Abstract
Background: Type A aortic dissection is a fatal disease. However, the role of endoplasmic reticulum stress-related genes (ERSRGs) in type A aortic dissection has not yet been fully clarified.
Methods: Differentially expressed genes in the aorta of type A aortic dissection patients were analyzed based on the GSE52093 database. The ERSRGs were downloaded from the GeneCards website. Functional enrichment analysis and protein–protein interaction analysis were performed on the acquired differentially expressed ERSRGs. The mRNA expression of the 5 top key differentially expressed ERSRGs was further explored in GSE153434 and clinical samples. Immune infiltration correlation analysis was performed on the validated key genes. Finally, we constructed regulatory networks of transcription factors, miRNAs, and chemicals.
Results: Twelve differentially expressed ERSRGs were identified, of which 8 genes were downregulated and 4 genes were upregulated. GeneMANIA was adopted to analyze these genes and their interacting proteins, and the results showed that the main function was calcium ion transport. Four key genes, ACTC1, CASQ2, SPP1, and REEP1, were validated in GSE153434 and clinical samples. The area under the ROC curve values for ACTC1, CASQ2, SPP1, and REEP1 were 0.92, 0.96, 0.89, and 1.00, respectively. ACTC1 and REEP1 correlated with multiple immune cells. Many transcription factors, microRNAs, and chemicals were identified with the potential to regulate these 4 key genes.
Conclusion: In this study, we identified 12 differentially expressed ERSRGs by analyzing the Gene Expression Omnibus database. Four key genes may influence the development of type A aortic dissection by regulating endoplasmic reticulum stress. These results expand our understanding of type A aortic dissection, and the 4 key genes are expected to be diagnostic markers and potential therapeutic targets.
Highlights
- The role of endoplasmic reticulum stress-associated genes (ERSRGs) in TAAD has not yet been fully clarified.
- Twelve differentially expressed ERSRGs in TAAD were identified by analyzing the GEO database.
- Four key genes (ACTC1, CASQ2, SPP1, and REEP1) were validated in GSE153434 and clinical samples, which are expected to be diagnostic markers and potential therapeutic targets of TAAD.
Introduction
Aortic dissection (AD) is a fatal disease caused by the rupture of the aortic intima, which allows high-speed blood from the aortic lumen to enter the aortic wall through the rupture of the intima, resulting in tearing of the middle layer of the aortic wall.1 The annual incidence rate of AD is 3.5-6 cases/100 000 people, but the mortality rate of AD is very high, often leading to sudden death.2 According to the Stanford classification method, AD can be divided into type A aortic dissection (TAAD) and type B aortic dissection (TBAD).3 Currently, the main treatment for TAAD is emergency surgery with artificial vessel replacement, whereas the main treatment for TBAD is interventional thoracic endovascular aortic repair (TEVAR).4 Mortality is higher in the cases of TAAD, especially in areas with poorer health care.5,
The endoplasmic reticulum (ER) controls the quality of the proteins it produces and oversees maintaining protein homeostasis, which is critical for cell survival. Protein imbalances can lead to a variety of diseases, including metabolic, oncological, neurodegenerative, and cardiovascular diseases.7,
In this study, we analyzed differentially expressed ERSRGs in TAAD based on the GEO database. Four key ERSRGs were identified and validated by quantitative reverse transcription PCR (qRT-PCR). The results of this study expand our understanding of TAAD and provide new potential diagnostic markers and potential therapeutic targets.
Methods
Artificial Intelligence Statement
We confirmed that we did not use artificial intelligence or any assisted technologies such as large language models in this paper.
Data Source
The Gene Expression Omnibus (GEO) database (
Analysis of Differentially Expressed Genes
We downloaded the dataset from the GEO database through the R software GEO query package using the limma package to standardize the data. The results of the analysis of differentially expressed genes (DEGs) were visualized by volcano plots using the ggplot2 R package,19 with the setting of |log fold change (FC)| >1 and an adjusted
Identification of Differentially Expressed Endoplasmic Reticulum Stress-Related Genes
Following the methods reported in the previous literature,21 we downloaded the ERSRGs from the GeneCards website, obtaining 1350 genes. These genes were intersected with differential genes obtained by GSE52093 to obtain differentially expressed ERSRGs in TAAD. Then, we plotted Venn plots.
