Abstract
Background
The metabolism of fatty acids is essential in the initiation and progression of cervical cancer. The objective of this research is to develop a prognostic model associated with fatty acid metabolism and to identify the FASN protein within this framework, with the intention of investigating its function in cervical squamous cell carcinoma (CESC) and its correlation with patient prognosis.
Methods
This prognostic model was developed using least absolute shrinkage and selection operator (LASSO) regression analysis on the training cohort derived from The Cancer Genome Atlas (TCGA) dataset. A nomogram for overall survival (OS) was effectively constructed by integrating the model risk scores with clinical characteristics. Furthermore, we analyzed various correlations involving pathway enrichment, therapeutic approaches, immune cell infiltration, and the prognostic model itself. The functional significance of the FASN gene was examined through Gene Ontology (GO) analysis, Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis, and gene set enrichment analysis (GSEA). In addition, we performed an in-depth assessment of the correlation between FASN expression and various clinical parameters sourced from the TCGA database, which encompassed tumor (T), node (N), and metastasis (M) stages, age, weight, and histological grade. Utilizing the “rms” R package, we constructed a nomogram model that combines clinical attributes with levels of FASN expression. Moreover, immunohistochemical analyses were performed to evaluate FASN expression in a cohort of 30 cases diagnosed with cervical cancer.
Results
We developed a prognostic model that includes eight genes: FASN, ACAT2, HMGCS1, IL4I1, ACAA2, SERINC1, CEL, and TP53INP2. The association between the altered expression of these genes and patient survival outcomes was confirmed through Kaplan-Meier survival analysis. Both univariate and multivariate Cox regression analyses revealed that FASN functions as an independent prognostic factor for OS within the TCGA cohort. Increased expression of FASN was associated with advanced T and N stages, higher clinical stages, older age, lower weight, greater histological grades, and a poorer prognosis. A nomogram was developed that integrates FASN expression with clinical variables to predict the likelihood of OS for patients at 1, 3, and 5 years. Additionally, immunohistochemical analysis indicated that FASN expression was significantly elevated in cervical cancer tissues when compared to adjacent non-cancerous tissues in a study involving 30 patients.
Conclusions
This study clarified a specific signature associated with fatty acid metabolism, specifically FASN, which is connected to both the initiation and progression of CESC. Furthermore, FASN may serve as a prognostic marker for individuals diagnosed with CESC, thus providing fresh insights for the formulation of clinical treatment strategies.
Keywords: FASN, cervical cancer, poor-prognosis, clinical stage, immune cell infiltration
Highlight box.
Key findings
• FASN is recognized as a valuable biomarker for screening, diagnosing, and assessing the prognosis of cervical neoplasms. In addition, a nomogram was created and validated using the FASN risk score to predict the overall survival (OS) rate of patients diagnosed with cervical neoplasms.
What is known and what is new?
• Elevated levels of FASN expression are linked to poor prognosis in several cancers, including liver and prostate cancer.
• The findings of this investigation demonstrate a notable elevation in the expression of FASN in instances of cervical neoplasms, which correlates significantly with more advanced pathological stages, infiltration of immune cells, and patient outcomes. These observations imply that FASN could function as a potential diagnostic biomarker and a promising therapeutic target for cervical cancer. In addition, a dynamic nomogram was constructed, incorporating FASN risk scores alongside clinical pathological parameters, to provide a thorough evaluation of the influence of FASN on the progression of cervical cancer. Moreover, it was noted that the levels of FASN protein are markedly increased in patients diagnosed with cervical neoplasms.
What is the implication, and what should change now?
• The potential of FASN to serve as a predictive biomarker for cervical neoplasms has been confirmed.
• The efficacy and safety of this nomogram need to be confirmed through further large-scale clinical trials.
Introduction
Cervical cancer, particularly cervical squamous cell carcinoma (CESC), represents a significant health challenge globally, being one of the most prevalent cancers among women (1,2). According to the World Health Organization, cervical cancer accounts for approximately 300,000 deaths annually, with a disproportionate impact on women in developing countries (3,4). Current treatment modalities for cervical cancer primarily include surgery, radiotherapy, and chemotherapy; however, these approaches often yield variable outcomes, particularly in the advanced stages of the disease, leading to poor prognoses (5). Consequently, there is an urgent need for research aimed at identifying novel biomarkers and therapeutic strategies to improve patient management and outcomes. This study focuses on the expression levels of FASN, which is upregulated in various tumor types and associated with tumor progression and prognosis. Investigating the role of FASN in CESC could provide valuable insights into its biological significance and potential clinical applications, thereby addressing the existing gaps in the understanding of cervical cancer pathophysiology and treatment efficacy.
Fatty acid metabolism serves as a vital component of cellular physiology, encompassing the synthesis, degradation, and functional roles of fatty acids in both energy metabolism and signal transduction (6). The implications of fatty acid metabolism are manifold, influencing energy supply, signal transduction, and immune modulation. Recent research has increasingly focused on the interplay between fatty acid metabolism and cervical cancer, indicating that disruptions in fatty acid metabolism may be intricately linked to the disease’s onset and progression. Particularly, the processes of fatty acid synthesis, uptake, oxidation, and their metabolic routes are crucial in the malignant advancement of cervical cancer cells. Evidence suggests a notable correlation between plasma-free fatty acid concentrations and the risk of developing cervical cancer. Specifically, levels of palmitic acid, docosahexaenoic acid, and total ω-3 fatty acids exhibit a negative correlation with cervical cancer risk, whereas arachidonic acid levels are positively associated, indicating that deviations in fatty acid metabolism could significantly contribute to cervical cancer development (7). In the context of cervical cancer, the abnormal activation of fatty acid biosynthesis pathways may facilitate tumor cell growth and survival (8). Furthermore, findings indicate that both monounsaturated and polyunsaturated fatty acids may augment the sensitivity of cervical cancer cells to radiotherapy, a phenomenon closely related to fatty acid metabolic pathways (9). Regarding metabolic reprogramming, cervical cancer cells frequently modify their fatty acid utilization to adapt to the tumor microenvironment. For instance, the overexpression of TM7SF2 has been linked to increased fatty acid accumulation, promoting the proliferation and migration of tumor cells, thereby suggesting that fatty acid metabolism may affect cancer progression via specific signaling pathways (10). Additionally, alterations in the expression of fatty acid transport proteins, such as FATP4 and CD36, have been closely associated with the advancement of cervical cancer and its precursors, providing new perspectives on the role of fatty acid metabolism in this malignancy (11). Consequently, fatty acid metabolism emerges as a promising area of research within the context of cervical cancer, necessitating further exploration.
