CircSAMD4A aggravates H/R‐induced cardiomyocyte apoptosis and inflammatory response by sponging miR‐138‐5p

Abstract Hypoxia/reoxygenation (H/R)‐induced myocardial cell injury is the main cause of acute myocardial infarction (AMI). Many proofs show that circular RNA plays an important role in the development of AMI. The purpose of this study was to investigate the role of circSAMD4A in H/R‐induced myocardial injury. The levels of circular SAMD4A (circSAMD4A) were detected in the heart tissues of AMI mice and H/R‐induced H9C2 cells, and the circSAMD4A was suppressed in AMI mice and H/R‐induced H9C2 cells to investigate its’ function in AMI. The levels of circSAMD4A and miR‐138‐5p were detected by real‐time quantitative PCR, and MTT assay was used to detect cell viability. TUNEL analysis and Annexin V‐FITC were used to determine apoptosis. The expression of Bcl‐2 and Bax proteins was detected by Western blot. IL‐1β, TNF‐α and IL‐6 were detected by ELISA kits. The study found that the levels of circSAMD4A were up‐regulated after H/R induction and inhibition of circSAMD4A expression would reduce the H/R‐induced apoptosis and inflammation. MiR‐138‐5p was down‐regulated in H/R‐induced H9C2 cells. circSAMD4A was a targeted regulator of miR‐138‐5p. CircSAMD4A inhibited the expression of miR‐138‐5p to promote H/R‐induced myocardial cell injury in vitro and vivo. In conclusion, CircSAMD4A can sponge miR‐138‐5p to promote H/R‐induced apoptosis and inflammatory response.

Non-coding RNA is a regulator of gene expression, including mi-croRNA, long non-coding RNA and circular RNA. 9 Previous studies found that long non-coding RNA ROR could promote H/R-induced cardiomyocytes apoptosis, which was associated with its increased phosphorylation of p38 and ERK1/2 and its combination with miR138 to up-regulate the expression of Mst1. 10,11 Recently, circular RNA has become increasingly prominent in the study of myocardial infarction.
Circular RNA can interact with specific microRNA to prevent the translation of microRNA, and this process is involved in biological processes such as cell proliferation, apoptosis, autophagy and pathological processes such as cardiovascular diseases, neurological diseases, cancer and other diseases. 3 Especially in cardiovascular diseases, the circular RNA Cdr1as inhibits miR-7a through PARP and SP1 and induces apoptosis of cardiomyocytes when myocardial infarction damage is aggravated. 12 Circular RNA MACF1 can regulate the miR-500b-5p/EMP1 pathway to inhibit the occurrence of AMI. 13 However, the role and mechanism of circSAMD4A in H/R-induced myocardial injury is not clear.
In this work, we first reported that circSAMD4A was raised in vivo and in vitro studies in AMI. Depressing the expression of circSAMD4A inhibited the apoptosis of cardiomyocytes and the expression of inflammatory cytokines, and we proved that circSAMD4A restrained the expression of miR-138-5p to promote cardiomyocytes apoptosis and inflammatory development in H/Rstimulated H9C2 cells and AMI mice. This finding may provide new ideas for the treatment of patients with AMI.

| Mice
The 30 male C57BL/6J mice used in this study were purchased from the Model Animal Research Institute of Nanchang University. They were completely randomly divided into a sham group (n = 6), AMI group (n = 6), AMI + si-NC group (n = 6), AMI + si-Circ group (n = 6) and AMI + si-Circ + anti-miR (n = 6). All animal studies meet the standards in the "Guidelines for Laboratory Animal Care and Use" published by the National Institutes of Health and are approved by the Animal Care and Use Committee.
In order to construct AMI mice model, firstly mice were anaesthetized by intraperitoneal injection of pentobarbital sodium (50 mg/kg, P3761, Sigma-Aldrich), then, mice were placed on the operating table, and connecting the ventilator after intubation and cut the left sternum of mice were cut to fully expose the heart. The left anterior descending coronary artery was ligated under a microscope to observe the discoloration of the myocardium and identify ischaemia. Adenovirus carrying shRNA against circSAMD4A was injected into mice by myocardial injection to construct a model of AMI + si-Circ group, and the corresponding control is AMI + si-NC group. Mice without ligation of the anterior descending branch of the left coronary artery were the sham group in this study. At 2 days after surgery, all mice were killed and hearts were collected and fixed with paraformaldehyde for subsequent studies.