Functional Enrichment Analysis and Protein–Protein Interaction
To explore the functions and metabolic pathways of differentially expressed ERSRGs, we conducted functional enrichment analysis using Metascape (version3.5) (
Immune Infiltration Analysis
Based on the GSE153434 dataset, immune infiltration in aortic wall tissue of TAAD patients was calculated by applying the R software CIBERSORT [v1.03] R package. Twenty-two markers of immune cells were provided by the CIBERSORTx website(
Construction of the Regulatory Network
Transcription factors (TFs) and microRNAs (miRNAs) have inhibitory and regulatory effects on target genes. Based on the NetworkAnalyst online database (
Clinical Specimen Collection and Quantitative Reverse Transcription Polymerase Chain Reaction Validation
The inclusion criteria for TAAD cases were as follows: clearly diagnosed TAAD by aortic CTA angiography and underwent aortic vascular replacement surgery, aged over 18 years old. The exclusion criteria were as follows: Combined connective tissue disease, severe infection, or malignant tumor. Inclusion criteria for coronary artery bypass grafting cases: those who underwent coronary artery bypass grafting surgery due to coronary heart disease. Exclusion criteria: combined connective tissue disease, severe infection, or malignant tumor. This study included a total of 24 clinical specimens of the ascending aorta to further validate the differentially expressed ERSRGs: 12 TAAD patients who underwent aortic vascular replacement (AD group) and 12 coronary artery bypass grafting patients (aortic wall tissue at the site of ascending aortic perforation for graft vessel) as a non-AD control group (NAD group). The study was performed in accordance with the Declaration of Helsinki. All study protocols and experiments were approved by the Ethics Committee of The First Affiliated Hospital of Wannan Medical College, and prior informed consent was signed by the participants.
Total RNA was extracted from aortic wall tissue using TRIzol reagent (Invitrogen Company), and mRNA reverse transcription was performed using Vazyme’s HiScript II Q RTsuperpMix for qPCR (+gDNA wiper) kit (R223-01). qRT-PCR was performed using the AceQ qPCR SYBR Green Master Mix kit (Q111-02103), with the primer sequences shown in
Statistical Analysis
Continuous variables are presented as the mean and standard deviation. The statistical analysis of bioinformatics data in this study was conducted using R software (
Results
Analysis of Differentially Expressed Genes
The analysis was based on the GSE52093 dataset. The normalized sample date is shown in
Identification of Differentially Expressed Endoplasmic Reticulum Stress-Related Genes
Twelve differentially expressed ERSRGs were obtained by taking the intersection of GSE52093 with the ERSRGs downloaded from the GeneCards website: ACTC1, CASQ2, REEP1, AKAP6, RYR2, JAK2, CAMK2G, WFS1, SERPINH1, CHEK1, CDKN3, SPP1, and CDKN3 (
Functional Enrichment Analysis
Based on the Metascape database, we conducted a functional enrichment analysis of the 12 ERSRGs obtained. The 5 most significantly enriched GO terms and KEGG pathways were identified: (1) cellular response to caffeine; (2) negative regulation of molecular function; (3) Signaling by receptor tyrosine kinases; (4) supramolecular fiber organization; and (5) regulation of growth (
PPI Analysis
GeneMANIA was used to analyze 12 differentially expressed ERSRGs and their interacting proteins, and the results are shown in
Validation in GSE153434
To evaluate the veracity of the differentially expressed ERSRGs in TAAD, we used GSE153434 to validate the expression levels of the top 5 (|log FC|) differentially expressed ERSRGs: ACTC1, CASQ2, CDKN3, SPP1, and REEP1. The expression levels of ACTC1, CASQ2, and REEP1 were significantly decreased in the AD group compared with the NAD group. In addition, the expression level of SPP1 was significantly increased in the AD group. However, the expression level of CDKN3 showed no significant difference between the 2 groups (
Validation in Clinical Samples
We examined the expression of ACTC1, CASQ2, SPP1, and REEP1 using qRT-PCR in the AD (n = 12) and NAD (n = 12) groups of clinical samples, and the results again demonstrated that ACTC1, CASQ2, and REEP1 were expressed at low levels in TAAD, while SPP1 was highly expressed in TAAD, and the difference was statistically significant (
Immune Infiltrate Analysis
To assess the correlation between the 4 validated ERSRGs and immune infiltrating cells, we analyzed the distribution ratio of 22 types of immune cells within the TAAD aortic wall tissue based on the GSE153434 dataset (
Construction of Regulatory Networks
Then, we built the gene–miRNA (
Discussion
Type A aortic dissection is a serious life-threatening disease that requires prompt diagnosis and emergency surgical treatment. However, the molecular mechanisms underlying its pathogenesis are still not fully elucidated. In this study, we included and analyzed 2 GSE datasets and identified 12 differentially expressed ERSRGs, and ACTC1, CASQ2, SPP1, and REEP1 were validated in clinical specimens. To further understand the possible role of these 4 differentially expressed ERSRGs, we analyzed their correlation with immune cell infiltration. Our study suggests that the 4 differentially expressed ERSRGs can serve as biomarkers for TAAD diagnosis and potential therapeutic targets.