The role of FASN in various cancers has garnered increasing attention. FASN is a key enzyme responsible for fatty acid synthesis and is overexpressed in many tumors, in which it is closely associated with tumor initiation and progression. In hepatocellular carcinoma, studies suggest that FASN promotes tumor metastasis by regulating cellular energy metabolism and influencing cell migration and invasion. For instance, the overexpression of FASN is significantly correlated with interactions between the enzyme and cytoskeletal proteins, which may promote tumor cell migration and invasion through epithelial-mesenchymal transition (12). Moreover, the downregulation of FASN can increase cellular sensitivity to hypoxia, thereby inducing apoptosis, highlighting its crucial role in the tumor microenvironment (13). In prostate cancer, the methylation pattern of FASN is negatively correlated with its expression levels, with high FASN expression linked to increased tumor invasiveness and poor prognosis (14). Inhibition of FASN not only reduces tumor cell proliferation but also significantly enhances sensitivity to chemotherapy, making FASN a promising therapeutic target (15). Additionally, FASN function is closely associated with intracellular signaling pathways. For example, FASN regulates the stability of β-catenin, which, in turn, affects the activation of the Wnt signaling pathway, a mechanism particularly prominent in prostate cancer cells (16). These mechanisms suggest that FASN plays multiple roles in cancer progression, involving not only lipid metabolism regulation but also its influence on cell signaling. However, research on FASN in cervical cancer is still in its early stages, and its potential as a therapeutic target warrants further exploration. Future studies should focus on elucidating the specific role of FASN in cervical cancer and developing targeted therapeutic strategies. The present study investigates the expression levels of FASN in CESC, a malignancy characterized by high incidence and mortality rates. Previous research has established a correlation between FASN upregulation and tumor progression across various cancer types, suggesting its potential role as a prognostic biomarker (17-19).
FASN is implicated in the treatment response of cervical cancer. Numerous investigations have indicated a strong correlation between FASN expression levels and factors such as treatment sensitivity, drug resistance, and overall prognosis. Specifically, in CESC, FASN influences tumor behavior via the Akt/mTOR signaling pathway, potentially affecting patients’ treatment responses (20). Inhibiting FASN has been shown to enhance the efficacy of chemotherapy agents. For example, the application of FASN inhibitors, such as orlistat, in in vitro studies has demonstrated a decrease in the viability of cervical cancer cells, alongside the induction of apoptosis, cell cycle arrest, and autophagy, suggesting that FASN inhibition may augment treatment responses (21). Notably, recent research has validated that targeting FASN with inhibitors like TVB-2640 can improve cisplatin sensitivity by promoting ferroptosis mediated by SLC7A11, thereby reversing cisplatin resistance in cervical cancer and exhibiting synergistic anti-tumor effects in both in vivo and in vitro settings (22). Furthermore, FASN expression is modulated by various molecular mechanisms; for instance, the targeting of FASN by miR-497-5p has been shown to inhibit the proliferation and invasion of cervical cancer cells, indicating that FASN may represent a viable therapeutic target that influences cellular responses to treatment interventions (23). Concurrently, FASN facilitates lymph node metastasis through cholesterol reprogramming and lymphangiogenesis, with its inhibition leading to reduced metastatic spread, thereby further highlighting the role of FASN in treatment resistance (24). Additional studies reinforce FASN’s potential as a therapeutic target; for example, computer-aided screenings of FASN inhibitors have revealed anti-proliferative effects in HeLa cells, underscoring the extensive applicability of FASN inhibition in oncological therapies (25). Notably, the association of FASN with clinical outcomes in CESC remains underexplored, highlighting a significant gap in the literature.
This study employs a multifaceted approach, integrating RNA sequencing, differential expressed genes (DEGs) analysis, prognostic model construction, and functional enrichment analysis to investigate the role of FASN in CESC. The advantage of this methodology lies in its ability to comprehensively analyze gene expression differences, thereby identifying potential biomarkers and establishing prognostic models that can enhance clinical decision-making. The primary objective of this research is to validate the efficacy of FASN as an independent prognostic factor and to explore its clinical significance in CESC. By elucidating the relationship between FASN expression and various clinical parameters, this study aims to contribute valuable insights into the molecular mechanisms underlying CESC progression and to identify novel therapeutic targets that could improve patient outcomes. We present this article in accordance with the TRIPOD reporting checklist (available at https://tcr.amegroups.com/article/view/10.21037/tcr-2025-1425/rc).
Methods
RNA‑sequencing data acquisition
We acquired RNA sequencing data about cervical cancer from The Cancer Genome Atlas (TCGA) and the Genotype-Tissue Expression project (GTEx), which had undergone processing via the Toil pipeline, in a standardized manner from the UCSC XENA platform (https://xenabrowser.net/datapages/) (26,27). The validation cohort was obtained from the Gene Expression Omnibus (GEO) database (https://www.ncbi.nlm.nih.gov/gds) and included comprehensive expression profile data from GSE138080. Furthermore, we compiled a comprehensive list of 151 genes linked to fatty acid metabolism, which are collectively referred to as fatty acid metabolism-related genes (FMRGs) (Table S1). These genes were meticulously curated from the ‘HALLMARK_FATTY_ACID_METABOLISM’ gene set available in the Molecular Signatures Database (MSigDB) (https://www.gsea-msigdb.org/gsea/msigdb).
DEGs analysis
The median expression level of FASN served as the threshold to distinguish DEGs across the two classifications (low-expression and high-expression) of FASN within CESC samples (HTSeq-Count). The analysis was conducted using the ‘DESeq2’ R package (28). For the selection of DEGs intended for subsequent functional enrichment analysis, the criteria were established as |log2 fold change (FC)| >1 alongside an adjusted P value of less than 0.05.
The construction of a prognostic model
The ‘sva’ package in R was utilized to eliminate batch effects present in the TCGA and GTEx datasets. To identify prognostic genes and develop a robust prognostic signature, univariate Cox regression analysis was performed via the ‘survival’ R package. The classic random forest algorithm was then employed for feature selection, facilitating the evaluation of the significance of prognostic-related genes with a P value threshold of <0.05. A comprehensive analysis was conducted on 151 genes associated with fatty acid metabolism utilizing the least absolute shrinkage and selection operator (LASSO) Cox regression technique, along with the application of 1,000-fold cross-validation, incorporating a predetermined random seed value of 2022. This analytical approach successfully pinpointed 28 potential prognostic variables pertinent to fatty acid metabolism, which serves to mitigate redundant data and diminish the risk of overfitting within the model. Following this, an intersection analysis was performed with differentially expressed genes, leading to the identification of 8 differential genes associated with fatty acid metabolism. Consequently, these 8 genes were chosen to develop a prognostic risk scoring model intended to forecast the OS of patients diagnosed with CESC. After establishing the median risk score, patients were stratified into high- and low-risk categories.
