Objective To analyze gene expression differences between human large cell lung cancer cells NCI-H460 and its acquired radioresistant subline NCI-H460R using transcriptomics, screen key genes associated with ferroptosis regulation, and explore the potential mechanism by which non-small cell lung cancer escapes ferroptosis during radiotherapy resistance.

Methods NCI-H460R cells were established through the fractionated irradiation of NCI-H460 cells (2 Gy per fraction, dose rate 0.786 Gy/min, total dose 60 Gy). Clonogenic assay was used to evaluate the survival ability of NCI-H460R cells and verify its radioresistant phenotype. RNA was extracted and subjected to high-throughput sequencing on the NovaSeq 6000 platform. Differentially expressed genes (DEGs) were screened with false discover rate <0.05 and |log₂ fold change (FC)| ≥ 1. Ferroptosis-related DEGs were identified by intersecting DEGs with the FerrDb ferroptosis database. Core genes were screened based on multiple indicators including log₂FC and gene expression abundance and verified by real-time fluorescence quantitative polymerase chain reaction (RT-qPCR). Student's t-test was used for comparison of measurement data between two groups, and two-way ANOVA was used for comparison among multiple groups and survival fractions at different dose points.

Results Clonogenic assay showed that compared with NCI-H460 cells, NCI-H460R cells exhibited increases in mean lethal dose from 1.109 Gy to 1.461 Gy, quasithreshold dose from 1.552 Gy to 2.131 Gy, and 37% survival dose from 2.661 Gy to 3.592 Gy, with a sensitizing enhancement ratio of 0.759, confirming enhanced radioresistance in NCI-H460R cells (F=20.31, P<0.001). Transcriptomic sequencing identified 2 936 DEGs, including 663 upregulated and 2 273 downregulated genes. Among the 80 ferroptosis-related DEGs, the following 5 core DEGs were finally screened: ceruloplasmin (CP), interleukin-1β, prostaglandin-endoperoxide synthase 2, arachidonic 15-lipoxygenase (ALOX15), and Krüppel-like factor 2 (KLF2). CP which involved in iron homeostasis was the most significantly upregulated gene (|log₂FC|=6.621). ALOX15, a key enzyme in lipid peroxidation, was markedly downregulated (|log₂FC|=9.199). RT-qPCR validation results showed that the messenser ribonucleic acid expression levels of these five core DEGs between NCI-H460R cells and NCI-H460 cells were all statistically significant (t=5.96–34.92, all P<0.01).

Conclusion The screened ferroptosis-related core DEGs in NCI-H460R cells suggest that dysregulated iron metabolism and lipid metabolism may serve as important mechanisms for cells to evade ferroptosis, which provide a basis and candidate targets for further investigating the role of ferroptosis in radioresistance.