| Cell culture and transfection
Rat cardiomyocyte H9C2 cells were cultured in DMEM (Gibco) supplemented with 10% FBS (Gibco). H9C2 cells in the control group were cultured at 37°C and 5% CO 2 . In order to construct a cell model of myocardial H/R injury, H9C2 cells were cultured in a hypoxic incubator (37°C, 95% N 2 and 5% CO 2 ) for 2 hours, and then, the cells were cultured with fresh medium and reoxidated for 4 hours at mixed gas environment (75% N 2 , 20% O 2 and 5% CO 2 ).
The circSAMD4A sequence was cloned into pcDNA3.1, and then, Lipofection 3000 reagent was used for cell transfection.
The Liposome 3000 reagent was used to transfect the miR-138-5p mimic and its negative control. The transfection time of cells without H/R treatment was 48 hours, but for the cells requiring H/R treatment, cells are treated with H/R 48 hours after transfection.

| Real-time quantitative PCR
Firstly, total RNA was extracted from cardiomyocytes or heart tissues with TRIzol reagent (Invitrogen), and then reversely transcribing total RNA into complementary DNA using a reverse transcription kit (TaKaRa). The expression of circSAMD4A

| Western blot
Proteins were extracted from cardiomyocytes or heart tissues with RIPA lysis buffer (Beyotime Biotechnology). Protein samples (60 μg) were separated by SDS-PAGE electrophoresis and transferred to PVDF membranes (Millipore). After blocking, the protein on the membrane was incubated with primary antibody Bcl-2 antibody (Cell Signaling Technology) and Bax antibody (Cell Signaling Technology) at 4°C overnight, and then incubated with HRP-conjugated secondary antibody the next day. Then FluorChemE imager (Alpha) was used for visualization, and the expression level of specific protein was normalized to GAPDH level.

| MTT
The cardiomyocytes were seeded in 96-well plates and incubated overnight at 37°C. Then, the cells were treated according to the instructions, 20 μL of MTT solution (5 mg/mL, Sigma-Aldrich) was added, incubated at 37°C for 4 hours. Then adding 100 μL of DMSO (Sigma-Aldrich) to each well. After the operation is completed, the absorbance was measured with a micro plate reader (BioTek Instrument) at 490 nm to determine the viability of the cells.

| ELISA analysis
The detection of IL-1β, TNF-α and IL-6 in cells was operated by ELISA kits (R&D Systems). The supernatants of cell culture medium were collected and then centrifuged (2458 g, 15 minutes). Following the instructions, each experiment was done for 3 times.

| Detection of apoptosis level
To investigate apoptosis, we applied the Annexin V-FITC ap- µL PA for 10 minutes under dark conditions at room temperature. Finally, the cells were detected by FACSCanto II flow cytometry (BD Biosciences).

| TUNEL assay
Apoptosis analysis of cardiomyocytes in heart slices was achieved using terminal deoxynucleotide transferase-mediated dUTP nick end labelling (TUNEL) staining method by in situ cell death detection kit (RocheFrance).
For the heart slices fixed with paraformaldehyde, firstly permeating them with 0.1% Triton X-100 for 2 minutes, then adding the TUNEL reaction mixture and incubating for 1 hour to stain the apoptotic cells. All nuclei were stained with DAPI. The three random fields were observed in each sample under the microscope, and the percentage of the number of positive apoptotic cells in the total number of nuclei were calculated.

| Dual-luciferase reporter detection
The RNAInter online tool (http://www.rna-socie ty.org/raid/search.html) was used to predict the binding site of the circSAMD4A and miR-138-5p.
The circSAMD4A fragment was cloned into pmirGLO vector (Promega) F I G U R E 1 A, Expression of circSAMD4A in sham group (n = 6), AMI group (n = 6) and AMI + si-Circ group (n = 6). B, The percentage of apoptosis, n = 6. C, Typical TUNEL staining that shows cardiac cell apoptosis, n = 6 to generate a reporter vector wild-type circSAMD4A (WT-circSAMD4A).
In order to generate the mutant circSAMD4A reporter vector (MUT-circSAMD4A), a site mutation is performed. Then, the vectors WT-circSAMD4A or MUT-circSAMD4A were co-transfected with miR-138-5p mimics and NC for 48 hours. The cells were lysed, the luciferase activity was detected using the Dual-Luciferase Reporter Assay Kit (Promega), and the firefly luciferase activity was normalized to Renilla luciferase activity.

| Data analysis
All data were analysed with GraphPad Prism 7.04 software (GraphPad Software, Inc), and all experiments were repeated 3 times independently. The experimental results are expressed in mean ± SD, and P < .05 indicates a significant difference between the data.

