Objective  To investigate the molecular mechanism underlying radiation resistance in non-small cell lung cancer (NSCLC) mediated by the nuclear factor-erythroid 2-related factor 2 (NRF2) gene via proteomic analysis, providing new insights for enhancing NSCLC sensitivity to radiotherapy.

Methods  The clustered regularly interspaced short palindromic repeat-associated protein 9 technology was employed to construct a NRF2 knockout (NRF2 KO) NSCLC A549 cell line, with the A549 wild-type (NRF2 WT) cells serving as controls. Total cellular proteins were collected and extracted, and Western blot was performed to verify the effectiveness of NRF2 gene knockout. Following single 8 Gy irradiation with ¹³⁷Cs γ-rays, lipid peroxidation levels were assessed by flow cytometry in both cell groups. Proteins were identified and quantitatively analyzed through 4D-Label-free quantitative proteomics mass spectrometry, and differentially expressed proteins were screened using bioinformatics methods. Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis was conducted to functionally analyze these differentially expressed proteins. Iron death-related genes were selected from the FerrDb database, and the intersection of differentially expressed protein-coding genes identified by bioinformatics methods was used to determine NRF2-associated ferroptosis genes in A549 cells. Comparisons between two groups of measurement data were performed using the independent samples t-test.

Results  Western blot results confirmed successful NRF2 gene knockout in the NRF2 KO cells. Compared with NRF2 WT cells, lipid peroxidation levels were significantly elevated in NRF2 KO cells after irradiation (t=8.142, P<0.05). Bioinformatics analysis revealed that, in contrast to NRF2 WT cells, a total of 90 differentially expressed proteins were identified in NRF2 KO cells post-irradiation, with 25 proteins upregulated and 65 proteins downregulated. KEGG pathway enrichment analysis revealed that several signaling pathways, including the Janus kinase-signal transducer and activator of transcription pathway and p53 signaling pathway, were upregulated in NRF2 KO cells following irradiation. Downregulated signaling pathways primarily involved metabolic pathways and adipocyte factor signaling pathways. Further analysis identified two NRF2-related ferroptosis genes in A549 cells: cyclin-dependent kinase inhibitor 1A and retinoblastoma 1.

Conclusion  Proteomic analysis revealed the potential mechanism by which the NRF2 gene mediates radiation resistance in NSCLC A549 cells, particularly through the ferroptosis pathway, providing new molecular targets for enhancing the sensitivity of radiotherapy.