Functional enrichment analysis
The analyses of Gene Ontology (GO)—encompassing biological processes (BPs), cellular components (CCs), and molecular functions (MFs)—alongside the Kyoto Encyclopedia of Genes and Genomes (KEGG, www.kegg.jp/kegg/kegg1.html) were performed utilizing the ‘ClusteProfiler’ R package (29,30).
Gene set enrichment analysis (GSEA)
The ‘ClusteProfiler’ R package was utilized to investigate the functional and pathway variations between two cohorts exhibiting distinct levels of FASN expression (31). Results of enrichment analyses that satisfied the thresholds of Padj <0.05 and FDR q-value <0.25 were considered to be statistically significant.
Immune infiltration analyses
An immune infiltration analysis of FASN was conducted utilizing single-sample gene set enrichment analysis (ssGSEA) through the ‘GSVA’ R package (32). As indicated earlier, the analysis incorporated 24 distinct types of infiltrating immune cells (33).
Prognostic model development
We conducted both univariate and multivariate Cox regression analyses to determine if FASN could act as an independent prognostic indicator. Clinical parameters, such as age, tumor (T) stage, node (N) stage, metastasis (M) stage, and clinical stage, were integrated into these analyses. Moreover, a nomogram along with a calibration plot was constructed utilizing the ‘rms’ package and the ‘survival’ package to forecast OS at 1-, 3-, and 5-year intervals (34). The model incorporated the same variables that were utilized in the Cox regression analyses. The calibration plot was assessed by contrasting the probabilities forecasted by the nomogram against the actual observed rates, with the diagonal line indicating the optimal predictive value. To evaluate the model’s ability to discriminate, the concordance index (C-index) was computed (35). Additionally, the receiver operating characteristic (ROC) curve was employed to measure the predictive accuracy of the nomogram.
Immunohistochemistry (IHC) staining
The evaluation of FASN protein levels in CESC and corresponding adjacent normal tissues was conducted utilizing IHC methodologies. The primary antibodies include FASN (1:500; Proteintech, Wuhan, China), KI67 (1:1,000; Zhongshan Golden Bridge Biotechnology Co., Beijing, China), D2-40 (1:1,000; Zhongshan Golden Bridge Biotechnology Co.), and CK7 (1:1,000; Zhongshan Golden Bridge Biotechnology Co.). Tissue samples were preserved in 4% paraformaldehyde and subsequently underwent a series of dehydration, embedding, and sectioning procedures by established protocols. The prepared slides were treated with primary antibodies overnight at a temperature of 4 ℃. Following this incubation period, the slides underwent three washes with public broadcasting service (PBS). A secondary antibody was then introduced and allowed to incubate for one hour at room temperature, shielded from light exposure. The nuclei were stained using hematoxylin. Ultimately, microscopic examination and image capture were carried out to facilitate the analysis of the findings.
Tissue specimens
A total of 30 tissue samples from CESC and their adjacent normal counterparts were obtained from the Department of Pathology at the Second Affiliated Hospital, Zhejiang University School of Medicine. Before undergoing surgical intervention, the CESC patients had not been subjected to any preoperative therapies, which included radiation, chemotherapy, or other oncological treatments. The study was conducted in accordance with the Declaration of Helsinki and its subsequent amendments. The study was approved by the Ethics Committee of the Second Affiliated Hospital, Zhejiang University School of Medicine, located in Hangzhou, China (approval No. 2023-1138). Informed consent was waived in this retrospective study.
Statistical analyses
All statistical evaluations and graphical representations were executed utilizing the R programming language (version 3.6.3). The expression levels of FASN were examined through the Wilcoxon rank-sum test for unpaired samples. Cox regression analyses were performed to determine hazard ratio (HR) alongside 95% confidence interval (CI) for various clinical characteristics, aiming to identify independent prognostic factors. Additionally, Kaplan-Meier survival analyses and log-rank tests were applied to assess survival distributions. A two-sided P value of less than 0.05 was deemed statistically significant.
Results
The genomic differences between normal cervix and CESC tissues
To develop a dependable prognostic signature, batch effects present in the TCGA and GTEx datasets were mitigated utilizing the ‘sva’ package in R. Systematic clustering effectively illustrated the genomic disparities distinguishing normal tissues from CESC tissues (Figure 1A). Principal component analysis (PCA) demonstrated a clear segregation between the control and tumor groups, exhibiting minimal overlap (Figure 1B). The volcano plot depicting differentially expressed genes underscored both the upregulation and downregulation of genes in CESC tissues when compared to normal tissues (Figure 1C).
Figure 1.
Identification of DEGs between normal and CESC groups within the TCGA and GTEx datasets. (A) Heatmap displaying prominently upregulated (red) and downregulated (blue) genes in tumor and normal groups under the threshold of |log2 (fold change)| >1 and P<0.05. (B) PCA analysis contrasting tumor (red) and normal (blue) groups. (C) Volcano plot portraying conspicuously upregulated (red) and downregulated (blue) genes in tumor and normal groups under the threshold of |log2 (fold change)| >1 and P<0.05. CESC, cervical squamous cell carcinoma; DEG, differential expressed gene; GTEx, Genotype-Tissue Expression project; PCA, principal component analysis; TCGA, The Cancer Genome Atlas.
Construction and assessment of the prognostic model based on FMRGs
To assess the prognostic significance of fatty acid-related genes in CESC, we established a prognostic risk model employing LASSO regression analysis. This model was constructed by selecting a total of 151 genes, which enabled the stratification of CESC patients into low- and high-risk categories based on OS outcomes through LASSO Cox regression analysis. As depicted in Figure 2A, an initial screening of FMRGs led to the identification of 28 genes suitable for the development of the prognostic risk model. The Venn diagram presented in Figure 2B highlights the existence of eight differentially expressed genes. The findings are supported by the gene heatmap illustrated in Figure 2C. Additionally, the waterfall plot depicted in Figure 2D, which outlines the mutation landscape of the 8 signature molecules, indicated that missense mutations were the most prevalent. To evaluate the prognostic relevance of these genes, univariate Cox regression analysis was conducted, the results of which are illustrated in Figure 2E. The findings revealed that patients exhibiting low expression levels of FASN, HMGCS1, and SERINC1 experienced a significant survival advantage in contrast to those with elevated expression levels of these genes. Furthermore, we investigated the differential mRNA expression of the eight FMRGs in both non-cancerous and tumor tissues, which demonstrated that four of the FMRGs—ACAT2, FASN, HMGCS1, and IL4I1—were markedly upregulated in CESC tissues, whereas ACAA2, SERINC1, and TP53INP2 exhibited diminished expression levels (Figure 2F). Ultimately, a risk score was computed for each sample, based on the expression levels and coefficients of the eight FMRGs incorporated into the model.
