ELISA)
Cardiomyocytes were seeded in 96-well plates. When the cells reached 70% confluence, the cell supernatant was harvested.

| Statistical analysis
All statistical analyses were performed with SPSS 21.0 software (IBM Corp.). The measurement data were expressed as the mean ± standard deviation. Comparisons between two groups were analysed using t test, whereas comparisons among multiple groups were performed using one-way analysis of variance. The enumeration data were presented as a ratio or percentage, and the comparisons were analysed by a chi-square test. P < .05 was considered as statistically significant.

| LINC00936 is selected as study subject
Based on the computer retrieval results from GEO database, we found that there were four MI-related data sets (GSE66360, GSE48060, GSE65705 and GSE97320). The GSE48060 data set without differentially expressed lncRNAs and the GSE65705 data set with only two non-MI control samples were excluded, while GSE66360 data set analysis revealed that LINC00936 showed increased expression in a largest fold in MI as compared to counterpart ( Figure 1A). Based on the GSE97320 data analysis, the expression of LINC00936 was also remarkably higher in the patients with MI when compared to the healthy controls ( Figure 1B). Therefore, LINC00936 was selected as the subject in the present study.

| NFKBIA is regulated by LINC00936 and implicated in the NF-κB signalling pathway
From the MEM website, four genes were obtained as putative targets of LINC00936. In addition, KEGG enrichment analyses in the WebGestalt website revealed that NFKBIA was implicated in the NF-κB signalling pathway ( Figure 1C). Among these 4 genes, NFKBIA was not only regulated by LINC00936, but also a central element in the NF-κB signalling pathway. NFKBIA played protective roles in myocardial injury by relieving inflammation through the inhibition of the NF-κB signalling pathway (Figure 2). In addition, the information retrieval of these 4 genes in MI revealed that the involvement of CXCL8, CD44 and JUNB in the functional mechanism of MI has been reported, 28

| Mouse ATP2B1-AS1 is evolutionarily conserved in mouse and human
The full-length nucleotide sequence of mouse ATP2B1-AS1 was edu/) revealed that it was mainly located in the mouse chromosome 10 ( Figure 3B). These results indicated that mouse ATP2B1-AS1 was evolutionarily conserved in both mouse and humans.

| The MI mouse model is successfully established
Electrocardiogram was used to examine the ST-segment elevation in the mouse model with MI. The results showed significant STsegment elevation and significant increases in amplitude of T-wave and lead II of ECG ( Figure 4A). Twenty-four and 48 hours after surgery, the UCG test was performed to evaluate cardiac function to ensure the induction of MI ( Figure 4B). Meanwhile, LVEDD, LVFS and LVESD were assessed before and after surgery and ejection fraction (EF), fractional shortening (FS) was evaluated. The results showed that EF, FS and left ventricular systolic diameter were remarkably reduced 48 hours after surgery. In the sham group, no statistically significant differences between before and after surgery were observed in LVEDD, LVESD, or LVFS. However, post-operative LVEDD and LVESD were increased in the MI group, compared with the pre-operative LVEDD and LVESD. Additionally, the ventricular anterior wall became thinner, and LVFS was reduced after operation (

| LINC00936 is highly expressed, NFKBIA is poorly expressed and the NF-κB signalling pathway is activated in the successful MI mouse model
Initially, morphology changes of cardiomyocytes were observed in the mice from both sham and MI groups. The results ( Figure S2A) showed that 2 weeks after MI, there was no infarction in the mice in the sham group, while a greyish-white area was obviously ob-

| Mouse ATP2B1-AS1 is highly expressed in myocardial fibroblasts and cardiomyocytes in MI mice
RT-qPCR was employed to analyse the mouse ATP2B1-AS1 expression in cardiomyocytes, myocardial fibroblasts, endothelial cells and PBMCs from heart tissues of MI mice. The results Figure S3) demonstrated that mouse ATP2B1-AS1 expression was higher in the myocardial fibroblasts and cardiomyocytes, and partial expression was detected in the endothelial cells and PBMCs.

