Identifying the Oxidative Stress-related Hub Genes in Dilated Cardiomyopathy by Bioinformatics Analysis

Introduction: Characterized as an idiopathic primary myocardial disorder, dilated cardiomyopathy (DCM) predominantly affects children and elderly adults. However, a lack of specific clinical symptoms and reliable biomarkers impedes timely diagnosis and rational clinical management of DCM. Methods: Whole-genome expression profiles (GSE120895, GSE9800) were retrieved from the Gene Expression Omnibus database via the GEOquery R package. A series of bioinformatic methods were employed, including DEG, GSVA, WGCNA, GO/KEGG enrichment, PPI, and immune infiltration analysis. Key biomarkers were validated by qRT-PCR in a Doxorubicin (DOX)-induced DCM model. Results: In total, 629 differentially expressed genes (DEGs) were screened out between DCM and control groups. Combined analysis of DEGs and WGCNA outputs identified 13 hub genes overlapping with oxidative stress-associated gene modules. Receiver Operating Characteristic (ROC) curve analyses confirmed that these hub genes exhibit favorable diagnostic efficiency for DCM. Functional enrichment results showed that these genes are mainly enriched in transmembrane transport and nucleotide metabolism pathways. Immune infiltration analysis indicated significantly elevated infiltration levels of five immune cell subsets in DCM myocardial tissues. In vivo experiments verified the significant upregulation of five core hub genes in DOX-induced DCM mice. By screening hub genes with diagnostic potency based on public transcriptome datasets and validating their expression alterations in a DOX-induced DCM animal model, this study provides partial experimental evidence to support the above bioinformatic outcomes. discussion: DCM is a heterogeneous cardiomyopathy characterized by ventricular enlargement and reduced myocardial contractile function[43,44]. Due to the lack of early diagnostic indicators, patients with DCM often lose the best opportunity for treatment, resulting in a poor prognosis. Oxidative stress is a critical factor in the development of DCM[45–47]. It significantly influences the progression of heart damage and remodeling by triggering the expression of numerous genes[48]. Therefore, analyzing and identifying the oxidative stress-related biomarkers has significant implications for improving prognosis for DCM patients[49,50]. In this study, 13 oxidative stress-related DEGs were identified by the intersection of WGCNA module genes and DEGs, which laid a foundation for subsequent mechanism studies. Further validation in a mouse DCM model confirmed the potential value of MVP, WISP1, FCN1, AMPD3, RARRES1, FTL, KRT14, these hub genes as biomarkers. Based on the expression of PCR and pathway enrichment, MVP, WISP1, FTL, and FCN1 genes were selected as the focus of in-depth study. MVP(Major Vault Protein) is the main component of cellular ribonucleoprotein particles located in the cytoplasm[51]. MVP is known to regulate several cellular processes including nucleocytoplasmic transport, signal transduction, cellular differentiation, cell survival, and immune responses[52]. MVP in macrophages specifically can promote SR-A-mediated TNF-α synthesis and apoptosis[53], and it can also prevent metabolic diseases via NF-κB signaling[54]. In this study, MVP performance showed its value as a potential biomarker. The ROC curve analysis showed that MVP had an AUC value of 0.812, indicating that MVP had a good ability to distinguish between patients with DCM and healthy controls, suggesting that MVP may play an important role in the early diagnosis of DCM. Further analysis showed that MVP expression was closely related to the infiltration of specific immune cells, especially with CD56dim natural killer cells (correlation coefficient R=0.683, p <0.001). This finding suggests that MVP may play a key role in the immune response and cell interactions in DCM.This is consistent with previous studies[55–57]. In the PCR results of animal models, the expression level of MVP in the left ventricular tissue of DCM mice was significantly higher than that in the control group. This result further confirmed the importance of MVP in the pathogenesis of DCM. Therefore, MVP not only shows good discriminative ability in distinguishing DCM patients from healthy populations, but also its correlation with immune cells provides a new perspective on understanding the pathogenesis of DCM. WISP1(Wnt Inducible Signaling Pathway Protein 1) matricellular protein, a target gene of the WNT signaling, has been shown to modulate immune cell behavior and ECM remodeling in various diseases[58–60]. The matricellular protein WISP1 is key components of the ECM milieu, playing crucial roles in regulating immune