Figure 2.
Prognosis value of fatty acid genetic risk model in CESC. (A) LASSO regression analysis identified signature genes. (B) Venn diagram of the FMRGs between LASSO genes and DEGs. (C) A heatmap depicting the expression levels of FMRGs within both normal and CESC cohorts. (D) Mutational profile of key molecules in 289 CESC tissue samples. (E) The forest plot illustrates the prognostic significance of genes associated with fatty acid. (F) Differential mRNA expression of 8 FMRGs associated with clinicopathological features in normal and tumor tissues. *, P<0.05; **, P<0.01; ***, P<0.001. CESC, cervical squamous cell carcinoma; CI, confidence interval; DEG, differential expressed gene; FMRG, fatty acid metabolism-related gene; HR, hazard ratio; LASSO, least absolute shrinkage and selection operator; TMB, tumor mutational burden; TPM, transcript per million.
Patients diagnosed with CESC were categorized into two distinct groups, high- and low-risk, determined by the median threshold of their risk scores. Those within the low-risk category demonstrated a markedly favorable OS in comparison to their high-risk peers. Figure 3A illustrates the analysis of risk scores, survival outcomes, and the expression levels of eight FMRGs across the various risk categories. The accompanying heatmap, which consolidates clinical parameters, indicates that elevated expression levels of FASN correlate with more advanced pathological stages of T and N (Figure 3B). Furthermore, Kaplan-Meier survival analyses revealed a significant association between heightened expression of FASN, ACAT2, HMGCS1, and SERINC1 and unfavorable prognostic outcomes in patients with CESC. Specifically, the increased expression of these genes was associated with diminished survival rates and an expedited progression of the disease, while the expression levels of other genes did not show notable differences in this regard (Figure 3C).
Figure 3.
Establishing the prognostic signature using the TCGA-CESC cohort. (A) Risk score and survival status of each patient. (B) The heatmap illustrates the correlation between 8 FMRGs and a range of clinical parameters. (C) The Kaplan-Meier survival curves demonstrate notable variations in OS linked to 8 FMRGs. CESC, cervical squamous cell carcinoma; CI, confidence interval; FMRG, fatty acid metabolism-related gene; HR, hazard ratio; M, metastasis; N, node; OS, overall survival; T, tumor; TCGA, The Cancer Genome Atlas.
FASN expression in various tumor tissues
To gain a deeper understanding of the influence of FASN expression on the prognosis of cancer patients, we conducted a univariate Cox regression analysis aimed at elucidating the association between FASN expression levels and OS across 33 different cancer types, as illustrated in Figure 4A. A notable finding was that elevated FASN expression was markedly correlated with unfavorable outcomes in patients diagnosed with adrenocortical carcinoma (ACC), bladder urothelial carcinoma (BLCA), CESC, and mesothelioma (MESO). Furthermore, FASN expression was found to be significantly heightened in several malignancies, particularly CESC. Interestingly, the downregulation of FASN in six distinct cancer types indicates a possible role in tumor suppression within these specific contexts (Figure 4B). Additionally, the heatmap correlation analysis provided further validation of the findings obtained from the univariate Cox regression analysis (Figure 4C).
Figure 4.
Expression levels of FASN across various tumor types. (A) The correlation between the expression levels of FASN and OS in patients with different cancer types was evaluated using univariate Cox regression analysis. (B) The expression levels of FASN in tumor tissues were compared to those in normal tissues across different types of cancer, utilizing data sourced from TCGA and the GTEx databases. (C) The correlation between the expression levels of FASN and OS in patients diagnosed with different cancer types was evaluated using heatmap analysis. *, P<0.05; **, P<0.01; ***, P<0.001. CI, confidence interval; GTEx, Genotype-Tissue Expression project; HR, hazard ratio; OS, overall survival; TCGA, The Cancer Genome Atlas; TPM, transcript per million.
Expression and prognosis of FASN-related proteins
A protein-protein interaction (PPI) network was constructed utilizing the STRING database to explore the interacting proteins associated with FASN, as depicted in Figure 5A. A total of ten genes (CAMKMT, KMT5A, KDM1A, TP53, HSP90AB1, HSP90AA1, H3C13, H3C12, SETD3, SETD7) demonstrated a significant correlation with the functionality of FASN. To assess the prognostic significance of these genes, a univariate Cox regression analysis was performed, and the outcomes are presented in Figure 5B. The results reveal that heightened expressions of SETD3 and SETD7 are significantly linked to adverse prognoses in patients diagnosed with CESC. Furthermore, there is a notable increase in the expression levels of TP53, HSP90AA1, and KDM1A in CESC, while the expressions of SETD7 and SETD3 show a significant decline (Figure 5C,5D). The waterfall plot illustrated in Figure 5E, which outlines the mutation landscape of the 7 pivotal molecules, indicated that missense mutations were the most prevalent.
Figure 5.
Expression of FASN-related genes and their prognostic significance. (A) A network diagram depicting the interconnections between FASN and various proteins sourced from the STRING database. (B) The forest plot demonstrates the predictive importance of genes linked to FASN. (C) A heatmap illustrating the expression of FASN-related genes in normal and CESC groups. (D) Differential mRNA expression of FASN-related genes in normal versus tumor tissues. (E) Mutational profile of key molecules in 289 cervical cancer tissue samples. *, P<0.05; **, P<0.01; ***, P<0.001. CESC, cervical squamous cell carcinoma; CI, confidence interval; HR, hazard ratio; TMB, tumor mutational burden; TPM, transcript per million.
