GEO Cohorts Research Articles (Page 1)

BackgroundNETO2 is a gene with potential prognostic value in various cancers, but its role in oral squamous cell carcinoma (OSCC) remains unclear. This study investigates NETO2 expression, clinical significance, and its association with the tumor immune microenvironment in OSCC.MethodsNETO2 expression in OSCC tissues was analyzed using public databases and correlated with prognosis via Kaplan–Meier survival analysis. A nomogram incorporating NETO2 and clinical factors was developed to predict survival, and its performance was assessed using ROC curves. Single-cell RNA sequencing (scRNA-seq) analyzed cell types and NETO2 expression across different cell populations. The relationships between NETO2 expression, tumor mutation burden (TMB), immune microenvironment, and drug sensitivity were explored, and molecular docking assessed NETO2's binding affinity with small-molecule compounds. The functional role of NETO2 in OSCC cell invasion, proliferation, and migration was validated experimentally.ResultsNETO2 was significantly overexpressed in OSCC tissues, and high expression correlated with shorter overall survival (OS) and progression-free survival (PFS). The nomogram model, developed using TCGA and GEO cohorts, demonstrated good predictive performance, with AUC values for 1-, 3-, and 5-year survival rates above 0.66 in both cohorts. scRNA-seq revealed elevated NETO2 expression in T cell clusters and significant associations with key immune signaling pathways, such as cytokine-cytokine receptor interactions and T cell receptor signaling. The high NETO2 expression group exhibited higher immune cell infiltration, increased potential for immunotherapy response, and differential sensitivity to various anticancer drugs. Molecular docking showed strong binding of NETO2 with compounds like ruxolitinib, paclitaxel, and docetaxel. Additionally, NETO2's role in OSCC cell invasion, proliferation, and migration was experimentally confirmed.ConclusionNETO2 is a significant prognostic biomarker in OSCC, potentially influencing tumor progression through modulation of the immune microenvironment. Its therapeutic targeting warrants further investigation.Supplementary InformationThe online version contains supplementary material available at 10.1186/s12885-025-15164-y.

BackgroundIncreasing evidence implicates the NF-κB/p65 signaling pathway in the progression of prostate cancer (PC) and the development of resistance to androgen deprivation therapy. This study aimed to investigate the potential role of UHRF1, a key epigenetic regulator, in modulating NF-κB activity, and to evaluate its association with clinicopathological features and clinical outcomes in PC patients.MethodsDifferentially expressed genes (DEGs) were identified using the GSE104749 dataset. UHRF1 expression was validated across TCGA and GEO cohorts and further confirmed in clinical specimens using Western blotting and immunohistochemistry. Survival outcomes were evaluated using Kaplan–Meier and Cox regression analyses. A prognostic model was built incorporating UHRF1, Gleason score, and PSA, with validation via ROC curves and nomogram. Functional studies assessed the impact of UHRF1 silencing or overexpression on cell proliferation, apoptosis, cell cycle, glucose metabolism, and NF-κB signaling. Co-immunoprecipitation was used to assess the physical interaction between UHRF1 and p65.ResultsHigher Gleason scores, advanced clinical stage, lymph node involvement, and distant metastases were all positively connected with UHRF1 expression, which was markedly overexpressed in PC tissues. High UHRF1 expression was associated with shorter overall survival (OS) and disease-free survival (DFS), and independently predicted biochemical recurrence (BCR). A prognostic model incorporating UHRF1 achieved a high predictive accuracy (C-index = 0.752), and the corresponding nomogram demonstrated strong reliability in individualized risk assessment. In vitro, UHRF1 promoted tumor progression by enhancing proliferation, inhibiting apoptosis, driving aerobic glycolysis, and regulating the cell cycle. Mechanistically, UHRF1 bound to p65, promoted its phosphorylation, and activated NF-κB signaling.ConclusionUHRF1 contributes to prostate cancer progression by driving tumor growth, metabolic reprogramming, and NF-κB activation. It holds promise as both a prognostic biomarker and a potential therapeutic target in PC.Supplementary InformationThe online version contains supplementary material available at 10.1186/s12885-025-15091-y.

Development and multi-cohort validation of a prognostic risk score model for oral squamous cell carcinoma based on a three-gene signature.

