CCR10通过NRF2抑制结直肠癌细胞铁死亡的机制研究
Study on the mechanism of CCR10 inhibiting ferroptosis of colorectal cancer cells through NRF2
徐昰栋 1刘子梅 1陈宁 2褚敬申 3姜晓星 2刘湘帆 2马俐君1
作者信息
- 1. 上海交通大学医学院附属同仁医院肿瘤科,上海 200336
- 2. 上海交通大学医学院医学技术学院,上海 200025
- 3. 上海交通大学医学院附属瑞金医院科技发展处,上海 200025
- 折叠
摘要
目的 探究C-C趋化因子受体10(C-C chemokine receptor 10,CCR10)在结直肠癌(colorectal cancer,CRC)中对细胞增殖和铁死亡的影响,并探究其影响铁死亡的机制.方法 在GEPIA数据库(Gene Expression Profiling In-teractive Analysis)共表达分析CCR10与铁死亡关键基因的相关性.构建慢病毒介导的CCR10过表达(CCR10-OE)、敲除(CCR10-KO)及其阴性对照(NC)稳转HCT116细胞,采用细胞活力(CCK8)、克隆形成和裸鼠成瘤实验检测CCR10过表达或敲除后的细胞增殖.利用CCK8法、流式细胞术检测细胞铁死亡和脂质过氧化,评估CCR10过表达及敲除后细胞对铁死亡诱导剂erastin和rsl3的耐受.利用WB检测CCR10过表达和敲除后铁死亡关键蛋白质的表达.在CCR10敲除细胞株瞬时转染核因子E2相关因子2(NF-E2-related factor 2,NRF2)质粒,鉴定铁死亡表型以及其下游分子溶质载体家族7成员11(solute Carrier Family 7 Member 11,SLC7A11)、谷胱甘肽过氧化物酶4(glutathi-one Peroxidase 4,GPX4)、铁重链蛋白1(ferritin Heavy Chain 1,FTH1)表达水平.结果 CCR10表达与铁死亡抑制基因 BCL-2(r=0.54,P＜0.001)、FTH1(r=0.18,P＜0.001)和 NRF2(r=0.31,P＜0.001)呈正相关,与铁死亡驱动基因 TP53(r=-0.46,P＜0.001)、TFRC(r=-0.45,P＜0.001)和 KEAP1(r=-0.29,P＜0.001)呈负相关.CCR10过表达促进CRC细胞增殖(P＜0.01),而CCR10缺失抑制CRC细胞增殖(P＜0.01)和小鼠皮下肿瘤生长(P＜0.001).CCR10过表达或缺失分别抑制或促进其对铁死亡的敏感性(P＜0.01),并影响转录因子NRF2表达.结论 CCR10基因可以通过上调转录因子NRF2表达而抑制erastin和rsl3诱导的CRC细胞铁死亡.
Abstract
Objective To investigate the effects of C-C chemokine receptor 10 on cell proliferation and ferroptosis in colorectal cancer(CRC),and to explore the mechanism of its effect on ferroptosis.Methods The correlation between CCR10 and key genes of ferroptosis was analyzed by co-expression in GEPIA database(Gene Expression Profiling Inter-active Analysis).Lentivirus-mediated CCR10 overexpression(CCR10-OE),knockout(CCR10-KO)and negative control(NC)were stably transfected into HCT116 cells.Cell viability(CCK8),clone formation and tumor formation in nude mice were used to detect the cell proliferation after CCR10 overexpression or knockout.Ferroptosis and lipid peroxida-tion were detected by CCK8 and flow cytometry to evaluate the tolerance of cells to ferroptosis inducers erastin and rs13 after CCR10 overexpression and knockout.WB was used to detect the expression of key proteins of ferroptosis after CCR10 overexpression and knockout.The plasmid of nuclear factor E2 related factor 2(NF-E2-related factor2,NRF2)was transiently transfected in CCR10 knockout cell line,and the phenotype of ferroptosis and the expression levels of solute Carrier Family 7 Member 11(SLC7A11),glutathione Peroxidase 4(GPX4),and ferritin Heavy Chain 1(FTH1)were identified.Results The expression of CCR10 was positively correlated with ferroptotic suppressor genes BCL-2(r=0.54,P＜0.001),FTH1(r=0.18,P＜0.001)and NRF2(r=0.31,P＜0.001),while negatively correlated with fer-roptotic driving genes TP53(r=-0.46,P＜0.001),TFRC(r=-0.45,P＜0.001)and KEAP1(r=-0.29,P＜0.001).Overexpression of CCR10 promoted the proliferation of CRC cells(P＜0.01),while the deletion of CCR10 in-hibited the proliferation of CRC cells(P＜0.01)and the growth of subcutaneous tumors in mice(P＜0.001).Overex-pression or deletion of CCR10 inhibits or promotes its sensitivity to ferroptosis(P＜0.01)and affects the expression of transcription factor NRF2,respectively.Conclusion CCR10 gene could inhibit ferroptosis induced by erastin and rs13 in CRC cells by up-regulating the expression of transcription factor NRF2.
关键词
结直肠癌/C-C趋化因子受体10/铁死亡/核因子E2相关因子2Key words
colorectal cancer/C-C chemokine receptor 10/ferroptosis/nuclear factor E2 related factor 2引用本文复制引用
基金项目
国家自然科学基金面上项目(81672335)
上海交通大学医工交叉研究基金(YG2022QN115)
出版年
2024
NETL
NSTL
万方数据