Identification of DEGs with FASN and functional enrichment analyses
A total of 1,184 DEGs were discerned between two classifications (low-expression and high-expression) of FASN, utilizing the thresholds of |log2FC| >1 and Padj <0.05. This analysis revealed 611 genes that were upregulated and 573 genes that exhibited downregulation (Figure 6A). Furthermore, the ten genes that exhibited the strongest positive and negative correlations with FASN are depicted in heat maps (Figure 6B,6C). To explore the signaling pathways linked to varying levels of FASN expression in CESC, GSEA was performed employing the MSigDB collection. Among the gene sets that were significantly enriched, eight distinct pathways were identified, which include the integrin-3 pathway, signaling mediated by SREBP, the PPAR signaling pathway, FGFRL1 modulation of FGFR1 signaling, FRS-mediated FGFR4 signaling, PPARα pathway, IRS-mediated signaling, and FRS-mediated FGFR1 signaling (Figure 6D,6E).
Figure 6.
Enrichment analyses from GSEA. (A) Volcano map of the DEGs based on the FASN expression levels. (B) The top 10 positively co-expressed genes of FASN are shown in heat maps. (C) The top 10 negatively co-expressed genes of FASN are shown in heat maps. (D) Bubble plot of GSEA results. (E) GSEA indicated that the high-FASN group was prominently enriched in certain pathways. DEG, differential expressed gene; FDR, false discovery rate; FPKM, fragments per kilobase of transcript per million mapped fragments; GSEA, gene set enrichment analysis; NES, normalized enrichment score.
The outcomes of GO functional analysis alongside KEGG enrichment analysis are summarized below. The BP that was highlighted includes positive regulation of hormone secretion, arachidonic acid metabolic processes, and the metabolism of fat-soluble vitamins. CC identified included axoneme, ciliary plasm, and intermediate filament cytoskeleton. As for MF, these encompassed hormone activity, serine hydrolase activity, and arachidonic acid epoxygenase activity. The KEGG pathways identified included linoleic acid metabolism, PPAR signaling pathway, and arachidonic acid metabolism (Figure 7A-7C, Table 1). These findings imply a potential involvement of FASN in biological functions and signaling pathways that are vital for patients with CESC.
Figure 7.
Functional enrichment analyses of FASN in CESC. (A) The lollipop chart illustrates the results of the GO and KEGG enrichment analyses. (B) The grid map depiction illustrates the results of the enrichment analysis concerning GO and KEGG. (C) The bubble plot depicts the findings from both GO and KEGG enrichment analyses. BP, biological process; CC, cellular compoment; CESC, cervical squamous cell carcinoma; GO, Gene Ontology; KEGG, Kyoto Encyclopedia of Genes and Genomes; MF, molecular function.
Table 1. Supplementary information of GO and KEGG analysis.
| Otology | ID | Description | P value |
|---|---|---|---|
| BP | GO:0046887 | Positive regulation of hormone secretion | <0.001 |
| GO:0019369 | Arachidonic acid metabolic process | 0.001 | |
| GO:0006775 | Fat-soluble vitamin metabolic process | 0.001 | |
| CC | GO:0005930 | Axoneme | <0.001 |
| GO:0097014 | Ciliary plasm | <0.001 | |
| GO:0045111 | Intermediate filament cytoskeleton | 0.009 | |
| MF | GO:0005179 | Hormone activity | <0.001 |
| GO:0017171 | Serine hydrolase activity | <0.001 | |
| GO:0008392 | Arachidonic acid epoxygenase activity | 0.001 | |
| KEGG | hsa00591 | Linoleic acid metabolism | <0.001 |
| hsa03320 | PPAR signaling pathway | 0.003 | |
| hsa00590 | Arachidonic acid metabolism | 0.004 |
BP, biological process; CC, cellular component; GO, Gene Ontology; KEGG, Kyoto Encyclopedia of Genes and Genomes; MF, molecular function.
Immune infiltration analyses in CESC
The infiltration of immune cells within tumors is instrumental in forecasting OS rates. The analysis indicated a positive correlation between the expression levels of FASN and the infiltration of Tem. Conversely, significant negative correlations were identified between FASN expression and the infiltration levels of pDC, iDC, cytotoxic lymphocytes, T lymphocytes, Th1 cells, DC, TFH, and B lymphocytes, all of which exhibited a notable reduction (Figure 8A-8C). The analysis of 24 distinct immune cell subtypes across varying levels of FASN expression indicated that aDC, B cells, cytotoxic lymphocytes, DC, iDC, pDC, T cells, Th1 cells, and TReg were all significantly diminished in the group with elevated FASN expression (Figure 8D).
Figure 8.
Association between FASN expression and immune infiltration in CESC. (A) Correlation between FASN expression and 24 immune cells. (B) The scatter plot demonstrates the relationship between FASN and various immune cell types. (C) The heatmap illustrates the correlation between FASN and different types of immune cells. (D) The infiltrating levels of 24 subtypes of immune cells in high and low FASN expression groups. *, P<0.05; **, P<0.01; ***, P<0.001; ns, P>0.05. CESC, cervical squamous cell carcinoma; TPM, transcript per million.
Association between FASN expression and clinical features
The clinical characteristics of CESC were examined about FASN expression levels, categorized into low and high expression groups. Within the high-expression cohort, there was a notable increase in the percentage of cases exhibiting advanced pathological T stage, pathological N stage, clinical stage, histological grade, OS events, disease-specific survival (DSS) events, and progression-free interval (PFI) events when compared to the low-expression group (Table 2). Furthermore, the levels of FASN expression were analyzed about various clinical attributes. The association between FASN expression and clinical factors was assessed utilizing Wilcoxon signed-rank tests. Increased FASN expression levels were correlated with more advanced T and N stages, later clinical stages, older patient age, decreased body weight, the existence of squamous cell carcinoma, elevated histological grades, and adverse outcomes regarding OS, PFI, and DSS (Figure 9A-9K).
Table 2. The clinicopathological features observed in CESC patients exhibiting high and low levels of FASN expression.