BackgroundOsteonecrosis of the femoral head (ONFH) is a challenging global health issue with an unclear pathogenesis, complicating the development of effective treatment strategies. Bone marrow edema (BME) is a critical imaging indicator of ONFH progression, yet its underlying mechanisms remain poorly understood.MethodsBioinformatics was employed to identify gene characteristic of BME in ONFH patients. Expression of PTGS2 was validated in these patients through Western blot and ELISA assays. Clinical relevance was assessed by analyzing the correlation between PTGS2 expression levels and pain severity as well as the timing of total hip replacement surgery. In addition to GSE74089, we externally validated PTGS2 in an independent GEO cohort (GSE123568, human serum; GPL15207) using single‐gene receiver operating characteristic (ROC) analysis.ResultsSix hundred and eighty‐eight overlapping targets were identified for ONFH and BME, with 15 key targets shared with XLGBC, including PTGS2, IGFBP3, MCL1, TNF, F7, PLA2G4A, PRKCA, MMP1, and PTGER3. GO and KEGG enrichment analyses indicated that XLGBC exerts its effects mainly through pathways related to inflammation, pain, angiogenesis, and bone metabolism, notably involving VEGF signaling, arachidonic acid metabolism, and MAPK pathways. Molecular docking revealed strong binding between XLGBC compounds and the target genes. ELISA results indicated that higher PTGS2 levels correlated with increased pain severity in ONFH patients, and Western blot analysis showed significantly elevated PTGS2 in ONFH patients compared to controls, with levels decreasing after XLGBC treatment. Patients with higher PTGS2 expression showed shorter times to hip replacement surgery, suggesting faster disease progression. In the external cohort (GSE123568), PTGS2 showed good diagnostic discrimination for ONFH versus controls (AUC = 0.86), supporting the robustness of our bioinformatics findings.ConclusionsPTGS2 is an important gene in ONFH with BME, influencing pain and disease progression. Monitoring PTGS2 expression may help to assess symptom severity and inform surgical timing in ONFH patients.

Background Breast cancer persists as a principal contributor to global cancer mortality, driven by heterogeneous molecular pathways. Necrosis by sodium overload, a recently characterized form of regulated cell death, remains underexplored in oncogenesis. This study investigates the pathobiological significance and therapeutic potential of NECSO-related genes in breast cancer, elucidating their mechanistic roles in tumor progression. Methods Multi-omics analyses were performed using transcriptomic data from TCGA and GEO cohorts ( n = 1, 217), we systematically evaluated seven NECSO-related genes. Advanced bioinformatics pipelines included differential expression analysis, immune subtype profiling, functional state correlation, protein interaction mapping, and survival analytics. Experimental validation involved immunohistochemical evaluation of clinical samples. Results Through multi-omics analysis of GEO and TCGA cohorts, we identified two sodium homeostasis-related genes, TRPM4 and SLC9A1, as consistently upregulated oncogenes in breast cancer, with significant diagnostic and prognostic relevance. Functional in vitro assays demonstrated that knockdown of either gene not only suppressed proliferation, colony formation, migration, and induced apoptosis in breast cancer cells, but also led to reduced expression of the sodium-calcium exchanger NCX1. Conclusion TRPM4 and SLC9A1 is a novel prognostic biomarker and potential therapeutic target in breast cancer. Dysregulated sodium homeostasis mediated by NECSO-related genes represents a targetable vulnerability in precision oncology.

ObjectiveOvarian cancer is one of the most lethal gynecological malignancies, with high recurrence rates and poor prognosis. Identifying molecular mechanisms underlying recurrence is critical for improving patient outcomes. This study aimed to develop a recurrence-related gene signature (RRGS) for prognosis and explore the role of Myeloid/Lymphoid or Mixed-Lineage Leukemia; Translocated To 6 (MLLT6), a key gene within the RRGS, in ovarian cancer progression and drug resistance.MethodsTCGA and GEO cohorts were analyzed to identify differentially expressed genes (DEGs) associated with recurrence. LASSO regression was applied to construct an eight-gene RRGS, which was validated in independent cohorts. Functional assays, including proliferation, migration, invasion, and drug resistance tests, were conducted in OVCAR3, TOV-21G and ES-2 cell lines to evaluate the role of MLLT6.ResultsThe RRGS effectively stratified patients into high- and low-risk groups, with high RRGS scores correlating with poorer overall survival and progression-free survival. Among the RRGS genes, MLLT6 was identified as the most significant prognostic marker. Functional assays showed that MLLT6 knockdown inhibited ovarian cancer cell proliferation, migration, invasion, and significantly enhanced Paclitaxel sensitivity by reducing IC50 values.ConclusionThe RRGS is a robust prognostic tool for ovarian cancer, and MLLT6 plays a critical role in tumor progression and drug resistance. Targeting MLLT6 may provide a novel therapeutic strategy to overcome Paclitaxel resistance and improve patient outcomes.Supplementary InformationThe online version contains supplementary material available at 10.1186/s13048-025-01791-3.

