Response of MRC-5 human lung fibroblasts to FLASH and conventional radiotherapy: A study on differential DNA damage and transcriptomic response.

Ultra-high dose rate FLASH radiotherapy can mitigate normal tissue toxicity while sustaining tumor control, yet the associated molecular mechanisms are still unclear. This study aimed to evaluate the differential DNA damage and transcriptomic responses of MRC-5 cells to FLASH versus conventional (CONV) irradiation. To address this, MRC-5 cells were exposed to X-rays irradiation at either FLASH dose rate or CONV dose rate. γH2AX/phosphorylated 53BP1 immunofluorescence was performed to detect early-stage DNA double-strand breaks. Transcriptome sequencing was implemented to characterize the transcriptomic expression profiles of the cells. Interaction patterns among differentially expressed genes (DEGs) were investigated via protein-protein interaction (PPI) network analysis. The expression levels of the screened core DEGs were validated via quantitative real-time PCR (RT-qPCR). Results showed that at high doses (8 Gy and 12 Gy), the FLASH group exhibited significantly fewer early-stage DNA double-strand breaks than the CONV group. Its transcriptomic profile was more analogous to that of nonirradiated cells, with differential regulation in DNA damage response (DDR)-related pathways; 9 core DEGs (CCNA2, CCNB1, CCNB2, RAD51, EXO1, BIRC5, BUB1, CDC6, CDC20) were screened out via PPI network analysis, and RT-qPCR verified that their downregulation was less extensive in the FLASH group relative to the latter. Collectively, X-ray FLASH irradiation alleviates MRC-5 cell damage by regulating DNA double-strand break repair and other DDR-related pathways, along with the nine core DEGs, providing a molecular basis for FLASH radiotherapy's normal tissue protective effect.