| Cardiomyocytes are successfully isolated from neonatal mice
The morphology of cardiomyocytes was observed and identified under a microscope. The results ( Figure S4) showed that the cardiomyocytes were initially oval-shaped, began to extend once the cells attached, forming a polygonal or spindle shape with a good refractive index. To confirm that the isolated cells were mouse cardiomyocytes, we used a neonatal mouse and observed the cTNT positive rate of cells isolated and cultured in vitro under a fluorescence microscope. The results revealed that the isolated cells emitted strong green fluorescence signals, suggesting that these isolated cells were cardiomyocytes.

| siRNA and overexpression plasmids are successfully delivered
The transfection efficiency of the siRNAs and overexpression plasmids in the mice was determined by RT-qPCR. The interference effect of siRNA-mouse ATP2B1-AS1-1 was the best among the three types of siRNA-mouse ATP2B1-AS1 ( Figure S5A), while siRNA-NFKBIA-3 was the best among the three types of siRNA-NFKBI ( Figure S5B). When compared with the NC group, an increased ATP2B1-AS1 expression was observed in the mouse ATP2B1-AS1 vector group but a down-regulated ATP2B1-AS1 expression observed in the siRNA-mouse ATP2B1-AS1 group ( Figure S5C), while the mRNA level of NFKBIA was obviously increased in the NFKBIA vector group but remarkably decreased in the siRNA-NFKBIA group (P < .05, Figure S5D), suggestive of successful transfection.

| Mouse ATP2B1-AS1 restrains the expression of NFKBIA and activates the NF-κB signalling pathway
The expression of ATP2B1-AS1 and the mRNA and protein levels of NFKBIA and NF-κB signalling pathway-related proteins in the transfected cardiomyocytes were determined by RT-qPCR ( Figure   S7A) and Western blot analyses ( Figure S7B-C). When compared with the blank and NC groups, the mouse ATP2B1-AS1 vector group and mouse ATP2B1-AS1 vector + NFKBIA vector group had significantly increased the expression of mouse ATP2B1-AS1 (P < .05), while the siRNA-mouse ATP2B1-AS1 group and siRNAmouse ATP2B1-AS1 + siRNA-NFKBIA group had decreased expression of mouse ATP2B1-AS1 (P < .05). Moreover, the NFKBIA vector group and siRNA-mouse ATP2B1-AS1 group had increased mRNA and protein levels of NFKBIA (P < .05), but the mouse ATP2B1-AS1 vector group and siRNA-NFKBIA group had decreased the mRNA and protein level of NFKBIA vs the blank and NC groups (P < .05). Consequently, either ATP2B1-AS1 overexpression or siRNA-mediated silencing of NFKBIA resulted in increased mRNA and protein levels of NF-κBp50, NF-κBp65 and IKKβ, but these parameters were decreased by either NFKBIA overexpression or siRNA-mediated silencing of ATP2B1-AS1 (P < .05). In contrast to the mouse ATP2B1-AS1 vector group, the mouse ATP2B1-AS1

F I G U R E 4
The successful establishment of MI mice is confirmed by ECG and UCG test. A, ECG of MI mice and sham-operated mice after surgery (the excitation and repolarization width is 100 ms; sensitivity is 10 mm/ mV; and chart speed is 50 mm/s); B, representative M-mode images for both sham-operated and MI mice by UCG. ECG, electrocardiograph; MI, myocardial infarction; UCG, ultrasonic cardiogram

| Mouse ATP2B1-AS1 suppresses, while NFKBIA promotes cardiomyocyte proliferation
The viability of the transfected cardiomyocytes was measured by MTT. As shown in Figure  As shown in Figure S8B, the cardiomyocytes were marked by cTNT, the viable cells were marked by ki67, and the cell nucleus