Ferroptosis and cellular senescence -Related Genes in Cervical Cancer: Mechanistic Insights from Multi-Omics and Clinical Sample Analysis.

Breast cancer (BRCA) remains the most common malignancy and the leading cause of cancer-related death among women worldwide. Recent studies have highlighted dysregulated copper homeostasis as a contributor to tumorigenesis, with cuproptosis-a copper-dependent form of regulated cell death-emerging as a potential therapeutic target. In this study, we systematically evaluated the prognostic significance of cuproptosis-related genes (CRGs) in BRCA by integrating multi-omics data from TCGA and GEO cohorts. Through weighted gene co-expression network analysis (WGCNA), we identified four key CRGs (CCDC24, TMEM65, XPOT, and NUDCD1) to construct a prognostic signature. The resulting risk score effectively stratified patients into high- and low-risk groups, with the high-risk group showing significantly worse overall survival, higher TP53 mutation frequency, and features of an immunosuppressive tumor microenvironment (TME). Functional enrichment analyses further implicated these genes in immune evasion and metabolic reprogramming pathways. Single-cell RNA sequencing (scRNAseq) confirmed heterogeneous expression of the signature genes across distinct cell populations, supporting their involvement in both tumor biology and immune modulation. Clinically, this CRG-based model provides a promising tool for individualized risk assessment and treatment planning. High-risk patients may benefit from intensified therapies or immunomodulatory strategies, while low-risk individuals could be considered for treatment de-escalation. In summary, our findings suggest that cuproptosis may play a regulatory role in BRCA progression and offer a novel, clinically actionable framework for prognostic stratification.

Background: Cancer stem cells (CSCs) are key drivers of tumorigenesis and metastasis. However, the precise roles of CSC-associated genes in these processes remain unclear. Methods: This study integrates cancer stem cell biomarkers and clinical data from The Cancer Genome Atlas (TCGA) specific to bladder cancer (BLCA). By combining differentially expressed genes (DEGs) from TCGA-BLCA samples with CSC-related biomarkers, we conducted comprehensive functional analyses and developed an 8-gene prognostic signature through Cox regression, least absolute shrinkage and selection operator (LASSO) analysis, and multivariate Cox regression. This model was validated with GEO datasets (GSE13507 and GSE32894), and the single-cell RNA seq dataset GSE222315 was subsequently analyzed to characterize the signature genes and elucidate their interactions. And a nomogram was created to stratify TCGA-BLCA patients into risk categories. The ‘oncoPredict’ algorithm based on the GDSC2 dataset assessed drug sensitivity in BLCA. Result: From the TCGA cohort, 665 CSC-related genes were identified, with 120 showing significant differential expression. The 8-gene signature (ALDH1A1, CBX7, CSPG4, DCN, FASN, INHBB, MYC, NCAM1) demonstrated strong predictive power for overall survival in both TCGA and GEO cohorts, as confirmed by Kaplan–Meier and ROC analyses. The nomogram, integrating age, tumor stage and risk scores, demonstrated high predictive accuracy. Additionally, the oncoPredict algorithm indicated varying drug sensitivities across patient groups. Based on retrospective data, we identified a novel CSC-related prognostic signature for BLCA. This finding suggests that targeting these genes could offer promising therapeutic strategies.