| Characteristics | Low expression of FASN (n=153) | High expression of FASN (n=153) | P value |
|---|---|---|---|
| Pathologic T stage, n (%) | 0.003 | ||
| T1 | 82 (33.7) | 58 (23.9) | |
| T2 | 38 (15.6) | 34 (14.0) | |
| T3 | 6 (2.5) | 15 (6.2) | |
| T4 | 1 (0.4) | 9 (3.7) | |
| Pathologic N stage, n (%) | 0.02 | ||
| N0 | 83 (42.6) | 51 (26.2) | |
| N1 | 27 (13.8) | 34 (17.4) | |
| Pathologic M stage, n (%) | 0.61 | ||
| M0 | 62 (48.8) | 54 (42.5) | |
| M1 | 5 (3.9) | 6 (4.7) | |
| Clinical stage, n (%) | 0.01 | ||
| Stage I | 93 (31.1) | 69 (23.1) | |
| Stage II | 32 (10.7) | 37 (12.4) | |
| Stage III | 18 (6.0) | 28 (9.4) | |
| Stage IV | 6 (2.0) | 16 (5.4) | |
| Histological type, n (%) | 0.49 | ||
| Adenocarcinoma | 26 (8.7) | 21 (7.0) | |
| Squamous cell carcinoma | 126 (42.0) | 127 (42.3) | |
| Histologic grade, n (%) | 0.006 | ||
| G1 | 15 (5.5) | 4 (1.5) | |
| G2 | 73 (26.7) | 62 (22.7) | |
| G3 | 50 (18.3) | 69 (25.3) | |
| OS event, n (%) | <0.001 | ||
| Alive | 130 (42.5) | 104 (34.0) | |
| Dead | 23 (7.5) | 49 (16.0) | |
| DSS event, n (%) | 0.002 | ||
| No | 134 (44.4) | 113 (37.4) | |
| Yes | 17 (5.6) | 38 (12.6) | |
| PFI event, n (%) | <0.001 | ||
| No | 130 (42.5) | 104 (34.0) | |
| Yes | 23 (7.5) | 49 (16.0) |
CESC, cervical squamous cell carcinoma; DSS, disease-specific survival; M, metastasis; N, node; OS, overall survival; PFI, progression-free interval; T, tumor.
Figure 9.
Association between FASN expression and clinical features. (A-J) The correlation between FASN expression and multiple clinical parameters—such as the T stage, N stage, clinical stage, age, weight, histological type, histologic grade, OS events, DSS events, and PFI events—have been explored. (K) The circular illustration illustrates the relationship between the expression levels of FASN and various clinical pathological features associated with CESC. *, P<0.05; **, P<0.01. CESC, cervical squamous cell carcinoma; DSS, disease-specific survival; FPKM, fragments per kilobase of transcript per million mapped fragments; M, metastasis; N, node; OS, overall survival; PFI, progression-free interval; T, tumor.
Relationship between FASN expression and prognosis
The clinical attributes, such as T stage, N stage, and FASN expression, were integrated into the nomogram model (Figure 10A). Importantly, the expression of FASN has demonstrated a capacity to improve the precision of survival probability forecasts at the 1-, 3-, and 5-year time points. Calibration plots demonstrated that the predicted probabilities aligned well with the actual observed outcomes (Figures 10B-10D). Additionally, we constructed time-dependent ROC curves and calibration plots for forecasting the OS rates at 1-, 3-, and 5-year durations (Figure 10E). Cox regression analysis revealed several significant predictors, including pathological T stage, pathological N stage, pathological M stage, clinical stage, histological grade, and FASN expression level, all of which were found to be significantly correlated with OS (Figure 10F). These risk factors were subsequently examined through multivariate Cox regression analysis. The findings indicated that FASN serves as an independent prognostic factor (HR =5.341, 95% CI: 1.763–16.178, P=0.003). The analysis of the ROC curve further validated the strong efficacy of our risk model, yielding AUC values of 0.942 and 0.708 (Figure 11A,11B). Furthermore, within the cohort of patients classified as T1&T2 stage, as well as those in clinical stages I&II and of younger age, a noteworthy survival benefit was observed in individuals demonstrating reduced expression of FASN (Figure 11C-11H). In contrast, higher levels of FASN expression were typically associated with lower DSS rates and abbreviated progression-free survival durations (Figure 11I,11J).
Figure 10.
Prognostic prediction model for FASN in CESC. (A) Nomogram for predicting 1-, 3-, and 5-year OS in patients with CESC. (B-D) Calibration plots for predicting 1-, 3-, and 5-year OS. (E) Time-dependent ROC curves and AUC values for predicting 1-, 3-, and 5-year OS. (F) Validation of Cox regression analyses for OS in CESC. AUC, area under the curve; CESC, cervical squamous cell carcinoma; CI, confidence interval; FPR, false positive rate; HR, hazard ratio; M, metastasis; N, node; OS, overall survival; ROC, receiver operating characteristic; T, tumor; TPR, true positive rate.
Figure 11.
The relationship between OS and the expression levels of FASN across different clinical subgroups of CESC. (A,B) The parameters were evaluated using ROC curves. (C-H) In patients classified with T1 and T2 stages, as well as clinical stages I and II, those who are younger and show reduced levels of FASN expression exhibit a notable advantage in survival rates. (I,J) Elevated levels of FASN expression were associated with unfavorable DSS and PFI outcomes in CESC. The data are presented as HR (95% CI) in (C-J). AUC, area under the curve; CESC, cervical squamous cell carcinoma; CI, confidence interval; DSS, disease-specific survival; HR, hazard ratio; OS, overall survival; PFI, progression-free interval; ROC, receiver operating characteristic; T, tumor.
Validation of FASN expression within the GEO dataset
The mRNA expression observed in the validation dataset (GSE138080) demonstrated consistency. An increase in FASN mRNA levels was noted in tumor samples (Figure 12A). The volcano plot distinctly depicts the genes that are differentially expressed. Additionally, the examination of the ROC curve reinforced the robust performance of the dataset (Figure 12B). Collectively, these results indicate that FASN is expressed at differential levels in CESC, exhibiting heightened expression levels (Figure 12C).
Figure 12.
Verification and ROC curve of the FASN by GEO datasets. (A) The volcano plot highlights genes exhibiting significant upregulation and downregulation. (B) The diagnostic significance of FASN in CESC was evaluated through the use of ROC curves. (C) The assessment of FASN mRNA expression was conducted utilizing the GSE138080 dataset. **, P<0.01. AUC, area under the curve; CESC, cervical squamous cell carcinoma; CI, confidence interval; FPR, false positive rate; GEO, Gene Expression Omnibus; ROC, receiver operating characteristic; TPR, true positive rate.
FASN expression levels in tissue samples of CESC patients
The examination conducted via hematoxylin and eosin (HE) staining indicated that the tumor cells associated with cervical adenocarcinoma displayed histological characteristics akin to moderately differentiated gastric-type adenocarcinoma. Conversely, the tumor cells identified in CESC exhibited features representative of non-keratinizing squamous cell carcinoma (Figure 13). KI67 serves as a crucial biomarker for evaluating tumor malignancy in both clinical and research settings. An elevation in KI67 expression is generally indicative of heightened cell division activity, accelerated proliferation, and increased invasiveness, all of which correlate with a poorer prognostic outlook. D2-40 is specifically utilized to delineate lymphatic vessel structures within tumor tissues, thereby functioning as a significant instrument for the staging of malignant tumors. CK7, a protein specific to epithelial cell intermediate filaments, is prominently expressed in adenocytic epithelium and its derived tumors. The results from immunohistochemical analysis demonstrated markedly elevated expression levels of FASN, KI67, and D2-40 in both cervical adenocarcinomas and squamous cell carcinomas when compared to normal cervical tissue (Figure 14A). In patients diagnosed with CESC, those presenting lower levels of FASN demonstrated significantly enhanced survival rates when contrasted with patients exhibiting high FASN expression levels (Figure 14B).
