低氧环境下人乳腺癌细胞系MCF-7转录调节因子-1表达变化及其机制  

ETS-1 expression changes in breast cancer MCF-7 cells induced by hypoxia and its mechanism

作　　者：刘颖[1] 乔如丽 尚里 张锋军[1] 焦扬驰 胡思畅 赵庆丽[1] LIU Ying;QIAO Ruli;SHANG Li;ZHANG Fengjun;JIAO Yangchi;HU Sichang;ZHAO Qingli(No.940 Hospital of Joint Logistics Support Force of People's Liberation Army,Lanzhou 730050,China)

机构地区：[1]中国人民解放军联勤保障部队第九四○医院,兰州730050

出　　处：《山东医药》2020年第24期45-49,共5页Shandong Medical Journal

基　　金：甘肃省自然科学基金(160RJZA170);甘肃省卫计委课题(GSWSKY2017-50)。

摘　　要：目的观察人乳腺癌细胞系MCF-7 E26转录特异性序列-1(Ets-1)表达变化,并探讨其机制。方法取对数生长期MCF-7细胞,使用氯化钴(CoCl2)诱导的化学性低氧培养环境以及缺氧小室培养环境模拟肿瘤细胞低氧微环境,通过过表达和敲降缺氧诱导因子-1α(HIF-1α)研究Ets-1基因的表达情况,通过荧光定量PCR(q-PCR)和蛋白印记实验(Western blot)方法分别研究低氧环境下Ets-1 mRNA和蛋白表达情况,使用免疫共沉淀(Co-IP)和免疫荧光染色(IF)方法研究不同情况下蛋白互作,使用免疫沉淀法研究Ets-1蛋白翻译过程中的乙酰化、泛素化和磷酸化。结果低氧刺激下,乳腺癌细胞系中Ets-1蛋白水平显著上升;低氧环境下Ets-1 mRNA相对表达量升高;过表达HIF-1α导致乙酰基转移酶p300活性增强;并且p300与Ets-1在细胞质中共定位;低氧刺激下,Ets-1的乙酰化水平升高,Ets-1乙酰化后与E3泛素连接酶COP-1相互作用减弱;p300抑制剂C646处理MCF-7细胞降低Ets-1蛋白水平,并增强其与COP-1相互作用。结论低氧微环境下培养的MCF-7细胞中Ets-1蛋白和mRNA表达升高,Ets-1表达升高的调控机制可能为低氧促进了Ets-1转录,同时HIF-1α使乙酰基转移酶p300活性增强,引起Ets-1乙酰化水平升高,乙酰化Ets-1与E3泛素连接酶COP-1相互作用受阻,进而降低其降解。Objective To observe the expression changes of E26 transformation specific sequence 1(Ets-1)in human breast cancer cell line MCF-7 under hypoxia and to explore the mechanism.Methods MCF-7 cells in the logarithmic phase were cultured in CoCl2-induced chemical hypoxia environment and hypoxia chamber culture environment to simulate the hypoxic microenvironment of tumor cells.The expression of Ets-1 gene was studied by overexpression and knockdown of hypoxia inducible factor-1α(HIF-1α).Fluorescence quantitative PCR(Q-PCR)and Western blotting were used to detect the expression of Ets-1 mRNA and protein in hypoxic environment.Immunoprecipitation(co IP)and immunofluorescence staining(If)were used to determine the protein interaction in different conditions.Co IP was used to study the acetylation,ubiquitination and phosphorylation of Ets-1 protein.Results Ets-1 protein and level in breast cancer cell line MCF-7 increased significantly under hypoxia.Overexpression of HIF-1αresulted in the increase of acetyltransferase p300 activity;p300 and Ets-1 were co-localized in cytoplasm;the acetylation level of Ets-1 increased under hypoxia stimulation,and the interaction between Ets-1 and E3 ubiquitin ligase COP-1 was weakened;c646,a p300 inhibitor,decreased Ets-1 protein level and enhanced its interaction with COP-1.Conclusions The expression of Ets-1 protein and mRNA increases in MCF-7 cells cultured in hypoxia microenvironment.The regulation mechanism of increased Ets-1 expression may be that hypoxia promotes Ets-1 transcription,meanwhile,HIF-1αincreases the activity of acetyltransferase p300,which leads to the increase of Ets-1 acetylation level;the interaction between acetylated Ets-1 and E3 ubiquitin ligase Cop-1 is blocked,and the degradation of Ets-1 is reduced.

关 键 词：低氧环境 乳腺癌 MCF-7细胞 E26转录特异性序列-1 

分 类 号：R341[医药卫生—基础医学]