BackgroundLung squamous cell carcinoma (LUSC), a therapeutically challenging non-small cell lung cancer (NSCLC) subtype with a poor prognosis, exhibits heterogeneous responses to immunotherapy. Cuproptosis, a recently discovered regulated cell death pathway, has been hypothesised to modulate the tumour immune microenvironment (TIME). Despite the well-established role of PDHA1 as a metabolic regulator, the specific mechanisms by which it interacts with GLS in cuproptosis-mediated immune-metabolic crosstalk remain to be elucidated in LUSC. The present study investigates the manner in which GLS/PDHA1 expression patterns influence TIME composition and contribute to the stratification of immunotherapy responsiveness.MethodsIt was determined that GLS and PDHA1 were the most significant copper oxidation-related genes, due to their highest absolute correlation with the ESTIMATE immune score. A consensus clustering analysis was conducted on a cohort of 501 TCGA-LUSC patients, with the objective of stratifying patients based on GLS/PDHA1 expression levels. Quantitative analysis of immune infiltration was performed using ESTIMATE, CIBERSORT, and ssGSEA methods. The pathway enrichment analysis was conducted using GSEA and WGCNA. A detailed analysis of 17,050 single-cell RNA sequencing (scRNA-seq) data from two LUSC patients was conducted, which revealed unique gene expression patterns. The validity of these findings was confirmed through the integration of four independent GEO cohorts (GSE181043/37745/43580/115457; n = 278).ResultsConsensus clustering delineated two subtypes:Cluster 1 (low GLS/high PDHA1) and Cluster 2 (high GLS/low PDHA1). Cluster two showed enhanced immune infiltration, characterized by: Elevated immune checkpoint expression and Enriched T-cell activation pathways. Validation across four GEO cohorts confirmed Cluster two conserved immune-hot phenotypewith elevated ESTIMATE stromal scores, reduced tumor purity, and activated immune subsets. scRNA-seq identified malignant epithelial cells as the hub of divergent GLS/PDHA1 expression (high GLS/low PDHA1), orchestrating cuproptosis-immunometabolic crosstalk.ConclusionGLS and PDHA1 have been proposed as potential prognostic markers for immunotherapy. Targeting cuproptosis has the potential to convert immunologically cold to hot tumours, thereby advancing precision immunotherapy.

BackgroundCrotonylation, a post-translational modification, is implicated in cancer progression, but its prognostic significance in clear cell renal cell carcinoma (ccRCC) remains unclear. This study aimed to demystify crotonylation heterogeneity and establish a robust prognostic model for ccRCC.MethodsUsing multi-omics approaches, we analyzed transcriptomic data from TCGA-KIRC and GEO cohorts (GSE40435, GSE167573, GSE29609). Crotonylation scores were calculated via ssGSEA, with related gene modules identified through WGCNA. We integrated 10 machine learning algorithms to develop a prognostic model. Immune microenvironment was profiled using Cibersort, mutation landscapes via maftools, and drug sensitivity through oncoPredict. Spatial transcriptomics and single-cell data were analyzed for expression patterns, validated by qRT-PCR in 786-O and HK-2 cell lines.ResultsDysregulation of 16/18 crotonylation-related genes was observed in ccRCC. WGCNA revealed crotonylation related modules significantly enriched in angiogenesis, calcium/Ras signaling, and cancer stemness pathways. A 5-gene prognostic model (PLCL1, DNASE1L3, CD248, CDH13, PDGFD) demonstrated robust stratification: High-risk patients showed poorer overall survival, higher Treg infiltration, elevated tumor mutation burden and increased sensitivity to several chemotherapy approaches like Cisplatin. Molecular docking identified diacetylmorphine as a potential therapeutic agent (binding energy: -7.278 kcal/mol with DNASE1L3). Spatial/single-cell analyses confirmed cell-type-specific gene expression and the diffferential expression between tumor and normal cell lines was validated by qRT-PCR.ConclusionThis study establishes a crotonylation-based prognostic model that effectively stratifies ccRCC risk and elucidates key mechanisms linking crotonylation heterogeneity to immune evasion, mutational burden, and metabolic reprogramming. The model offers clinical utility for personalized therapy selection.Supplementary InformationThe online version contains supplementary material available at 10.1186/s12894-025-01914-4.

Pseudoautosomal regions (PARs), located at the ends of sex chromosomes, harbor genes that may play a role in tumor pathology by regulating cell proliferation and the immune microenvironment. Gastric cancer (GC) is a prevalent and molecularly heterogeneous malignancy of the digestive system. However, studies on the role of PARs-related genes in GC are limited. This study focuses on the PARs gene CSF2RA and its regulatory role in GC progression through the JAK2/STAT3 signaling pathway. Differentially expressed PARs-related genes associated with GC were identified using multi-omics datasets(including TCGA-STAD and four GEO cohorts). Functional enrichment, immune infiltration, and drug sensitivity analyses, combined with in vitro (using MKN45 and AGS cell lines) and in vivo experiments, were conducted to validate the role of CSF2RA in GC. Additionally, RT-qPCR, Western blot, immunofluorescence, and co-immunoprecipitation assays were performed to investigate the interaction between CSF2RA and JAK2 and their activation of downstream signaling pathways. CSF2RA was found to be highly expressed in GC and associated with poor prognosis. It was significantly enriched in the JAK/STAT signaling pathway and closely related to immune cell infiltration. Furthermore, CSF2RA promoted GC cell proliferation and metastasis by activating the JAK2/STAT3 pathway. The JAK inhibitor Ruxolitinib effectively reversed the tumor-promoting effects of CSF2RA and demonstrated significant inhibitory effects in both in vitro and in vivo experiments. This study is the first to reveal the tumor-promoting mechanism of CSF2RA in GC, demonstrating its role in facilitating tumor progression via the JAK2/STAT3 signaling pathway. The potential therapeutic value of Ruxolitinib was validated in both cell-based and xenograft tumor models. Future research should further explore the upstream regulatory mechanisms of CSF2RA and its dynamic role in the immune microenvironment to advance precision treatment strategies for GC.