| Mouse ATP2B1-AS1 enhances, while NFKBIA inhibits cardiomyocyte apoptosis
The cell cycle distribution ( Figure S9A) and apoptosis ( Figure S9B) of the transfected cardiomyocytes were detected by flow cytometry.
When compared with the blank and NC groups, the proportion of cells in the G1 phase and the apoptosis rate were progressively increased, but the proportion of cells in the S phase was decreased in the mouse ATP2B1-AS1 vector and siRNA-NFKBIA groups (P < .05).
By contrast, the proportion of cells in the G1 phase and the apoptosis rate were decreased, and the proportion of cells in the S phase was increased in the siRNA-mouse ATP2B1-AS1 and NFKBIA vector groups (P < .05). When compared with the mouse ATP2B1-AS1 vector group, the mouse ATP2B1-AS1 vector + NFKBIA vector group showed reduced proportion of cells in the G1 phase and apoptosis rate but an increased proportion of cells in the S phase (P < .05).
When compared with the siRNA-mouse ATP2B1-AS1 group, the siRNA-mouse ATP2B1-AS1 + siRNA-NFKBIA group showed increased proportion of cells in the G1 phase and apoptosis rate but a decreased proportion of cells in the S phase (P < .05). These results indicated that mouse ATP2B1-AS1 promoted cell apoptosis, but NFKBIA inhibited cell apoptosis, and the overexpression of NFKBIA could reverse apoptosis which induced by enhancement of mouse ATP2B1-AS1 in cardiomyocytes.

| Mouse ATP2B1-AS1 promotes, while NFKBIA inhibits the release of inflammatory cytokines
The levels of inflammatory cytokines in the transfected cardiomyocytes were determined by ELISA. When compared with the blank and NC groups, the mouse ATP2B1-AS1 vector and siRNA-NFKBIA   Table 3). These results indicated that mouse ATP2B1-AS1 promoted the release of inflammatory cytokines, but NFKBIA suppressed the release of inflammatory cytokines, and the overexpression of NFKBIA could reverse the up-regulation of inflammatory cytokines caused by the overexpression of mouse ATP2B1-AS1.

