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

doi:10.3969/j.issn.1004-616x.2025.02.003

摘要/Abstract

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射线照射可能通过上调HaCaT细胞内E2F5蛋白表达促进细胞铁死亡，从而降低细胞的存活能力。

关键词: E2F转录因子5, X射线, 人角质形成细胞, 铁死亡

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

中图分类号:

[R146]

田晓丹, 王成芳, 曲功霖, 邵帅, 苟巧. 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[J]. 癌变·畸变·突变, 2025, 37(2): 105-112.

TIAN Xiaodan, WANG Chengfang, QU Gonglin, SHAO Shuai, GOU Qiao. Effect of E2F transcription factor 5 in X-ray-induced ferroptosis of human keratinocytes in vitro[J]. Carcinogenesis, Teratogenesis & Mutagenesis, 2025, 37(2): 105-112.

参考文献

[1] YANG X J, REN H R, GUO X M, et al. Radiation-induced skin injury:pathogenesis, treatment, and management[J]. Aging, 2020, 12(22):23379-23393.
[2] SINGH M, ALAVI A, WONG R, et al. Radiodermatitis:a review of our current understanding[J]. Am J Clin Dermatol, 2016, 17(3):277-292.
[3] 陆蒋惠文, 张舒羽, 张杰, 等. 放射性皮肤损伤的治疗进展[J]. 国际放射医学核医学杂志, 2021, 45(7):461-469.
[4] 黄佳榕. "三黄肤康油"防治乳腺癌急性放射性皮炎的疗效评价与机制探讨[D]. 广州:广州中医药大学, 2023.
[5] TANG D L, CHEN X, KANG R, et al. Ferroptosis:molecular mechanisms and health implications[J]. Cell Res, 2021, 31(2):107-125.
[6] LIU L H, LIAN N, SHI L Q, et al. Ferroptosis:Mechanism and connections with cutaneous diseases[J]. Front Cell Dev Biol, 2023, 10:1079548.
[7] WANG X Y, LU Y X, CHENG X C, et al. Local multiple-site injections of a plasmid encoding human MnSOD mitigate radiation-induced skin injury by inhibiting ferroptosis[J]. Curr Drug Deliv, 2024, 21(5):763-774.
[8] 冯亚辉, 蒋胜, 涂文玲, 等. 电离辐射对皮肤细胞铁死亡的影响及其抑制剂Ferrostatin-1的防护作用研究[J]. 中华放射医学与防护杂志, 2021, 41(8):602-608.
[9] SUN M Y, JI Y T, ZHANG G J, et al. Posttranslational modifications of E2F family members in the physiological state and in cancer:Roles, mechanisms and therapeutic targets[J]. Biomed Pharmacother, 2024, 178:117147.
[10] MALGUNDKAR S H, HASSAN N A, AL BADI H, et al. Identification and validation of a novel long non-coding RNA (LINC01465) in ovarian cancer[J]. Hum Cell, 2023, 36(2):762-774.
[11] WANG Z Z, ZHANG H H, LI F, et al. Knockdown of RNA-binding protein IMP3 suppresses oral squamous cell carcinoma proliferation by destabilizing E2F5 transcript[J]. Aging, 2024, 16(2):1897-1910.
[12] TANG Y, GAO G, XIA W W, et al. METTL3 promotes the growth and metastasis of pancreatic cancer by regulating the m6A modification and stability of E2F5[J]. Cell Signal, 2022, 99:110440.
[13] 张瑞丹, 杨丽莉, 孙全富, 等. 一例职业性放射性皮肤癌的诊断与劳动能力鉴定[J]. 中华放射医学与防护杂志, 2020, 40(7):540-542.
[14] WILSON V G. Growth and differentiation of HaCaT keratinocytes[J]. Methods Mol Biol, 2014, 1195:33-41.
[15] NIELSEN S, BASSLER N, GRZANKA L, et al. Proton scanning and X-ray beam irradiation induce distinct regulation of inflammatory cytokines in a preclinical mouse model[J]. Int J Radiat Biol, 2020, 96(10):1238-1244.
[16] 李园. Connexin26调控辐射诱导皮肤免疫趋化因子CCL27的作用机理研究[D]. 合肥:安徽医科大学, 2021.
[17] ZHU W, XU J, GE Y Y, et al. Epigallocatechin-3-gallate (EGCG) protects skin cells from ionizing radiation via heme oxygenase-1(HO-1) overexpression[J]. J Radiat Res, 2014, 55(6):1056-1065.
[18] 郭倪君, 林忠宁, 林育纯. 铁死亡信号介导抗肿瘤作用的细胞器调控机制研究进展[J]. 癌变·畸变·突变, 2020, 32(2):149-154.
[19] XU Y J, JIA B W, LI J, et al. The interplay between ferroptosis and neuroinflammation in central neurological disorders[J]. Antioxidants, 2024, 13(4):395.
[20] LINTEL H, ABBAS D B, LAVIN C V, et al. Transdermal deferoxamine administration improves excisional wound healing in chronically irradiated murine skin[J]. J Transl Med, 2022, 20(1):274.
[21] 杨莉, 苟巧. E2F转录因子4在肿瘤中的研究进展[J]. 癌变·畸变·突变, 2024, 36(6):506-509.
[22] FAN Z W, KONG M, MIAO X L, et al. An E2F5-TFDP1-BRG1 complex mediates transcriptional activation of MYCN in hepatocytes[J]. Front Cell Dev Biol, 2021, 9:742319.
[23] QI J C, YANG Z, LIN T, et al. CDK13 upregulation-induced formation of the positive feedback loop among circCDK13, miR-212-5p/miR-449a and E2F5 contributes to prostate carcinogenesis[J]. J Exp Clin Cancer Res, 2021, 40(1):2.
[24] SHENG J Q, LUO Y M, LV E J, et al. LINC01980 induced by TGF-beta promotes hepatocellular carcinoma metastasis via miR-376b-5p/E2F5 axis[J]. Cell Signal, 2023, 112:110923.
[25] LI S B, YANG X F, LI W Q, et al. Comprehensive analysis of E2F family members in human gastric cancer[J]. Front Oncol, 2021, 11:625257.
[26] THOMAS R J, OLEINIK N, PANNEER SELVAM S, et al. HPV/E7 induces chemotherapy-mediated tumor suppression by ceramide-dependent mitophagy[J]. EMBO Mol Med, 2017, 9(8):1030-1051.