Figure 13.
Application of HE staining for examining normal cervical tissue, cervical squamous cell carcinoma, and cervical adenocarcinoma. The level of magnification is 200-fold. HE, hematoxylin and eosin.
Figure 14.
Validation of FASN expression in clinical samples from patients with CESC. (A) The expression levels of the FASN protein were elevated in CESC tissues when contrasted with normal tissues. (B) Kaplan-Meier plot of the survival outcomes of patients with CESC, categorized by high or low FASN expression levels. The technique employed for staining is known as IHC staining. The level of magnification is 200-fold. CESC, cervical squamous cell carcinoma; CI, confidence interval; HR, hazard ratio; IHC, immunohistochemistry.
Discussion
Cervical cancer, especially CESC, poses a considerable public health issue worldwide, ranking among the most commonly diagnosed cancers in females (1). The World Health Organization estimates that cervical cancer is responsible for nearly 300,000 fatalities each year, predominantly affecting individuals in developing nations (36). Current treatment modalities, such as surgical intervention, radiotherapy, and chemotherapy, demonstrate inconsistent effectiveness, particularly in the later stages of the disease, resulting in unfavorable prognoses for patients. Consequently, there is a pressing necessity for research focused on the discovery of new biomarkers and therapeutic strategies to enhance patient outcomes. This investigation seeks to fill this significant void by exploring the function of FASN in CESC, which may shed light on its underlying mechanisms and prospective clinical applications. In this research, we aimed to clarify the role of FASN in CESC through a multifaceted approach that incorporated RNA sequencing, DEG analysis, the construction of prognostic models, and functional enrichment assessments. The methodology employed in this study is particularly beneficial as it facilitates a thorough exploration of gene expression fluctuations, the identification of promising biomarkers, and the development of prognostic frameworks. Our main goal was to confirm the effectiveness of FASN as an independent prognostic marker and to investigate its clinical relevance in CESC. The results of this research not only advance the comprehension of FASN’s role in CESC but also open avenues for further studies into its therapeutic possibilities and biological consequences.
The pathways uncovered via GSEA offer an essential understanding of the biological mechanisms that govern FASN expression in CESC. Notably, the PPAR signaling pathway is particularly significant, as it plays a crucial role in the regulation of lipid metabolism and cellular differentiation (37). PPARs, or peroxisome proliferator-activated receptors, are a class of nuclear receptor proteins that regulate the expression of genes associated with lipid balance and inflammatory responses when activated by fatty acids or their metabolites (38). Dysregulation of peroxisome proliferator-activated receptor (PPAR) signaling has been associated with multiple types of cancer, one of which is cervical cancer, where it potentially affects both tumor proliferation and the process of metastasis (39). The correlation between FASN and the PPAR pathway indicates that elevated FASN expression could foster a metabolic milieu that supports tumor advancement, thereby underscoring the therapeutic potential of targeting this pathway. Additionally, our analysis reveals that the SREBP-mediated signaling pathway is another crucial pathway of interest. SREBPs, functioning as transcription factors, are essential for lipid synthesis and the regulation of cellular cholesterol balance (40). The stimulation of SREBPs results in the enhanced expression of genes that play critical roles in the biosynthesis of fatty acids and cholesterol. These metabolic pathways are frequently exploited by neoplastic cells to facilitate their rapid growth and ensure their survival (41). The association observed between FASN and SREBP signaling in CESC suggests that FASN could function not merely as an indicator of aggressive disease but also as an active participant in the metabolic reprogramming inherent to cancerous cells. The role of this pathway highlights the critical nature of lipid metabolism within the realm of cancer biology, implying that strategies aimed at modulating SREBP activity might prove advantageous for the treatment of CESC. Furthermore, the integrin-3 pathway, which demonstrated significant enrichment, is recognized for its role in facilitating cell adhesion and signaling within the tumor microenvironment (42). Integrins serve as transmembrane receptors that enable interactions between cells and the extracellular matrix, thereby affecting various cellular processes such as migration, proliferation, and survival. The interaction between FASN and integrin signaling pathways may play a significant role in the invasive characteristics of CESC. Specifically, an increase in FASN expression could facilitate a more aggressive phenotype by engaging integrin-mediated signaling pathways.
The presence of immune cells infiltrating tumors serves as a significant determinant of OS outcomes in CESC. Our investigation demonstrated a positive association between the expression levels of FASN and the infiltration of Tem, indicating that elevated FASN levels may promote a more vigorous immune response. Effector memory T cells are crucial components of the adaptive immune system, enabling swift reactions to antigens that have been previously recognized (43,44). The existence of these cells within the tumor microenvironment is frequently linked to a more favorable prognosis, given their capability to proficiently identify and eradicate cancerous cells (45). Consequently, the identified association between the expression of FASN and the infiltration of tumor-associated macrophages may suggest a plausible mechanism by which FASN affects tumor advancement and influences patient prognoses. On the other hand, our results revealed noteworthy negative correlations between FASN expression and a range of immune cell types, such as pDC and cytotoxic lymphocytes. Plasmacytoid dendritic cells are recognized for their capability to synthesize type I interferons, thereby serving an essential function in the initiation of immune responses targeting tumors (46). The observed decrease in pDC infiltration in tumors exhibiting high levels of FASN expression may indicate a potential immune evasion mechanism utilized by the tumor, which could be associated with unfavorable prognostic outcomes. In addition, the reduced presence of cytotoxic lymphocytes—crucial for the direct elimination of cancer cells—further emphasizes the immunosuppressive milieu promoted by increased FASN expression. This association underscores the significance of FASN, not merely as a metabolic regulator, but also as a modulator of immune responses within the tumor microenvironment. Moreover, the notable inverse relationship between FASN expression and Treg infiltration merits further exploration. TReg cells are recognized for their ability to dampen immune responses and sustain tolerance, frequently facilitating tumor progression by suppressing effective anti-tumor immunity (47). The relationship between the expression of FASN and the infiltration of TReg may shed light on the mechanisms underlying immune evasion in CESC. A comprehensive understanding of the role FASN plays in modulating the composition of immune cell populations within the tumor microenvironment could lead to innovative therapeutic approaches designed to bolster anti-tumor immunity, thereby potentially enhancing patient outcomes in cases of CESC. These revelations not only deepen our comprehension of the tumor microenvironment but also underscore the promise of targeting FASN as a viable therapeutic strategy to improve immune-mediated tumor control. Additional research is necessary to clarify the specific mechanisms through which FASN influences immune cell dynamics and to investigate its viability as a therapeutic target in CESC.
