Advanced Glycation End Products' Receptor DNA Methylation Associated with Immune Infiltration and Prognosis of Lung Adenocarcinoma and Lung Squamous Cell Carcinoma

Background Advanced glycation end products' receptor (AGER) is a multiligand receptor that interacts with a wide range of ligands. Previous studies have shown that abnormal AGER expression is closely related to immune infiltration and tumorigenesis. However, the AGER DNA methylation relationship between prognosis and infiltrating immune cells in LUAD and LUSC is still unclear. Methods AGER expression in pan-cancer was obtained by using the UALCAN databases. Kaplan–Meier plotter showed the correlation of AGER mRNA expression levels and clinicopathological parameters. The protein expression levels for AGER were derived from Human Protein Atlas Database Analysis. The copy number, somatic mutation, and DNA methylation of AGER were presented with UCSC Xena database. TIMER platform and TISIDB website were used to show the correlation between AGER expression and tumor immune cell infiltration level. Results The expression level of AGER was significantly reduced in lung adenocarcinoma (LUAD) and lung squamous cell carcinoma (LUSC). Low expression of AGER was significantly correlated with histology, stage, lymph node metastasis, and tumor protein 53 (TP53) mutation and could be used as a potential indicator of poor prognosis of LUAD and LUSC. Moreover, AGER expression was positively correlated with the infiltrating immune cells. Further analysis showed that copy number variation (CNV), mutation, and DNA methylation were involved in AGER downregulation. In addition, we also found that hypermethylated AGER was significantly correlated with tumor-infiltrating lymphocytes. Conclusion AGER may be a candidate for the prognostic biomarker of LUAD and LUSC related to tumor immune microenvironment.


Introduction
Cancer as a major public health problem, the morbidity and mortality have risen sharply worldwide, placing a heavy burden on the public health system. In 91 of 172 countries, cancer is the frst or second leading cause of death before age 70 [1][2][3]. Due to the typical early clinical symptoms that are not obvious and the limitations of diagnostic methods, the vast majority of patients with lung cancer is diagnosed at a later stage [4]. Over the past few decades, thanks to the eforts of clinical and scientifc researchers, breakthroughs have been made in the diagnosis and treatment of lung cancer [5,6]. Terefore, the 5-year survival rate of patients diagnosed with lung cancer is not satisfactory, only an astonishing 15%, while the prognosis of individuals diagnosed with advanced disease is even worse [7]. Hence, screening for potential lung cancer gene therapy targets and prognostic markers is particularly important.
Advanced glycation end products (AGEs) refer to a group of heterogeneous macromolecules that are produced by post-translational modifcation of proteins through nonenzymatic glycation, lipids, aging, and nucleic acids [8]. AGEs provide the bridge between intracellular and extracellular damage through the advanced glycation end products' receptor (AGER), also known as the receptor for advanced glycation end products (RAGE). AGER protein is a multiligand receptor that interacts with a wide range of ligands, including AGEs, β-sheet fbrils, S100 proteins (S100B, S100P, S100A4, S100A6, S100A8/9, and S100A11-13), high mobility family protein-1, and prion [9,10]. AGER expression plays a central role in the neurodegeneration, retinal microvascular dysfunction, and thymic hyperplasia via the toll-like receptor 4 and AGE/ AGER signaling pathways [10]. Nevertheless, AGER expression can be induced under certain pathological conditions (including high glucose, reactive oxygen species, hypoxia, proinfammatory mediators, or AGER itself ) [8,10].
Tumor microenvironment (TME) comprises a complex milieu of nonmalignant cells including vascular vessels, fbroblasts, extracellular matrix, and immune infltrates, which can interact closely with tumor cells and afect tumor growth and metastasis [11]. Immune infltration plays a central role in the tumor microenvironment, especially tumor-infltrating lymphocytes [12]. Previous studies have shown that abnormal AGER expression is closely related to immune infammatory response and tumorigenesis [13]. Other related studies also showed that AGER expression and mutation play an important role in brain disease, esophageal cancer, breast cancer, gastric cancer, prostate, melanoma, and endometrial carcinoma [14][15][16][17][18][19][20][21][22][23][24]. Some reports have also been made on AGER in non-small-cell lung cancer (NSCLC). Excellent studies have shown that low expression of AGER signifcantly reduced the median survival time of LUAD patients [25,26]. To clarify the mechanism of action between AGER in NSCLC, Yang et al. [27] verifed the function of AGER in modulating the tumor microenvironment via miR-182-5p/NF-κB axis mediating the malignant phenotypes of NSCLC. During the occurrence and metastasis of lung cancer, AGER's signifcance has been demonstrated in the progression, angiogenesis, and immune cell infltration mediated by lysophosphatidic acid [28]. Although AGER has multitudinous functions in the tumor microenvironment, numerous mechanisms are still unclear, especially the potential mechanism between DNA methylation and lymphocyte infltration in LUAD and LUSC.
In our work, Tumor Immune Estimation Resource (TIMER), Gene Expression Profling Interactive Analysis (GEPIA), UALCAN, and Kaplan-Meier plotter databases were used to demonstrate AGER expression level and its correlation with the prognosis. Furthermore, we used the TIMER network resource to explicate the associations of AGER and important components of the tumor microenvironment (tumor-infltrating immune cells). We also explained the relativity between tumor-infltrating immune cells and prognosis. In addition, we further explored the potential molecular mechanism of AGER imbalance including CNV, somatic mutation, and DNA methylation. Furthermore, we clarifed that the high degree of AGER DNA methylation was obviously related to infltrating lymphocytes. Tus, we raise a possible regulatory mechanism of AGER DNA methylation and tumor-infltrating lymphocytes which infuence prognoses of LUAD and LUSC to some extent.

