摘要
目的通过体内外实验验证溴结构域PHD指转录因子(BPTF)通过调节胶质瘤细胞中溶质载体家族40成员1(SLC40A1)表达影响胶质瘤发展和铁死亡的机制。方法U87MG细胞分为sh-NC组、sh-BPTF组、sh-BPTF+ov-NC组和sh-BPTF+ov-SLC40A1组,采用慢病毒建立稳定转染的细胞系,采用qRT-PCR和Western blot验证转染效率。CCK-8和平板克隆形成实验检测细胞增殖能力;Transwell检测细胞迁移和侵袭能力;细胞经皮下注射裸鼠并检测肿瘤生长情况;相应试剂盒检测细胞中活性氧(ROS)水平、铁含量、丙二醛(MDA)含量和还原性谷胱甘肽/氧化型谷胱甘肽(GSH/GSSG)比值以评价细胞铁死亡情况;免疫共沉淀(Co-IP)实验用于验证BPTF和c-Myc蛋白相互作用;染色质免疫共沉淀(ChIP)实验用于验证BPTF和c-Myc结合SLC40A1启动子;双荧光素酶报告基因实验用于验证BPTF影响SLC40A1转录。结果敲减BPTF降低胶质瘤细胞中SLC40A1表达,抑制细胞体外增殖、迁移、侵袭和体内肿瘤生长,增加细胞中铁含量、ROS水平和MDA含量并降低细胞中GSH/GSSG比值。过表达SLC40A1逆转敲减BPTF对胶质瘤细胞增殖、迁移、侵袭和体内肿瘤生长的抑制作用,降低细胞中铁含量、ROS水平和MDA含量并增加细胞中GSH/GSSG比值。胶质瘤细胞中BPTF与c-Myc蛋白相互作用。SLC40A1启动子存在c-Myc的潜在结合位点,且BPTF和c-Myc蛋白结合SLC40A1启动子。敲减BPTF降低SLC40A1启动子的转录活性,且结合位点突变后敲减BPTF不影响SLC40A1启动子的转录活性。结论BPTF可能通过与c-Myc相互作用上调其下游靶基因SLC40A1表达,从而抑制胶质瘤细胞铁死亡并促进胶质瘤发展。
Objective To verify the mechanism of bromodomain PHD finger transcription factor(BPTF)affecting develop-ment of glioma and ferroptosis by regulating the expression of solute carrier family 40 member 1(SLC40A1)in glioma cells by in vitro and in vivo experiments.Methods U87MG cells were divided into sh-NC group,sh-BPTF group,sh-BPTF+ov-NC group and sh-BPTF+ov-SLC40A1 group.Cell lines were stably transfected by lentivirus,and the transfection efficiency was verified by qRT-PCR and Western blotting.Cell proliferation ability was detected by CCK-8 and plate clone formation test.Cell migra-tion and invasion ability were detected by Transwell.Cells were injected subcutaneously into nude mice to detect the tumor growth.To evaluate the ferroptosis of cells,the reactive oxygen species(ROS)level,iron content,malondialdehyde(MDA)con-tent and reduced glutathione/oxidized glutathione(GSH/GSSG)ratio in cells were detected by corresponding kits.Co-immuno-precipitation(Co-IP)experiment was used to verify the interaction between BPTF and c-Myc protein.Chromatin immunoprecipi-tation(ChIP)experiment was used to verify that BPTF and c-Myc combined with SLC40A1 promoter.Double luciferase reporter gene experiment was used to verify the effect of BPTF on SLC40AI transcription.Results After BPTF knockdown,the expres-sion of SLC40A1 in glioma cell was decreased,cell proliferation,migration,invasion in vitro,and tumor growth in vivo were in-hibited,iron content,ROS level and MDA content in cells were increased,and GSH/GSSG ratio in cell was de-creased.Overexpression of SLC40Al reversed the inhibitory effect of BPTF on the proliferation,migration,invasion and tumor growth in vivo,decreased the iron content,ROS level and MDA content in cells,and increased the GSH/GSSG ratio in cells.There was an interaction between BPTF and c-Myc proteins in glioma cells.A potential binding site of c-Myc in SLC40A1 promoter was verified,BPTF and c-Myc protein bound to SLC40A1 promoter.BPTF knockdown reduced the transcriptional ac-tivity of SLC40A1 promoter,and BPTF knockdown after binding site mutation did not affect the transcriptional activity of SLC40A1 promoter.Conclusion BPTF may upregulate the expression of its downstream target gene SLC40A1 by interacting with c-Myc,thereby inhibiting ferroptosis in glioma cells and promoting glioma progression.
作者
林志仁
潘艳玲
朱燕兴
Lin Zhiren;Pan Yanling;Zhu Yanxing(Department of Radiation Oncology Therapy,Haikou People's Hospital,Haikou 570208,China;Department of Oncology Chemotherapy,Haikou People's Hospital,Haikou 570208,China)
出处
《华中科技大学学报(医学版)》
北大核心
2025年第4期507-512,534,共7页
Acta Medicinae Universitatis Scientiae et Technologiae Huazhong
基金
海南省自然科学基金青年基金项目(No.819QN389)。