The survival analysis performed in this study highlights the significant prognostic value of FASN expression in CESC. The AUC values for survival predictions at 1, 3, and 5 years were determined to be 0.685, 0.742, and 0.636, respectively, thereby reinforcing the model’s predictive capability. These findings align with previous studies that have recognized FASN as a vital factor affecting tumor progression and patient outcomes in various cancers, such as clear cell renal carcinoma and bladder cancer (48,49). The multivariate Cox regression analysis revealed that the expression of FASN, along with various clinical parameters including pathological T and N staging, serves as a noteworthy predictor of OS. The HR linked to FASN expression was determined to be 5.341, indicating that heightened levels of FASN are associated with more than a fivefold increase in the risk of adverse outcomes. This finding reinforces its potential role as a crucial biomarker for patient stratification according to prognosis. This result is consistent with other studies that have similarly highlighted the prognostic significance of metabolic enzymes within cancer research (50). Calibration plots demonstrated that the predicted survival probabilities closely corresponded with the actual observed outcomes, indicating the model’s potential applicability in clinical settings for guiding treatment decisions. Incorporating FASN expression into clinical protocols may improve the accuracy of prognostic assessments and facilitate the development of personalized treatment strategies. This discovery positions FASN as an independent prognostic marker, highlighting its prospective role as a biomarker for patient stratification in clinical practice. The inclusion of FASN expression levels in clinical decision-making could enhance the management of CESC, directing therapeutic approaches and tracking disease progression. Future research endeavors should focus on validating these findings across larger, multicenter cohorts and exploring the mechanisms by which FASN influences tumor biology and patient survival. Collectively, the results of this study contribute to an expanding body of evidence that underscores FASN’s significance as a biomarker in CESC, with potential implications for patient management and outcomes.
Additionally, histopathological analysis of CESC specimens revealed signs of fibrosis and infiltration by inflammatory cells, both of which are commonly associated with tumor progression. The presence of fibrosis and inflammatory cells can significantly alter tumor dynamics by enabling the remodeling of the extracellular matrix, thereby creating a microenvironment that supports tumor growth and metastatic dissemination. The identified association between FASN expression and these histological changes suggests that FASN may play a crucial role in modulating the tumor microenvironment, subsequently influencing tumor progression and patient prognosis. These findings are significant as they indicate that FASN not only serves as a marker of poor prognosis but may also be integral to the histopathological characteristics of CESC. A thorough understanding of the interactions between FASN expression and histological alterations could provide valuable insights into the mechanisms underlying tumor invasiveness, ultimately informing the development of targeted therapies. Future investigations should prioritize elucidating the specific molecular pathways through which FASN influences cellular and tissue structures, along with its interactions with the immune microenvironment, to fully harness its potential as a therapeutic target in CESC.
The constraints of this research primarily arise from the absence of thorough validation through empirical laboratory experiments, which may compromise the dependability of our conclusions. Nonetheless, the investigation by Du et al. indicated that the inhibition of FASN expression can markedly reduce the proliferation and migratory capacity of HeLa cells (24). Moreover, the relatively limited sample size may restrict the broader applicability of the results, as a larger cohort could yield more definitive insights concerning the prognostic significance of the identified FMRGs. These considerations underscore the imperative for additional validation and investigations within larger, more diverse populations to substantiate the clinical significance of the proposed genetic risk prediction model. Future studies should incorporate laboratory experiments to elucidate the roles of these genes in various biological functions, thereby offering a more comprehensive understanding of the underlying biological mechanisms involved. Additionally, subsequent research should strive to encompass larger and more heterogeneous sample populations to augment the reliability and practical application of the model.
In conclusion, the findings of this research emphasize the critical importance of FASN in CESC, indicating its promise as both a prognostic biomarker and a target for therapy. The thorough examination of gene expression, prognostic modeling, and immune response reveals the complex roles that FASN plays in tumor biology. Significantly, the link between elevated FASN expression and poor clinical outcomes, along with its relationship to immune cell infiltration patterns, implies that FASN may affect tumor progression through mechanisms of immune evasion. Additionally, the exploration of key signaling pathways related to FASN offers a basis for future therapeutic approaches aimed at regulating its function. Although there are certain limitations in this study, such as the lack of experimental validation and the possibility of batch effects, the results provide valuable insights into CESC and lay the groundwork for subsequent research into the clinical significance and therapeutic prospects of FASN.
Conclusions
The findings suggest that elevated levels of FASN expression in CESC are associated with heightened disease severity, poor prognostic outcomes, and abnormalities in immune cell infiltration. It is anticipated that the identification of FASN will enhance individualized treatment strategies for CESC patients and support more informed choices in clinical practice.
Supplementary
The article’s supplementary files as
Acknowledgments
We acknowledge the contributions from TCGA, GTEx, GEO databases.
Ethical Statement: The authors are accountable for all aspects of the work in ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved. The study was conducted in accordance with the Declaration of Helsinki and its subsequent amendments. The study was approved by the Ethics Committee of the Second Affiliated Hospital, Zhejiang University School of Medicine, located in Hangzhou, China (approval No. 2023-1138). Informed consent was waived in this retrospective study.
Footnotes
Reporting Checklist: The authors have completed the TRIPOD reporting checklist. Available at https://tcr.amegroups.com/article/view/10.21037/tcr-2025-1425/rc
Funding: The present study was funded by grants from the National Natural Science Foundation of China Youth Fund Project (No. 82302903), the Medical Health Science and Technology Project of Zhejiang Provincial Health Commission (No. 2025KY938), and Lianyungang Traditional Chinese Medicine Hospital “Zhao Huanan” Youth Science and Technology Fund Project (No. Lzyq2401).
Conflicts of Interest: All authors have completed the ICMJE uniform disclosure form (available at https://tcr.amegroups.com/article/view/10.21037/tcr-2025-1425/coif). The authors have no conflicts of interest to declare.
Data Sharing Statement
Available at https://tcr.amegroups.com/article/view/10.21037/tcr-2025-1425/dss
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