Tumor Immune Estimation Resource (TIMER) Database
Analysis. Te TIMER database is a feature-rich resource. Te TIMER algorithm is used to systematically analyze the relationship between gene expression of diferent cancer types and tumor-infltrating immune cells. Te abundance of six tumor-infltrating cells was assessed [29]. Te TIMER website is used to illustrate the diferential expression of AGER in normal and tumor tissues in diverse malignant tumors. Moreover, we analyzed the relationship between AGER and 6 types of tumor-infltrating immune cells in the "Gene" module. We also used this site to investigate the relationship between gene expression level and immuneinfltrating cells in LUAD and LUSC.

Gene Expression Profling Interactive Analysis (GEPIA).
GEPIA is a newly developed database that provides customizable functions with RNA sequencing expression data form 9736 tumors and 8587 normal samples. It is a useful network resource for visualization of gene expression based on Te Cancer Genome Atlas (TCGA) and Genotype Tissue Expression (GTEx) data [30]. We showed various expression levels of AGER in normal and tumor tissues in diferent tumors. In LUAD and LUSC, normal and tumor tissues were used to detect the expression level of AGER. In addition, the survival module contributes to clarify the relationship between AGER expression and prognosis.

Human Protein Atlas Database Analysis. Te Human
Protein Atlas is an efcient and open database that allows free access by academic researchers and provides a reference for exploring the human proteome [31,32]. We focused on Pathology Atlas, which shows the impact of protein levels for the survival of patients with cancer. We screened protein expression in LUAD and LUSC through immunohistochemistry in the pathology module.

UALCAN Database
Analysis. UALCAN is a fully functional, friendly, and interactive network resource, mainly used to analyze cancer omics data. By linking multiple databases, the expression analysis of genes, proteins, and epigenetics can be quickly realized. Tese resources enable researchers to efciently lock interesting targets and valuable information [33]. We used the UAL-CAN web resource to verify the results between AGER and various clinicopathological parameters including pathology, cancer stages, nodal metastasis status, and TP53 mutation status of lung cancer and calculated the P value. Genetics Research

2.5.
Kaplan-Meier Plotter Database Analysis. Kaplan-Meier plotter downloads gene expression data, recurrence-free, and overall survival information through links to GEO, EGA, and TCGA and then meta-analyzes the prognostic value of a specifc gene. Its database has been able to assess the impact of more than 50,000 genes (mRNA, miRNA, and protein) on the survival rate of 21 cancer types and is a commonly used tool for bioinformatics analysis [34]. Kaplan-Meier plotter web resources were used to verify the correlation between diverse clinical results and the expression of AGER in LUAD and LUSC. We showed a prognostic analysis of AGER expression in distinct immune cell subsets with this web.
2.6. PrognoScan Database Analysis. Te PrognoScan database is a publicly available cancer microarray dataset with clinical annotation function, which can be used as an online analysis tool to evaluate the biological relationship between gene expression and prognosis. A systematic meta-analysis can be performed on multiple datasets. It is a powerful platform for evaluating potential tumor markers and treatment targets. Its existence will certainly promote cancer research [35]. Tis database was used to illustrate the efects of abnormal AGER expression on the prognosis in lung cancer, LUAD, and LUSC.