responses[59]. In this study, the expression of WISP1 highlighted its potential value as a biomarker. ROC curve analysis showed that the AUC value of WISP1 was 0.791, indicating that the gene had a good discrimination ability and could effectively distinguish patients with DCM from healthy controls. This result emphasized the possible key role of WISP1 in the diagnosis of DCM. Furthermore, through real-time quantitative PCR analysis of the mouse model, we observed that the expression level of WISP1 in the left ventricular tissue of DCM mice was significantly higher than that in the control group. This finding further supports the importance of WISP1 in the development of heart disease, suggesting its potential role in cardiac remodeling and apoptosis processes. Previous studies have shown WISP1 is a key molecule in various disease development, which is involved in oxidative stress, apoptosis and autophagy[61–63]. These findings make WISP1 a promising biomarker for DCM diagnosis and prognosis, and further studies are warranted to explore its specific mechanisms and clinical application potential. FTL(Ferritin Light Chain) as the light chain of iron storage protein has been widely regarded as one of the regulators of iron metabolism for a long time[64,65]. In this study, the performance of FTL showed its value as a potential biomarker. The ROC curve analysis revealed that the AUC value of FTL was 0.771, indicating its strong ability to differentiate between patients with DCM and healthy controls. This finding underscores the importance of FTL in DCM diagnosis and suggests its potential for significant clinical application. Further experiments showed that, through real-time quantitative PCR, the expression level of FTL in the left ventricular tissue of DCM mice was significantly higher than in the control group. The increase of FTL may reflect the response of cardiomyocytes to iron overload and its associated oxidative damage[66–68]. This finding suggests that the upregulation of FTL may be closely related to the development of DCM, especially in terms of iron metabolism imbalance and oxidative stress response. FCN1(Ficolin-1), a protein involved in the immune response, plays a crucial role by binding to pathogens and activating the complement system[69–71]. In this study, FCN1 was significantly expressed, indicating its potential as a biomarker. The ROC curve analysis indicates that the AUC value of FCN1 is 0.764, indicating its strong ability to differentiate between patients withDCM and healthy controls. This finding highlights the potential of FCN1 in the early diagnosis of DCM and suggests its critical role in disease progression. Additionally, real-time quantitative PCR analysis in animal models revealed that the expression level of FCN1 in the left ventricular tissue of DCM mice was significantly higher than in the control group. This finding suggests that the upregulation of FCN1 may be associated with the enhanced inflammatory response in DCM, and further speculate its immunomodulatory role in cardiopathy[72]. Future studies can further explore the functional mechanism of FCN1 and its application potential in DCM treatment, in order to provide new strategies to improve the prognosis of this disease. In this study, our analysis of the signaling pathways of key genes MVP and WISP1 showed that these two genes were closely associated with several significantly downregulated pathways, including HALLMARK_OXIDATIVE_PHOSPHORYLATION (p<0.0001), HALLMARK_ FATTY_ACID_METABOLISM (p<0.01), and HALLMARK_BILE_ACID_METABOLISM (p <0.05). The downregulation of these pathways suggests that the dysregulation of energy metabolism and lipid metabolism may be complementary in the pathogenesis of DCM, supporting the metabolic abnormality theory of DCM. Furthermore, in the GO pathways associated with oxidative stress, the gene expression levels of secretory granule lumen also showed upregulation of key genes. In the disease group, the expression of MVP, FTL, and FCN1 was significantly elevated, suggesting that these genes may play a crucial role in the body's response to oxidative stress. This high expression likely reflects the adaptive response of myocardial cells during the progression of DCM, although persistent high levels of oxidative stress can still lead to cell damage. According to Zhou Jun's research[73], the BMP signaling pathway and the interaction between cytokines and their receptors play a crucial role in the development of DCM. The results of DEGs also indicate that cell apoptosis, hypoxia, heme metabolism, and epithelial-mesenchymal transformation pathways are closely linked to DCM. Prior studies suggest that cell apoptosis and metabolic disorders may be significantly associated with the pathological progre