| D ISCUSS I ON
Although treatment and prevention of MI have achieved great progress in the past few years, patients with MI still face a modest risk of heart failure due to the failed compensation of apoptotic cardiomyocytes. 8 The whole-genome transcriptome analysis has showed that many cardiac-specific lncRNAs involve in many unique functions and regulations related to myocardial remodelling, myocardial regeneration and heart function. 34 This study was conducted to investigate the role of mouse ATP2B1-AS1 in MI based on its related bioinformatics information. The results conclusively suggested that the overexpression of NFKBIA induced by silencing of mouse ATP2B1-AS1 may protect against MI by inhibiting the NF-κB signalling pathway in mice.
Initially, poor expression of NFKBIA but high expression of mouse ATP2B1-AS1 was witnessed in MI. It has indicated that lncRNAs not only exert a great influence on various pathobiologies of human diseases, but also regulate gene expression at the epigenetic, transcriptional and post-transcriptional levels. 35 However, only a few lncRNAs have been reported to be involved in the cardiovascular system. 36 LncRNA metastasis-associated lung adenocarcinoma transcript 1 (MALAT1) has been found to be up-regulated in MI. 9 Differentially expressed lnRNAs are determined in MI and co-expressed with differentially expressed mRNAs, and their interactions are implicated in pathogenesis mechanism of MI. 37 Silencing of ATP2B1-AS1 can suppress renal interstitial fibrosis and oxidative stress in chronic renal failure. 10 In this study, mouse ATP2B1-AS1 silencing was suggested to inhibit the cardiomyocyte apoptosis and inflammation, suggesting a potential of ATP2B1-AS1 in protection against MI. In this study, based on a target prediction programme and the dual-luciferase activity determination, we found that mouse ATP2B1-AS1 targeted NFKBIA. In addition, a reduction in MI susceptibility has been shown to be related to the −94 ins/del ATTG NFKB1 gene variant. 38 We speculated that ATP2B1-AS1 might target NFKBIA to play a regulatory role in MI.
In addition, we found that mouse ATP2B1-AS1 silencing or NFKBIA overexpression resulted in reduced levels of NF-κBp50, NF-κBp65 and IKKβ, suggestive of blockade of the NF-κB signalling pathway.
The activation of NF-κB was triggered by IκBα degradation via phosphorylation by the IκB kinase complex. 39 NF-κB activation has been found in other different heart diseases, including cardiac hypertrophy, diabetic cardiomyopathy, myocardial infarction, ischaemia-reperfusion injury and heart failure. 40 The signalling pathways that mediate NF-κB activation could be divided into canonical pathways (mainly p50-RelA) and non-canonical (mainly p52-RelB) pathways. 41 The canonical pathway is related to a great variety of stimuli, including both endogenous and exogenous ligands and physical and chemical stress plethora. 42 The activation of IKK could result in the phosphorylation of IκBα at two N-terminal serines to trigger its proteasomal degradation and ubiquitination, thus causing the nuclear translocation of NF-κB complexes, including p50/RelA and p50/c-Rel dimers. 43 The non-canonical NF-κB pathway mainly relies on p52/RelB NF-κB complex activation by inducing the processing of p100, which is a molecule that acts as both an inhibitor of RelB and precursor of p52. 44 The disruption of the NF-κB signalling pathway contributes to reduced TA B L E 3 Expression of inflammatory cytokines in transfected myocardial cells in different groups after transfection (pg/mL) post-infarct myocardial remodelling. 45 In addition to impairing the function of NF-κB by inhibiting its binding to DNA, NFKBIA also suppresses NF-κB by masking the nuclear localization signals of NF-κB protein and allows it to remain inactive in the cytoplasm. 11 Our findings mainly suggested that ATP2B1-AS1 targeted NFKBIA and consequently activated the NF-κB signalling pathway.
Of crucial importance, this study demonstrated that NFKBIA overexpression restrained the cardiomyocyte apoptosis and the production of inflammatory cytokines, while enhancing cardiomyocyte proliferation through the blockade of the NF-κB signalling pathway. NF-κB most likely acts as a key mediator of immune and inflammatory responses and is involved in stress responses as well as the regulation of cell proliferation and apoptosis. 46 Another study has revealed that the reduced NF-κB activity is resulted from the overexpression of IkB could attenuate post-infarct remodelling and improve the cardiac function. 47 Additionally, NFKBIA has been demonstrated to be associated with altered NF-κB activities, thus mediating carcinogenesis in nasopharyngeal carcinoma. 48 A published article has demonstrated that mouse ATP2B1-AS1 overexpression significantly inhibits multiple myeloma cell growth and has a negative impact on genes involved in the mTOR signalling pathway. 49 Regarding our results, NFKBIA could reverse both the inhibition of cardiomyocyte viability and the up-regulation of the inflammatory cytokines expression which was triggered by mouse ATP2B1-AS1.
In conclusion, this study provides evidence that the knocking down mouse ATP2B1-AS1 may protect against MI-induced cell apoptosis and inflammation by inhibiting the NF-κB pathway through the up-regulation of NFKBIA expression ( Figure S10). However, due to the limited time, space and study subjects, this study was not highly comprehensive for translation into clinical application. Extensive studies should be conducted to further investigate the multiple target sites, as well as the clinical target therapy related to mouse ATP2B1-AS1 in the future.

ACK N OWLED G EM ENTS
We would like to show our sincere appreciation to the reviewers for their critical comments regarding this article.

CO N FLI C T O F I NTE R E S T S
None.

AUTH O R CO NTR I B UTI O N
Kai-You Song, Xian-Zhao Zhang, Feng Li and Qing-Rong Ji designed the study. Kai-You Song collated the data, designed and developed the database, carried out data analyses and produced the initial draft of the manuscript. Xian-Zhao Zhang, Qing-Rong Ji and Feng Li contributed to drafting the manuscript. All authors have read and approved the final submitted manuscript.

DATA AVA I L A B I L I T Y S TAT E M E N T
The data that support the findings of this study are available from the corresponding author upon reasonable request.