Introduction
Myocardial infarction (MI) is one of the most common cardiovascular events worldwide [
1‐
3]. Although advances in interventional cardiology and pharmacological strategies have led to a decline in all-cause mortality from MI over the past few decades, MI remains one of the most common causes of morbidity and mortality all over the world [
2,
3]. MI is characterized as myocardial cell ischemia and hypoxia due to coronary artery occlusion, then, the inflammation and cell apoptosis was triggered [
4]. The onset of MI, hypoxia leads to cardiomyocytes damage and necrosis, the damaged cardiomyocytes release pro-inflammatory chemokines to recruit inflammatory cells to MI zone and clear necrotic cardiomyocyte [
4,
5]. However, the myocardial reperfusion following percutaneous coronary intervention (PCI) exacerbates the pro-inflammatory response and myocardial injury [
1].
A larger number of studies have demonstrated the release of damage-associated molecular patterns or DAMPs (such as ATP, mtDNA, RNA, and HMBGB1) induce a pro-inflammatory response following MI, which mediates cardiomyocyte death via cytokines, mitochondrial dysfunction and inflammasome formation [
4,
6,
7]. Therefore, it’s necessary to explore the mechanism of inflammation and apoptosis induced by myocardial hypoxia to find new targets for mitigating myocardial injury after MI. Previously studies showed the early growth response gene family (EGR) was play key roles in inflammation and apoptosis in physiology and pathophysiology [
8,
9]. In human, the EGR1 and EGR2 were highly expressed in cardiomyocytes, the EGR1 were demonstrated play important roles in myocardial injury via oxidative stress, ferroptosis and apoptosis signaling pathways in MI process [
9,
10], but the roles of EGR2 in MI remain unrevealed.
Early growth response 2 (EGR2) is one of the early growth response gene families, contains three cyc2-His2 zinc fingers binding to the same cognate GC-rich consensus DNA binding motif of 28–30 amino acids [
11]. EGR2 is expressed in many tissue and different cell types, and plays key roles in response to inflammation, apoptosis and tissue damage [
11]. In immune cells, study has demonstrated that EGR2 regulates the inflammatory responses of PD-1 high MP CD4 T cells and maintains their adaptive immune fitness [
12]. Meanwhile, the EGR2 expression in T cells is mediated through IFNγ/STAT1 and IL-6/STAT3 signaling pathway [
13]. EGR2 has been reported to directly activate the expression of pro-apoptotic proteins of Bcl-2 family, BNIP3L and BAK, which activates caspase families, such as caspase-3, caspase-8 and caspase-9 [
14]. In cancer, NFAT2 inhibits the growth of hepatocellular carcinoma by inducing EGR2 expression [
15]. In the MI process, the miR-150 plays a cardioprotective role by directly repressing EGR2 expression in cardiomyocytes [
16]. A recent study showed that the inhibition of long non-coding RNA MIAT ameliorates myocardial dysfunction induced by myocardial infarction via MIAT/miR-10a-5p/EGR2 axis [
17]. Although EGR2 is involved in inflammation and apoptosis, the function of EGR2 in the MI process remains unclear.
In the present study, we analyzed the gene expression in an animal model of MI and attempted to identify the hub genes and potential therapeutic targets. We downloaded the gene expression data for heart tissue of mouse with MI and sham control from Gene Expression Omnibus (GEO) database and screened the differentially expressed genes (DEGs). Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway functional enrichment analysis was performed to underlying the pathway changes of MI compared with sham controls. Protein–protein interaction (PPI) network analysis was used to find the potential hub-genes in MI, and the EGR2 was highlighted in the top20 Hub-genes. We then validated the expression and revealed the function of EGR2 in hypoxia induced human cardiomyocyte cell line. Our data suggested the EGR2 is a potential therapeutic target for the treatment of myocardial injury in MI.
Methods
Data sources
The datasets of GSE71906 was downloaded from the GEO database (
http://www.ncbi.nlm.nih.gov/geo/). A total of 12 samples were collected in the GSE71906 dataset, 6 samples from MI zone of mouse heart and 6 from normal sham control hearts, and all the samples were obtained post 8 h of surgery. GPL8321 platform was used for GSE71906 sequencing ([Mouse430A_2] Affymetrix Mouse Genome 430A 2.0 Array).