TISIDB Database Analysis.
TISIDB is an open, free, and useful database. It integrates data from multiple public databases including UniProt, Gene Ontology (GO), Drug-Bank, PubMed, and TCGA. It aims to clarify the interaction between tumors and immune cells and is a valuable resource for cancer immunology research and treatment [36]. To illustrate the potential relationship between AGER and tumor-infltrating lymphocytes (TILs), 28 TILs were used to analyze the association with AGER in diferent tumor sites in the TISIDB database. Besides, we also demonstrated the correlation between AGER DNA methylation and tumorinfltrating lymphocytes via this platform.

UCSC Xena Database
Analysis. UCSC Xena database provides interactive online visualization of seed cancer genomics datasets, which can support online analysis of a variety of genomics, proteomics, phenotype, and clinical annotation data. It has included more than 50 cancer-type related data and is a user-friendly database [37]. In the study, gene expression, copy number, somatic mutation, and DNA methylation were presented in this database. Details of the probe cohorts for detecting AGER DNA methylation and the level of methylation are also displayed.
2.9. Statistical Analysis. TIMER, Kaplan-Meier plotter, PrognoScan, GEPIA, and UALCAN network resources were used for AGER expression verifcation. Te survival curve based on the Kaplan-Meier plotter and GEPIA was presented using HR and P or P values from a log-rank test. SPSS 25.0 (SPSS, Inc., Chicago, IL) was used for data analysis. For two-group comparison, Student's t-test method was used. Two-tailed P < 0.05 was considered statistically signifcant.

AGER Expression Level Is Downregulated in LUAD and LUSC Patients.
Te AGER expression level in diferent cancer types was elaborated using the TIMER web database. Lower expression of AGER was revealed in breast invasive carcinoma (BRCA), thyroid carcinoma (THCA), kidney chromophobe (KICH), LUAD, and LUSC compared with corresponding normal tissues. On the contrary, in bladder urothelial carcinoma (BLCA), cholangiocarcinoma (CHOL), esophageal carcinoma (ESCA), head and neck squamous cell carcinoma (HNSC), kidney renal clear cell carcinoma (KIRC), kidney renal papillary cell carcinoma (KIRP), liver hepatocellular carcinoma (LIHC), and stomach adenocarcinoma (STAD) compared with the control group, AGER showed a high expression trend (Figure 1(a)). In the UALCAN database, the result showed that the expression level of AGER in the normal lung tissue was signifcantly higher than that in LUAD and LUSC (Figure 1(b)). Tis result indicates that AGER may act as a signifcant part in the biological process of lung cancer.
Furthermore, the expression level of AGER in lung cancer samples and adjacent tissues is obtained from GEPIA online resource. AGER expression was signifcantly decreased in LUAD and LUSC (Figure 1(b)). Te same result was also verifed in the UALCAN database. Further studies showed that the expression of AGER in tumor histology, stage, lymph node metastasis, and TP53 mutation was signifcantly increased in normal tissues, and it was low in LUAD and LUSC tumor tissues (Figures 1(c) and 1(d)).

AGER Protein Presented Low Expression in LUAD and
LUSC Tissues. Protein expression levels in LUAD and LUSC obtained and visualized with the Human Protein Atlas database. We then established a scoring system whereby high levels of positive AGER expression received 3 points, moderate levels received 2 points, low levels received 1 point, and no expression received 0 points. Te results indicated that AGER exhibited moderate positive expression in all 4 normal tissues. In addition, there were 0, 0, 8, and 12 cases of high, medium, low levels positive, and undetected staining in LUAD and 0, 2, 9, and 9 cases in LUSC (Figures 2(a) and 2(b)), respectively. It was observed that in both LUAD and LUSC, the expression of AGER was signifcantly decreased in tumor tissues, as shown in Figure 2(c).