BackgroundOsteosarcoma is a primary malignant tumor, characterized by its high incidence and recurrence rate in children and adolescents. Ferroptosis, an iron-dependent form of regulated cell death, has recently been recognized as a potential therapeutic vulnerability in cancer treatment. However, its prognostic significance and underlying regulatory mechanisms in osteosarcoma remain largely unexplored.Materials and methodsWe constructed a prognostic model based on 12 ferroptosis-related genes using LASSO regression and validated across independent GEO cohorts (GSE21257 and GSE39055). We identified hub genes via machine learning algorithms (SVM, RF, XGBoost, BORUTA) and single-cell RNA sequencing. The exosomal transfer of COX4I2 protein from CAFs to 143B osteosarcoma cells was evaluated by Western blot, confocal microscopy, and transmission electron microscopy. Ferroptosis indicators, including Fe2+, MDA, ACSL4, and ROS levels, were assessed in vitro. We performed tumorigenicity assays in vivo in nude mice to validate biological function.ResultsThe ferroptosis-based risk model exhibited robust prognostic performance. We identified COX4I2 as a stromal hub gene, highly enriched in cancer-associated fibroblasts (CAFs). Functional experiments demonstrated that exosome-mediated delivery of COX4I2 suppressed ferroptosis in osteosarcoma cells and enhancd cell proliferation and mitochondrial integrity. Studies in vivo further revealed that overexpression of exosomal COX4I2 markedly promoted tumor growth while inhibiting ferroptosis.ConclusionThese findings underscore the potential of exosomal COX4I2 as a biomarker and therapeutic target for ferroptosis-based interventions in osteosarcoma.

BackgroundThe tumor microenvironment (TME) is highly complex and significantly influences cancer prognosis and drug sensitivity. Tumor-associated neutrophils (TANs) play a key role in the TME. In this study, we aimed to investigate the TANs-related markers in colorectal cancer (CRC) and develop an integrated signature for prognostic stratification.MethodsThe CRC single-cell RNA sequencing (scRNA-seq) data and RNA-seq data were obtained from TCGA and GEO. A risk score was calculated based on the 18 TAN-associated genes identified in CRC by scRNA-seq data and LASSO regression. Prognosis, stromal and immune infiltration landscape, metabolism, and treatment response were then investigated in the low- and high-risk score clusters using RNA-seq data.ResultsPatients with a High-risk score had a significantly worse survival outcome than those with a Low-risk score (p < 0.0001). The prognostic predictive potency of the risk score was validated in both the TCGA validation cohort (p < 0.0001) and the GEO cohort (p < 0.00015). The areas under the curves of 1-, 3-, and 5-year survival were 0.76, 0.74, and 0.70 in the TCGA training set; 0.78, 0.68, and 0.78 in the TCGA validation set; and 0.65, 0.64, and 0.62 in the GEO set. The risk score was related to T, N, and M stages. A prognostic nomogram was constructed, and the predictive accuracy was assessed by calibration curve analysis. Decision curve analysis showed the clinical utility of the nomogram. Furthermore, the High-risk score cluster was significantly associated with the levels of cancer-associated fibroblasts, as well as activity in the transforming growth factor-β and WNT pathway. In depth, the High-risk score cluster exhibited lower levels of amino acid, tricarboxylic acid, and nucleotide metabolism, as well as poorer responses to chemotherapeutic agents such as 5-fluorouracil.ConclusionThis novel TAN subtype, based on 18 prognostic-related genes, could provide new insights into the prognostic stratification and treatment options for CRC.

Elucidating the correlation between polychlorinated dibenzo-p-dioxins and prostate cancer progression: Insights from gene expression and molecular docking.

Integrated multi-omics analysis identifies SELENOP and PKMYT1 as immune-metabolic hub genes in breast cancer.