| Increased levels of circSAMD4A in cardiomyocytes in AMI
To clarify the role of circSAMD4A in AMI, this study firstly estab-  Figure 1A, the levels of circ-SAMD4A were significantly higher in AMI group than sham group.
There is no evident difference in circSAMD4A expression levels between AMI group and AMI group transfected with si-NC. However, the circSAMD4A concentration was significantly decreased after transfected si-CircSAMD4A with AMI group. The results showed that circSAMD4A was overexpressed in AMI.

| circSAMD4A can promote apoptosis of cardiomyocytes in vivo
In order to explore the function of circSAMD4A in AMI, we observed the apoptosis of cells by TUNEL staining in this study. Compared with sham group, the numbers of cardiomyocytes apoptosis in AMI group were significantly increased, but inhibiting the circSAMD4A in mice with AMI significantly reduced the numbers of cardiomyocyte apoptosis, as shown in Figure 1B. And it was found that the numbers of TUNEL staining positive cells in AMI group were higher than sham group, and the numbers of TUNEL staining positive cells in AMI + si-Circ group were lower than AMI + si-NC group, as shown in Figure 1C. These data indicated that circSAMD4A could regulate cardiomyocytes apoptosis.

| Inhibiting circSAMD4A suppresses H/Rinduced cardiomyocyte apoptosis and inflammatory response in H9C2 cells
In

| CircSAMD4A targets miR-138-5p in H9C2 cells
The bioinformatic method predicts that circSAMD4A binds to miR-138-5p. The binding sequence was shown in Figure 3A. And dual-luciferase report test was used to verify the hypothesis, and it was found that the relative luciferase activity of miR-138-5p mimic was significantly reduced in WT-circSAMD4A compared with the NC group, while there was no significant change in relative luciferase activity in MUT-circSAMD4A, as shown in

| CircSAMD4A promotes cardiomyocyte apoptosis and inflammatory response by inhibiting the expression of miR-138-5p in vitro
In order to study the mechanism of circSAMD4A on cardiomyocytes, as shown in Figure 3C, we observed that the levels of miR-

| CircSAMD4A suppresses miR-138-5p to induce cardiomyocyte apoptosis in vivo
Else, we conducted the vivo experiments to validate the interactions between circSAMD4A and miR-138-5p. As shown in Figure 5A,B, the relative miR-138-5p expression was significantly up-regulated but the cell apoptosis ratio was clearly decreased after knocking out the circSAMD4A in AMI mice, which further treated with anti-miR evidently could decrease the concentration of miR-138-5p and augment the ability of cell apoptosis. The similar apoptosis results were also shown in Figure 5C by TUNEL staining. The findings showed that circSAMD4A could target miR-138-5p to support cardiomyocyte apoptosis in AMI mice.

| D ISCUSS I ON
AMI is a type of disease that causes serious damage to human life and health, and it imposes a huge economic burden on the development of individuals and society. 14 It is important to elucidate the molecular mechanism in AMI development.
In this study, we tested the level of circSAMD4A in AMI mice and H/R-stimulated H9C2 cells. 138-5p is included in these microRNAs. 6 Wang et al 16 17 Previous studies reported that the levels of IL-1β were closely related with the impaired myocardial function, and IL-1β might promote adverse cardiac remodelling during the acute phases of myocardial function and inhibit systolic/diastolic function F I G U R E 5 A, The miR-138-5p expression in AMI + si-NC (n = 6), AMI + si-Circ group (n = 6) and AMI + si-Circ + anti-miR group (n = 6). B, The cell apoptosis in AMI + si-NC (n = 6), AMI + si-Circ group (n = 6) and AMI + si-Circ + anti-miR group (n = 6). C, Typical TUNEL staining that shows cardiac cell apoptosis in AMI mice (bar = 50 µm) during the chronic phases of myocardial function. 18,19 The levels of TNF-α increased in the local infarct myocardium, which contributed to AMI and cause myocardial cell apoptosis. 8,20 IL-6 contributed to the development of infarct size during the early stage of AMI ischaemia-reperfusion injury. 21,22 In the present study, we found that the concentration of IL-1β, TNF-α and IL-6 was significantly different between control group and H/R-induced H9C2 cells, and inhibiting the expression of circSAMD4A in H/R-stimulated H9C2 cells would ameliorate changed levels of IL-1β, TNF-α and IL-6.
After further research, it was found that the circSAMD4A could Overall, we demonstrated that the circSAMD4A can regulate the expression of miR-138-5p to participate in H/R-induced cardiomyocyte apoptosis and inflammatory response. CircSAMD4A may be a new direction to explore the treatment of patients with AMI.

ACK N OWLED G EM ENTS
Mice used in this study were provided by the Model Animal Research Institute of Nanchang University. All laboratory members will be thanked to discuss this study.

CO N FLI C T O F I NTE R E S T
The authors confirm that there are no conflicts of interest.