Te Prognostic Value of AGER Was Verifed Based on
Kinds of Clinicopathological Features. To understand the relationship between AGER and prognostic value in more detail, we investigated the correlation between AGER mRNA expression and clinicopathological features using Kaplan-Meier database. Interestingly, low expression AGER was associated with poor overall survive (OS) only in American Joint Committee on Cancer (AJCC) stage T2 of lung cancer patients ( Figure 3). Ten, the correlation   between AGER expression and poor OS was observed in AJCC stage N0 population ( Figure 3). Ten, low AGER expression was evidently associated with poor OS in both males and females ( Figure 3). Moreover, we clarifed that low expression AGER represented worse OS in both smoking and nonsmoking patients ( Figure 3). Ten, low AGER expression was obviously related with poor OS in lung cancer patients with negative surgical margins ( Figure 3). Furthermore, we observed that in patients who received chemotherapy or radiotherapy, low level of AGER indicated worse OS, but without statistical signifcance ( Figure 3).
Tese results indicate that the prognostic value of low expression of AGER for lung cancer is meaningful.

Relativity Analysis between Low Level AGER and Infltrating Immune Cells in LUAD and LUSC.
Tumor-infltrating immune cells can be used independently to predict the status of tumor sentinel lymph node metastasis and prognosis [38]. We elaborated the correlation between AGER expression and 6 types of immune cells which is default in the database, including B cells, CD4 + T cells, CD8 + T cells, neutrophils, macrophages, and dendritic cells with TIMER database. Te results showed that AGER correlated with infltration of B cells, CD4 + T cells, CD8 + T cells, neutrophils, macrophages, and dendritic cells. In addition, both LUAD and LUSC reached the same conclusion ( Figure 5(a)). We investigated the correlation between AGER expression level and 28 tumor immune infltrating lymphocyte subtypes. Tose results demonstrated that AGER was linked to 21 and 20 diferent lymphocyte subtypes in LUAD and LUSC, respectively ( Figure 5(b) and Table 1). Especially, it is signifcantly related to activated B cell, macrophage, natural killer cell, efector memory, CD8 + T cell, and T follicular helper cell, both in LUAD and LUSC ( Figure 5(c)).

Prognostic Value of AGER Expression in LUAD and LUSC
Based on Diverse Immune Cells. Our research indicated AGER expression related to the immune infltration of LUAD and LUSC. In addition, low level of AGER was involved with the poor prognosis of lung cancer. Terefore, we intended to investigate whether AGER might impact the prognosis of LUAD and LUSC through immune infltration to some extent. We reported that LUAD patients with low AGER levels in enriched B cells, CD4 + memory T cells, eosinophils, macrophages, mesenchymal stem cells, natural killer T cells, regulatory T cells, and type 1 T helper cells had poor prognosis ( Figure 6(a)). Interestingly, the high AGER level in LUSC-enriched Basophils, Eosinophils, macrophages, Type 1 T helper cells, and Type 2 T helper cells cohort had a worse prognosis (Figure 6(b)). Te data suggest that diferent expression levels of AGER may afect the immune infltration cells of diverse subtypes of lung cancer, such as LUAD and LUSC, ultimately infuencing their prognosis.

CNV, Mutation, and DNA Methylation Analysis of AGER Gene in LUAD and LUSC.
We further explored the expression, CNV, gene mutation, and DNA methylation levels of AGER in LUAD and LUSC through UCSC Xena database. Heatmap analysis revealed a correlation between AGER mRNA expression and CNV and gene mutation and DNA methylation in LUAD (Figure 7(a)) and LUSC (Figure 7(b)). Simultaneously, the heatmap also indicated that AGER DNA methylation levels in LUAD and LUSC were higher than normal tissues (Figures 7(a)  and 7(b)).

AGER DNA Methylation Was Obviously Related to Tumor
Immune Infltrating Lymphocyte Subtypes. We have clarifed that AGER displays high level of DNA methylation in LUAD and LUSC. We utilized UCSC Xena to establish the correlation between AGER DNA methylation and immuneinfltrating lymphocytes. Te signal intensity of DNA methylation is detected by various probe cohorts and then expressed in the form of β value. Any β value of 0.6 or higher is considered fully methylated, while β value of 0.2 or lower is considered completely unmethylated. A β value between 0.2 and 0.6 is partially methylated [39]. In LUAD, out of the 25 probes, complete DNA methylation was observed in 22 probes while 3 showed partial DNA methylation (Figure 8(a) upper). Consistently, 23 probes were detected in LUSC suggesting complete DNA methylation, while 2 showed partial DNA methylation (Figure 8(a) lower). TISIDB was utilized to further investigate the relationship between AGER and tumor-infltrating lymphocytes. Tose results exhibited that it was signifcantly related to active CD4 cells, active CD8 cells, memory B cells, natural killer T cells, and type 2 T helper cells both in LUAD and LUSC (Figure 8(c)). Tis indicates a possible association between the DNA methylation of AGER and tumor-infltrating immune lymphocytes.

