Bioinformatic Identification and Preliminary Validation of TP53, TGFB1, and NFE2L2 as Potential Ferroptosis-Related Regulators in Pancreatic Adenocarcinoma

Shouying Li,1 Chunfang Zhang,1 Shengxiang Lv,1 Zhimei Zhang,1 Dazhou Xu,1 Tonglei Xu1,2 1Department of Gastroenterology and Department of Pathology, Lianyungang Clinical College of Nanjing Medical University/ The First People’s Hospital of Lianyungang, Lianyungang, Jiangsu, People’s Republic of China; 2Department of Hepatobiliary and Pancreatic Surgery, Zhongda Hospital, Southeast University, Nanjing, People’s Republic of ChinaCorrespondence: Dazhou Xu, Email [email protected] Tonglei Xu, Email [email protected]: Pancreatic adenocarcinoma (PAAD) is a highly malignant tumor with very poor prognosis for patients. Ferroptosis, as a novel cell death mechanism, brings a new direction for PAAD therapeutic research.Methods: Ferroptosis-related differentially expressed genes were identified by bioinformatic analysis of the FerrDb, TCGA, and GTEx databases. A series of analyses, including functional enrichment, protein-protein interaction (PPI) networking, survival analysis, and immune infiltration profiling, were performed to screen for and characterize hub genes. Subsequently, the expression of key hubs—NFE2L2 (NRF2), TGFB1, and TP53—was validated in PDAC patient tissues and cell lines using qRT-PCR and Western blot. Their biological functions were investigated via shRNA-mediated knockdown in BxPC-3 cells, followed by assays for cell migration (wound healing, transwell) and ferroptosis (ROS, MDA, GSH/GSSG).Results: Bioinformatic analysis identified 221 ferroptosis-related DEGs. NFE2L2, TGFB1, and TP53 emerged as core hub genes whose high expression correlated with poor patient survival and specific immune infiltration patterns. Experimental validation confirmed that NRF2, TGFβ 1, and TP53 proteins were significantly upregulated in PDAC tissues compared to adjacent normal tissues. Functionally, knockdown of NFE2L2 and TGFB1 suppressed PDAC cell migration, whereas TP53 knockdown enhanced it. Inversely, NFE2L2 knockdown promoted ferroptosis (increased ROS and MDA, decreased GSH), while knockdown of both TGFB1 and TP53 inhibited ferroptosis.Conclusion: TP53, TGFB1, and NRF2 are key ferroptosis-related regulators in PAAD, influencing tumor progression, ferroptosis sensitivity, and immune contexture. These findings provide new insights and potential targets for ferroptosis-based therapies in PAAD.Keywords: pancreatic adenocarcinoma, ferroptosis, PPI, hug genes, GEPIA