Endoplasmic reticulum stress is closely associated with tumors, myocardial injury, and other diseases. Previous studies have demonstrated that ATF3 attenuates myocardial injury by regulating SPHK1 through the ERS pathway.26 In colon cancer, oridonin increases cancer cell death via TP53-promoted ERS.27 In this study, 4 differentially expressed ERSRGs, ACTC1, CASQ2, SPP1, and REEP1, were identified and validated. In cardiomyocytes and skeletal muscle, calsequestrin 2 (CASQ2) was found in the intracellular endoplasmic reticulum or sarcoplasmic reticulum and binds to calcium.28 Catecholaminergic polymorphic ventricular tachycardia can be caused by homozygous point mutations in CASQ2.29 CASQ2 controls the intracellular calcium concentration, which in turn modulates muscular contraction and activates cardiomyocytes. Additionally, ryanodine receptor 2 (RYR2) allows CASQ2, the main calcium reservoir protein in the heart, to release calcium into the cytosol in response to changes in calcium concentration.30 RYR2 activity in myocytes is linked to electrical and contractile failure in the arrhythmogenic heart of an elderly person via mitochondrial function, calcium homeostasis, and excitation–contraction coupling.31 In the present study, we found lower expression of CASQ2 in TAAD patients and further validated this finding in clinical specimens. Our results suggest that alterations in calcium channels may influence the development of TAAD.
SPP1, also known as osteopontin, has previously been linked to degenerative aneurysmal illness, including abdominal aortic aneurysms,32 and has been demonstrated to play a role in a variety of blood vessel diseases.33 SPP1, for example, is produced during inflammation and binds to transmembrane ligands to influence tissue remodeling pathways.34 Furthermore, SPP1 participates in cellular migration, proliferation, apoptosis, and macrophage chemotaxis.35 Previous research has linked SPP1 to abdominal and thoracic aneurysmal illness by upregulating matrix proteinases via NF-B, increasing tissue degradation.36,
Expression-enhancing protein 1 (REEP1) is one of the proteins that make up mitochondria-associated endoplasmic reticulum membranes (MAM). Abnormalities in MAM proteins often lead to the incidence and progression of associated disorders such as diabetic kidney disease, neurodegenerative diseases, and type 2 diabetes mellitus.39 The correlation between REEP1 and TAAD has not been previously reported, and we believe that the downregulation of REEP1 may be related to the development of TAAD, which needs to be further investigated in future experiments.
Vascular inflammation is a major contributor to the development of AD. Many studies have shown that a cascade of cytokines and inflammatory cells plays a role in disease progression. For example, the cytokine IL33 is an efficient AD diagnostic marker.40 In addition to cytokines, inflammatory cells pass across the compromised endothelial tight connections, causing vascular inflammation and, eventually, AD.41 Endoplasmic reticulum stress is associated with a variety of pathological conditions associated with chronic inflammation, which can trigger inflammatory pathways and proinflammatory stimuli such as Toll-like receptor ligands, reactive oxygen species, and cytokines. These proinflammatory signals can initiate ERS and lead to activation of the unfolded protein response, which further amplifies the inflammatory response.42 Immunoinflammation-related mechanisms play an important role in AD disease, for example, in animal models, neutrophil-secreted MMP9 can induce AD.43 Previous studies have shown that aortic arterial wall remodeling depends on complex interactions between cells, proinflammatory mediators, and MMPs, which are regulated by immune responses.44,
However, this study still has some limitations. First, clinical validation with large samples was lacking, and second, animal models were not constructed to study the specific molecular mechanisms of these genes. These limitations will be solved in our future research work.
Conclusion
In this study, we identified 12 differentially expressed ERSRGs by analyzing the GEO database. Four key differentially expressed ERSRGs may influence the development of TAAD by regulating ERS. These results expanded our understanding of TAAD, and the 4 genes are expected to be diagnostic markers and potential therapeutic targets.
Footnotes
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