Discussion
In recent decades, lung cancer has emerged as the primary cause of cancer-related deaths on a worldwide scale. Lung cancer is divided into non-small-cell lung cancer and small cell lung cancer according to the pathological type. Among them, NSCLC accounts for 85% of all lung cancer [1,3,40]. Terefore, it is imperative to focus on improving the level of diagnosis and treatment of NSCLC. Despite the promising results of immune checkpoint inhibitors in the treatment of lung cancer, the efcacy has not matched the anticipated outcomes [41]. Tus, it is essential to explore the mechanism of immunotherapy and identify promising prognostic biomarkers for lung cancer. Our research suggested that the expression of AGER was signifcantly downregulated in LUAD and LUSC using bioinformatics analysis of GEPIA, TIMER, and UALCAN databases (Figure 1(a)). At the same time, the protein level has also been further verifed. Consistent with the conclusion reached at the gene level that AGER has lower expression in LUAD and LUSC (Figures 2(a) and 2(b)). Tese results were aggregated into valuable information and further showed that AGER may play the role of tumor suppressor involved in the occurrence of lung cancer. Ten, the clinical prognostic signifcance of

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AGER in patients with LUAD and LUSC was reported. Te downregulation of AGER was signifcantly correlated with tumor histology, stage, lymph node metastasis, and TP53 mutation of LUAD and LUSC patients (Figures 1(c) and  1(d)). In addition, Kaplan-Meier survival analysis presented that overexpression AGER was notable live longer than those patients with low AGER expression ( Figure 4). Hence, AGER has the potential to serve as a valuable prognostic biomarker for patients with NSCLC. Increasingly substantial evidence demonstrates that LUAD and LUSC exhibit distinguishable characteristics in numerous aspects, comprising gene expression profle,  biological behavior, molecular pathological features, clinical features, and therapeutic responses [42]. Compared with LUAD, LUSC is usually associated with smoking and infammatory diseases. Generally speaking, LUSC grows more slowly than LUAD during the same period, and the volume of the mass is smaller, but most patients have the tendency of early metastasis [43]. Tere are also signifcant diferences between LUAD and LUSC in the gene mutation spectrum. Previous reports indicated that mutations of epidermal growth factor receptor (EGFR) gene are the commonest type of NSCLC patients. Te frequency of EGFR mutations is 27% and 9% in LUAD and LUSC, respectively [44]. In addition, studies have shown that there are also great differences in mRNA, protein expression, signal transduction pathway, and DNA methylation mode between LUAD and LUSC [45][46][47]. Tese fndings provide valuable experience and research basis for explaining the molecular mechanism of LUAD and LUSC. Similarly, in the research, we also found that AGER had diferences in gene, protein level, and prognosis in LUAD and LUSC. In terms of protein expression level, AGER in LUSC was higher than that in LUAD ( Figure 2). Tis also proved that low expression of AGER was related to a worse prognosis, consistent with previous research outcomes. In addition, we only observe that low expression AGER was related to OS in LUAD, but not in LUSC (Figure 3). Tis may be related to the diferent datasets selected by the database for analysis. Moreover, a limited number of samples may also have contributed to bias in the results. Tis also further verifed the heterogeneity of LUAD and LUSC. Immune cells have irreplaceable involvement in cancer progression and aggressiveness [48]. It is considered to be an important determinant of prognosis and the efcacy of immunotherapy [49]. In previous meaningful studies, immunohistochemical experiments showed that downregulating the AGER could signifcantly upregulate angiogenesis (CD34), leukocyte (CD45), and macrophage (F4/80) markers level. Further research pointed out that lysophosphatidic acid (LPA) induces proliferation, migration, colonization, and tumor microenvironment via RAGE and downstream protein kinase B (PKB) pathways [28]. In nontumor studies, it has been confrmed that AGER interacts with immune cells [50].
Valuable study had pointed out that in diabetic mouse models, RAGE was involved in tissue repair related to infammatory damage. In-depth study has shown that RAGE downregulates the expression of pro-repair infammatory genes in ischemic muscle and lowers the number of macrophages [51]. Similarly, we reported that low expression levels of AGER in LUAD and LUSC were linked to reduced infltration of B cells, CD4 + T cells, CD8 + T cells, neutrophils, macrophages, and dendritic cells ( Figure 5(a)). Moreover, we disclosed the correlation analysis between AGER and 28 tumor-infltrating lymphocytes ( Figure 5(b) and Table 1). It should be emphasized that it was signifcantly related to activated B cell, macrophage, natural killer cell, efector memory, CD8 + T cell, and T follicular helper cell, both in LUAD and LUSC ( Figure 5(c)). Moreover, AGER has a partial impact on the survival time of LUAD and LUSC patients by immune cell infltration (Figures 5(a) and 5(b)).
Tis indicates that AGER could potentially be targeted for immune-related therapy in cases of lung cancer. Epigenetics plays a key role in the regulation of gene expression [52]. Epigenetic regulation of genes can enable organisms to quickly adapt to changes in the new environment to obtain characteristics that are benefcial to themselves. It should be noted that epigenetic disorders can trigger the repression of tumor suppressor genes or the stimulation of oncogenes, ultimately serving as a contributing factor to tumor development and progression. As a common epigenetic phenomenon of tumors, DNA methylation features can be used as biomarkers for the prognosis and diagnosis of diferent cancer types and provide more optimized strategies for cancer treatment. Te medical benefts of it are gaining broad recognition [53]. In our research, we discovered that AGER expression exhibited strong correlation with CNV, somatic mutations, and DNA methylation (Figures 6(a) and 6(b)). Furthermore, we clarifed AGER was related to the tumor-infltrating lymphocytes of LUAD and LUSC patients. In addition, our subsequent    investigation revealed signifcant abnormalities in the DNA methylation status of these two types of cancers. Tis leads us to propose a hypothesis whether there is a mutual regulatory relationship between tumor-infltrating lymphocytes and DNA methylation. Subsequent studies showed that highly DNAmethylated AGER in LUAD was closely correlated with active CD4 cells, active CD8 cells, memory B cells, natural killer T cells, and type 2 T helper cells. Similar phenomena could also be observed in LUSC. However, the potential mechanism of tumor immune microenvironment and AGER DNA methylation still needs to be further investigated. As for the relevance between AGER and lung cancer, this study has provided a new vision and expanded our understanding of the mechanisms that contribute to the development of lung cancer. However, it is undeniable that this study also has some limitations. Firstly, we focused on LUAD and LUSC in NSCLC, while SCLC, lung sarcoma, and other types of tumors were not involved. In fact, diferent pathological types of tumors exhibit signifcant variations in biological behavior and prognosis. Secondly, infltrating immune cells, a major participant in the tumor microenvironment, have a variety of types and complex mechanisms. Tus, a hierarchical analysis is required to thoroughly explore their functions. Overall, the downregulation of AGER implies the critical role in the occurrence and development of lung cancer.

