Abstract: OBJECTIVE：To investigate the role of E2F transcription factor 5 (E2F5) in X-ray-induced ferroptosis of human keratinocyte，HaCaT. METHODS：HaCaT cells were irradiated with 0，2.5，5，10 and 20 Gy X-rays. Cell survival rates were detected by the CCK-8 method at 24，48 and 72 h after irradiation. Expression levels of E2F5，ferroptosis marker cyclooxygenase-2 (COX-2) and related protein glutathione peroxidase 4 (GPX4) were detected by Western blot at 24 h. For cells irradiated with 10 Gy X-ray，expression levels of E2F5，COX-2 and related protein GPX4 were detected by Western blot at 24，48 and 72 h，levels of ferrous ion and lipid peroxidation were detected by flow cytometry at 24 and 48 h，GSH (glutathione) levels were measured by GSH kit at 48 and 72 h，and colony formation was measured by colony formation assay at 12 d. Small interfering RNA (siRNA) transfection technique was used to silence E2F5 expression in the 10 Gy irradiated cells. Four groups of cells were set up：unirradiated blank control，irradiated control，negative control，and E2F5 silencing. Survival rate of these cells at 72 h after irradiation was detected by CCK-8，expression levels of COX-2 protein at 24 h was detected by Western blot，levels of ferrous ion 20 h and lipid peroxidation levels at 24 h were detected using flow cytometry，and colony formation ability was detected by colony formation assay at 12 d. RESULTS：The survival rates of the HaCaT cells decreased at 24 h after 2.5-20 Gy X-ray irradiation and 48 and 72 h after 5-20 Gy X-ray irradiation (P<0.05 or P<0.01). The protein expression of E2F5 increased at 24 h after 2.5-10 Gy X-ray irradiation. At 24 h after 2.5-20 Gy X-ray irradiation，COX-2 protein expression increased，GPX4 protein expression decreased (P<0.05 or P<0.01). At 48 and 72 h after 10 Gy X-ray irradiation，the expression of E2F5 and COX-2 protein increased，the expression of GPX4 protein decreased，and the intracellular GSH content decreased. At 24 and 48 h after 10 Gy X-ray irradiation，the levels of ferrous ions and lipid peroxidation increased，and the colony-forming ability decreased at 12 d (P<0.05 or P<0.01). After 10 Gy X-ray irradiation and compared with the negative control group，the survival rates of the E2F5 silenced group increased at 72 h after irradiation，the expression levels of COX-2 protein and lipid peroxidation decreased 24 h after irradiation，the level of ferrous ion decreased 20 h after irradiation，and the colony formation ability increased at 12 days after irradiation (P<0.05 or P<0.01). CONCLUSION：X-ray irradiation promoted ferroptosis by up-regulating E2F5 protein expression in HaCaT cells，thereby reducing cell viability.

Key words: E2F transcription factor 5, X-rays, human keratinocytes, ferroptosis