In this study, we investigated PSD3, CD274 (PD-L1), and TNFSF18 as potential immune-related biomarkers in esophageal squamous cell carcinoma (ESCC) using integrative transcriptomic and experimental approaches. CD274 and TNFSF18 were consistently up-regulated in ESCC across both TCGA and GEO datasets, while PSD3 showed significantly higher expression in TCGA but no significant difference in the GEO cohort. Only PSD3 demonstrated a significant association with overall survival, with higher expression correlating with improved prognosis. Interestingly, despite its favorable prognostic value, PSD3 functionally promoted ESCC cell proliferation, invasion, and migration in vitro, while inversely regulating PD-L1 expression. Conversely, heterozygous knockout of PD-L1 in KYSE150 cells impaired tumor aggressiveness. Co-immunoprecipitation revealed a direct physical interaction between PSD3 and PD-L1, suggesting a regulatory axis with implications for immune evasion. These findings position PSD3 as a context-dependent immuno-oncogenic factor and a potential therapeutic target in ESCC.

BackgroundOsteosarcoma (OS), the leading primary malignancy of bone in adolescents, is known for its aggressive metastatic behavior and poor responsiveness to conventional therapies. As a lipid-mediated post-translational modification, protein palmitoylation has gained attention for its pivotal role in modulating oncogenic signaling pathways and facilitating tumor immune escape. However, its prognostic value and functional role in OS remain unclear.MethodsTranscriptomic and clinical data from the TARGET and GEO cohorts were used to identify palmitoylation-related prognostic genes. The palmitoylation-related prognostic signature (PPS) was constructed using univariate Cox and LASSO regression. The model was validated by GSE39058 and assessed via survival analysis, ROC curves, and nomogram construction. Functional enrichment (GO, KEGG, GSVA) and immune infiltration analyses were performed. Single-cell expression profiles were explored using the TISCH2 database, and the predictive value of PPS for immunotherapy response was evaluated in the IMvigor210 cohort.ResultsA three-gene PPS (ZDHHC3, ZDHHC21, ZDHHC23) was identified and shown to independently predict survival in OS. Elevated PPS levels correlated with unfavorable clinical outcomes, diminished immune cell presence, and suppressed immune checkpoint molecule levels. Functional analysis revealed enrichment of oncogenic and immunosuppressive pathways in the high-PPS group. Single-cell analysis confirmed PPS gene expression in malignant and immune cells. In the IMvigor210 cohort, high PPS predicted worse response to anti-PD-L1 immunotherapy.ConclusionsThis study establishes a novel palmitoylation-related prognostic signature in osteosarcoma, which reflects tumor aggressiveness and immune evasion. PPS holds promise as both a stratification indicator and an intervention point for osteosarcoma treatment.Supplementary InformationThe online version contains supplementary material available at 10.1007/s12672-025-03364-8.

Identification and validation of a hypoxia- and immune-related prognostic signature for pancreatic cancer

R-loops, RNA-DNA hybrid structures, play essential roles in maintaining genomic stability and regulating transcription. This study aims to identify key R-loop regulatory genes as prognostic markers for LUAD and explore their associations with immunotherapy response and drug sensitivity, supporting personalized treatment strategies. We integrated 1771 R-loops genes with differentially expressed genes in LUAD. Through univariate Cox, LASSO, and multivariate Cox analyses, we constructed an R-loops prognostic risk score (R-loops Score) and validated it in three independent GEO cohorts. Correlations with clinical variables, immune features, and drug response were examined. Key genes were further evaluated by qPCR and Western blot in LUAD cell lines and tumor tissues. Patients with high R-loops Scores had significantly poorer overall survival compared with low-score patients. A nomogram combining the R-loops Score and clinical factors achieved AUCs of 0.732, 0.713, and 0.719 for predicting 1-, 2-, and 5-year OS, respectively. Pathway enrichment indicated that high-score tumors were enriched in cell cycle regulation, phase separation, and epithelial-mesenchymal transition. High R-loops Scores were associated with male sex, advanced stages, immune evasion and immunotherapy resistance, but increased chemotherapy and targeted therapy sensitivity. EIF3B was further validated as a key gene through analysis of a local patient cohort. The R-loops Score represents a promising prognostic tool for LUAD, offering valuable insights into survival outcomes, immune characteristics and drug responsiveness. Notably, qPCR and Western blot validation consistently confirmed EIF3B as a key gene, further supporting their potential as biomarkers. These results support future research and serve as a reference for the personalized precision treatment of LUAD.