Data preprocessing of DEGs
The Uniform manifold approximation and projection (UMAP) plot and volcano plot were obtained by GEO2R (
https://www.ncbi.nlm.nih.gov/geo/geo2r). The dataset of GSE71906 was download with the CEL format, and the gene expression profiling analysis were preprocessed using Robust Multichip Average algorithm in the “affy” and “affyPLM” packages within Bioconductor (
http://www.bioconductor.org) by a R × 64 4.1.1 software (R Foundation for Statistical Computing, Vienna, Austria). After correcting for background, and performing quantile normalization, the DEGs analysis was performed with the 13,015 genes. 235 genes were met the adjusted p value < 0.05 and logFC > 1 (upregulated genes) or logFC < −1 (downregulated genes), and were considered as DEGs for the subsequent analysis, the unqualified genes were discarded.
GO analysis and KEGG pathway enrichment analysis
Gene Ontology (GO) analysis is a functional analysis associating DEGs with GO categories, and involve cell composition (CC), biological process (BP)and molecular function (MF). Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis offers biological pathways of DEGs genes related to diseases. The DEGs were used for GO and KEEG analysis by an online biological function database, Database for Annotation, Visualization and Integrated Discovery (DAVID,
http://david.ncifcrf.gov), The enrichment index with p < 0.05 was considered to be statistically significant. The enrichment plots were constructed by ImageGP.
Protein–protein interaction (PPI) network construction and hub genes identification
To explore possible protein–protein interaction network interactions, the DEGs were mapped to the Search Tool for the Retrieval of Interacting Genes/ Proteins database (STRING,
https://string-db.org/). The PPI pairs were extracted with a minimum required interaction score > 0.4. The interactions were download from STRING and used to construct a PPI network by Cytoscape software platform (3.9.0). The nodes with higher connectivity degree were considered important in maintaining the stability of the PPI network, therefore, the CytoHubba plugin for Cytoscape software was used to indentify the top20 hub genes.
Cell culture
The human cardiomyocyte cell line AC16 and HEK293T cell line were purchased from the American Type Culture Collection (ATCC). Cells were cultured in DMEM medium (Invitrogen, Carlsbad, USA) with 10% FBS (Invitrogen) under 5% CO2 at 37 °C. For hypoxia treatment, the AC16 cells were maintained in a hypoxic incubator (N2 94%, O2 1% and CO2 5%) for 12 h in medium deprived of serum and glucose.
Stable cell line construction
The full-length sequence of human EGR2 consensus CDS region was cloned and insert to pHAGE vector which containing a Flag tag to construct the EGR2 expression vector pHAGE-Flag-EGR2. The pHAGE-Flag-EGR2 or the empty pHAGE-Flag vector was co-transfected into HEK293T with lentivirus packaging plasmids, psPA × 2 (12260, Addgene) and pMD2.G (12259, Addgene) to obtain the lentivirus. Then, the lentivirus was used to infect AC16 cells with polybrene (10 μg/mL). 48 h later, the EGR2 stable overexpressed cell line were screened by 2 μg/mL puromycin (A1113803; Gibco, USA), and the expression was verified by Western blot analysis. Primers: Forward 5′-TCGGGTTTAAACGGATCCATGGCATGATCAACATTGAC-3′, Reverse 5′-GGGCCCTCTAGACTCGAGAGGTGTCCGGGTCCGAGAGG-3′.
EGR2-knockout cell line generation
The EGR2-Knockout cardiomyocyte were generated using a pX459 vector (62988, Addgene). The human EGR2-sgRNA was synthesized and cloned into pX459, then, the recombinated vector was transfected into AC16 cells. Homozygous EGR2-knockout cells were generated from a monoclonal line after puromycin selection. The target sequence for human EGR2 sequence was 5′-CAATCCGTAACTTTACCCTG-3′.
