E2F转录因子5在X射线诱导的体外培养人角质形成细胞铁死亡中的作用  

Effect of E2F transcription factor 5 in X-ray-induced ferroptosis of human keratinocytes in vitro

作　　者：田晓丹 王成芳 曲功霖[1] 邵帅[1] 苟巧[1] TIAN Xiaodan;WANG Chengfang;QU Gonglin;SHAO Shuai;GOU Qiao(Key Laboratory of Radiological Protection and Nuclear Emergency,National Institute for Radiological Protection,Chinese Center for Disease Control and Prevention,Beijing 100088,China)

机构地区：[1]中国疾病预防控制中心辐射防护与核安全医学所,辐射防护与核应急中国疾病预防控制中心重点实验室,北京100088

出　　处：《癌变.畸变.突变》2025年第2期105-112,共8页Carcinogenesis,Teratogenesis & Mutagenesis

基　　金：国家自然科学基金(81000862)。

摘　　要：目的:探讨E2F转录因子5(E2F5)在X射线诱导的人角质形成细胞HaCaT铁死亡中的作用。方法:HaCaT细胞经0、2.5、5、10、20 Gy X射线单次照射,照后24、48和72 h用CCK-8法检测细胞存活率,照后24 h用Western blot法检测细胞内E2F5、铁死亡标志蛋白环氧合酶-2(COX-2)和相关蛋白谷胱甘肽过氧化物酶4(GPX4)的表达水平;用10 Gy X射线照射HaCaT细胞,Western blot法检测照后24、48和72 h细胞内E2F5、COX-2和相关蛋白GPX4的表达水平,流式细胞术检测照后24和48 h细胞内亚铁离子和脂质过氧化水平,谷胱甘肽(GSH)试剂盒检测照后48和72 h细胞内GSH水平,集落形成实验检测照后12 d细胞的集落形成能力;采用小干扰RNA(siRNA)转染技术使HaCaT细胞内E2F5蛋白表达沉默,并经10 Gy X射线照射,设未照射空白对照、照射对照、无义序列、E2F5沉默4组,CCK-8法检测受照后72 h HaCaT细胞存活率,Western blot法检测受照后24 h HaCaT细胞内COX-2蛋白的表达水平,流式细胞术分别检测受照后20 h HaCaT细胞内亚铁离子平均荧光强度和受照后24 h HaCaT细胞内脂质过氧化水平,集落形成实验检测受照后12 d HaCaT细胞的集落形成能力。结果:2.5~20 Gy X射线照射后24 h,以及5~20 Gy X射线照射后48和72 h,HaCaT细胞存活率降低(P<0.05或P<0.01);2.5~10 Gy X射线照射后24 h,细胞内E2F5蛋白表达增强;2.5~20 Gy X射线照射后24 h,细胞内COX-2蛋白表达增强,GPX4蛋白表达减弱(P<0.05或P<0.01);10 Gy X射线照射后48和72 h细胞内E2F5和COX-2蛋白均表达增强,GPX4蛋白表达减弱,细胞内GSH含量降低,照后24和48 h细胞内亚铁离子和脂质过氧化水平升高,照后12 d HaCaT细胞的集落形成能力降低(P<0.05或P<0.01)。10 Gy X射线照射后,与无义序列组相比,E2F5沉默组细胞在照后72 h的存活率升高,照后24 h COX-2蛋白表达水平和脂质过氧化水平降低,照后20 h亚铁离子水平降低,照后12 d的集落形成能力增强(P<0.05或P<0.01)。结论:X射�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

关 键 词：E2F转录因子5 X射线 人角质形成细胞 铁死亡 

分 类 号：R146[医药卫生—公共卫生与预防医学]