Conclusion
Te gene and protein expression of AGER in LUAD and LUSC was downregulated, and it was obviously related to the prognosis. After adjusted by tumor purity, AGER showed a signifcant association with the tumorinfltrating lymphocytes. Further analysis showed that AGER DNA methylation may be correlated with tumor-infltrating lymphocytes, especially CD4 + T cells, active CD8 + T cells, memory B cells, natural killer T cells, and type 2 T helper cells. Consequently, our study provides insight into a novel role of AGER expression and DNA methylation in tumor immune infltration. AGER could be a potential prognostic biomarker of LUAD and LUSC related to tumor-infltrating lymphocytes. It should be noted that we recognize the following limitations in our research: our research fndings were primarily obtained through bioinformatics analysis, without undergoing any additional experimental validation. Data obtained from diferent laboratories, platforms, and equipment may exhibit some variations. Numerous databases lack a consistent standard for integrating data and ensuring data quality during collection, resulting in the presence of bias. In fact, the database adopts strict standards and reasonable algorithms when incorporating relevant data to maximize the availability of data. In addition, our results are primarily based on bioinformatics analysis and have not been subjected to additional experimental validation. However, the data in the database are also compiled and analyzed based on the collection of clinical samples and various clinicopathologic features, providing a certain reference value. Furthermore, due to infuences such as gene regulation and gene interactions, there are complex and intricate interactions between molecules and cells within organisms. Our results only indicate a correlation between AGER and other clinical pathological features but do not elucidate its regulatory relationship. Further exploration can be conducted through additional experiments to establish specifc regulatory mechanisms. We need to recognize the limitations of bioinformatics analysis clearly, which is a prerequisite for improving the efciency and accuracy of our research.