Reverse transcription-quantitative polymerase chain reaction (RT-qPCR)
Total RNA from AC16 was extracted using TRIzol reagent (Invitrogen, CA, USA) according to the manufacturer’s protocol. RNA was converted to cDNA using the HiScript II Q RT SuperMix for qPCR (+ gDNA wiper) (R223-01, Vazyme, China). qPCR was conducted using ChamQ SYBR qPCR Master Mix (#Q311-02, Vazyme, China). GAPDH were used as endogenous control, all samples were run in triplicate. The data analyses were performed with SDS 2.2.2 software. Primers: EGR2, Forward 5′-TCAACATTGACATGACTGGAGAG-3′, Reverse 5′-AGTGAAGGTCTGGTTTCTAGGT-3′; GAPDH, Forward 5′-GGAGCGAGATCCCTCCAAAAT-3′, Reverse 5′-GGCTGTTGTCATACTTCTCATGG-3′; IL-6, Forward 5′-ACTCACCTCTTCAGAACGAATTG-3′, Reverse 5′-CCATCTTTGGAAGGTTCAGGTTG-3′; IL-1b, Forward 5′-ATGATGGCTTATTACAGTGGCAA-3′, Reverse 5′-GTCGGAGATTCGTAGCTGGA-3′; CCL2, Forward 5′-CAGCCAGATGCAATCAATGCC-3′, Reverse 5′-TGGAATCCTGAACCCACTTCT-3′; TNF, Forward 5′-CCTCTCTCTAATCAGCCCTCTG-3′, Reverse 5′-GAGGACCTGGGAGTAGATGAG-3′; BAX, Forward 5′-CCCGAGAGGTCTTTTTCCGAG-3′, Reverse 5′-CCAGCCCATGATGGTTCTGAT-3′; BAD, Forward 5′-CCCAGAGTTTGAGCCGAGTG-3′, Reverse 5′-CCCATCCCTTCGTCGTCCT-3′; BCL2, Forward 5′-GGTGGGGTCATGTGTGTGG-3′, Reverse 5′-CGGTTCAGGTACTCAGTCATCC-3′.
Western blot assay
Total protein was extracted by RIPA lysis buffer (#P0013C, Beyotime Institute of Biotechnology, China) and quantified using a Pierce BCA Protein Assay Kit (#P0012S, Beyotime Institute of Biotechnology, China). The total protein was electrophoresed by 10% SDS-PAGE and transferred to PVDF membranes (#AR0136-04, BOSTER, China). The membranes were blocked in non-fat dry milk (~ 5%) for 60 min at room temperature and incubated with special primary antibodies at 4 °C overnight. After washing, the membranes were incubated with peroxidase-conjugated secondary antibody (anti-rabbit) at room temperature for 60 min. Protein expression levels were visualized using enhanced chemiluminescence kit (#P0020; Beyotime Institute of Biotechnology, China). Images were visualized with a ChemiDoc MP Imaging System (Bio-Rad, Hercules). Primary antibodies: EGR2 antibody, (#ab245228, dilution 1:1000, Abcam, UK); GAPDH antibody (#A19056, dilution 1:1000, ABclonal, China).
Statistical analysis
All data were presented as mean ± standard deviation (SD) of three independent experiments with the software GraphPad Prism 7. Significance was assessed by one-way ANOVA followed by Tukey’s test. All the statistics were performed in SPSS 22.0 (SPSS Inc., Chicago, Illinois, USA). A p value < 0.05 was considered statistically significant.
Discussion
MI is a common cause of mortality worldwide because of the irreversible damage to the non-renewable cardiomyocytes, and aggravates cardiac inflammation and cell apoptosis [
18]. However, the pathogenesis of MI remains poorly understood. To explore the changes of gene expression and identify the hub genes in MI, we screened and analyzed a microarray dataset from MI mice heart tissue.
Compared with the previous studies, our investigation provides new insights into the pathogenesis of MI.
Bennardo M et al. produced the GSE71906 dataset, the authors established the mice MI models within a 2-h time window either shortly after lights on or lights off, respectively, to observe the early remodeling response at 8 h after infarction [
19]. They found that the day-night dependence of gene expression and inflammatory responses in the remodeling murine heart post-myocardial infarction [
19]. In the present study, we explored global genetic changes and attempted to figure out the hub genes in mice after MI. We first identified the 235 significant DEGs between MI and sham control samples. Most of the top 20 upregulated genes were reported related to inflammation and apoptosis. For instance, IL6 is often used as an evaluative indicator for inflammatory response [
20] and there have been many studies of it related to MI [
21]. FOS is a subunit of activator protein-1 (AP-1) and plays an important role in inflammation and cell apoptosis [
22‐
24]. In addition, the AP-1 significantly increased in IM tissues [
25,
26] and involved in the process of cardiac injury after MI [
27]. We then conducted GO and KEGG pathway enrichment using the upregulated DEGs. As expected, the analysis showed that the pathways related to inflammation and cell apoptosis.
The PPI and hub-gene analysis exhibited the key genes in the MI process, and two members of the EGR family were highlighted. Many previous studies have revealed the function of EGR1 in the MI process.
Bhindi R et al
. found EGR1 is a key contributor to myocardial ischemia reperfusion injury [
28], and targeting EGR1 by DNA-zymes reduced the infarct size following myocardial ischemia reperfusion [
29]. Some scholars have pointed out that EGR1 is a key player in myocardial cell injury in MI process [
9], and it was supported by later research [
10,
30‐
32]. Although
Tang Y and
Cao X revealed the miRNA150/10a-5p-EGR2 axis play roles in MI [
16,
17], the function of EGR2 in MI remains unclear.
Previous study suggested EGR2 was a pro-apoptotic gene.
Unoki M and
Nakamura Y found that EGR2 induces apoptosis in a large proportion of these lines by altering the permeability of mitochondrial membranes, releasing cytochrome c and activating caspase-3/8/9 by directly transactivates expression of BNIP3L and BAK [
14]. In tumor, study have showed EGR2 knockdown promotes gastric cancer cell growth and inhibited their apoptosis [
33],
Wang J et al
. revealed the EGR2 mediated the function of NFAT2 in inhibition of the invasion and malignancy of hepatocellular carcinoma [
15].
Zeng T et al
. found the EGR2 was upregulated through lncRNA-AF113014-miR-20a axis and inhibits the proliferation of hepatocellular carcinoma cells [
34]. In Hirschsprung's disease, EGR2 may mediated the downregulated miR-140-5p to promote apoptosis in SH-SY5Y cells [
35]. In the present study, we generated the EGR2 overexpression and knockout cell lines based on AC16 cells and established the MI cell model in vitro. Our data showed the EGR2 was upregulated in myocarcytes induced by hypoxia, the excessively overexpressed EGR2 facilitates the hypoxia-induced pro-inflammatory and pro-apoptotic genes expression in myocarcytes. Meanwhile, these phenotypes were reversed in EGR2 knockout myocarcytes. Therefore, these data indicated the hub gene, EGR2, deteriorates cardiac injury by aggravating inflammation and cell apoptosis in the MI process. Thus, our study identified the role of EGR2 in myocardial apoptosis.
Inflammation post-MI has been the focus of cardiovascular research as it influences the remodeling process of the ischemic heart, which critically determines the clinical outcome of MI patients [
36]. As we know, inflammation is critical for initiation of the natural wound healing process, in the MI, an appropriate inflammatory response helps scar formation of heart tissue and recovery of cardiac function. However, it is commonly known that inflammation is a double-edged sword. Several studies have shown that high levels of inflammation post-MI leads to an increase of scar volume and lead to poor ventricular remodeling and deterioration of cardiac function [
36,
37]. Thus, controlling the intensity of the inflammatory response after MI is particularly important for the recovery of cardiac function.
Hausenloy et al. showed a multi-targeted approach and combining anti-inflammatory agents offering a better approach to reducing MI size in STEMI patients [
38].
Arslan et al. revealed anti-inflammation by TLR2 antibodies reduces MI size in both small and large animal AMI models [
39,
40]. What’s more, studies showed that genetic and pharmacological inhibition of the NLRP3 inflammasome may reduce MI size and prevent adverse LV remodeling [
41,
42]. In the present study, EGR2 knockout exhibited an anti-inflammation role in hypoxia-induced cardiomyocytes, thus, the EGR2 may offer a new potential target for controlling the inflammatory response post-MI. However, the effect of EGR2 on scar formation and myocardial fibrosis after MI should be focused in the future.
Publisher's Note
Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.