饥饿诱导三阴性乳腺癌细胞株MDA-MB-231的自噬应答观察  

Starvation-induced autophagy response in triple negative breast cancer MDA-MB-231 cells

作　　者：刘宇坤 胡杨 卯升义 许安永 邹迪 熊喆 成文敏 赵红业[1,2,3,5] LIU Yukun;HU Yang;MAO Shengyi;XU Anyong;ZOU Di;XIONG Zhe;CHENG Wenmin;ZHAO Hongye(Dali University,Dali 671000,China;不详)

机构地区：[1]大理大学药学与化学学院,大理671000 [2]云南省动物基因编辑与体细胞克隆技术重点实验室 [3]云南农业大学植物保护学院 [4]云南农业大学动物科学技术学院 [5]云南省异种器官移植工程研究中心

出　　处：《山东医药》2020年第18期21-24,共4页Shandong Medical Journal

基　　金：国家自然科学基金资助项目(81872452,31660656);云南省科技计划第二批项目资助项目(2016FA010)。

摘　　要：目的观察饥饿诱导三阴性乳腺癌细胞株MDA-MB-231的自噬应答情况,并探讨其相关分子机制。方法将MDA-MB-231细胞分为四组,对照组加入DMEM培养液正常培养;Baf-A1组在DMEM培养液中加入100 nM的Baf-A1;EBSS(饥饿)组加入EBSS培养液;EBSS+Baf-A1(自噬阻断)组在EBSS培养液中加入100 nM的Baf-A1,四组均处理8 h后,分别采用实时荧光定量法和Western blotting法检测各组微管相关蛋白轻链3(LC3B)、自噬相关蛋白7(ATG7)和自噬相关蛋白4A(ATG4A)的mRNA及蛋白。结果与对照组比较,EBSS组LC3B、ATG7 mRNA相对表达量升高,ATG4A mRNA相对表达量降低;与EBSS组比较,EBSS+Baf-A1组LC3B mRNA相对表达量升高、ATG7 mRNA相对表达量降低,与Baf-A1组对比,EBSS+Baf-A1组中LC3B mRNA相对表达量增加,ATG7和ATG4A mRNA相对表达量降低。与对照组比较,EBSS组LC3BⅡ/LC3BⅠ升高,ATG4A蛋白表达降低;与EBSS组比较,EBSS+Baf-A1组LC3BⅡ/LC3BⅠ增加,ATG7蛋白表达降低;与Baf-A1组相比,EBSS+Baf-A1组LC3BⅡ/LC3BⅠ增加,ATG7蛋白表达降低。结论EBSS处理的MDA-MB-231细胞自噬水平升高,细胞自噬体形成增多,在此过程中EBSS可能通过增加ATG7 mRNA的表达量增加细胞自噬水平。Objective To investigate the autophagy response of MDA-MB-231 cells(a three negative breast cancer cell line)induced by starvation and its molecular mechanism.Methods We divided MDA-MB-231 cells into four groups.In the control group,DMEM medium was added to the cells for normal culture;in the Baf-A1 group,100 nm Baf-A1 was added to the DMEM medium;in the EBSS(starvation)group,EBSS medium was added;in the EBSS+Baf-A1(autophagy blocking)group,100 nm Baf-A1 was added to the EBSS medium.After 8 hours of treatment,the mRNA and protein expression levels of microtubule-associated protein light chain 3(LC3B),autophagy-related gene 4A(ATG4A)and autophagy-related gene 7(ATG7)were detected by real-time fluorescence quantitative method and Western blotting,respectively.Results Compared with the control group,the relative mRNA expression of LC3B and ATG7 in the EBSS group increased,while the mRNA expression of ATG4A decreased,the relative expression of LC3B mRNA increased and ATG7 decreased in the EBSS+Baf-A1 group Compared with the EBSS group.The expression of LC3B mRNA in the EBSS+Baf-A1 group increased,and the expression of ATG7 and ATG4A mRNA decreased compared with that of the the Baf-A1 group.Furthermore,we found the protein ratio of LC3BII/LC3BI increased and the ATG4A decreased in the EBSS group compared with those of the control group.The LC3BII/LC3BI increased and the ATG7 protein expression decreased in the EBSS+Baf-A1 group compared with those of the EBSS group.The LC3BII/LC3BI increased and the ATG7 protein expression decreased in the EBSS+Baf-A1 group compared with those of the Baf-A1 group.Conclusion EBSS can induce the autophagy in MDA-MB-231 cells and promote the formation of autophagosome by increasing the ATG7 mRNA expression.

关 键 词：细胞自噬 饥饿 微管相关蛋白轻链3 自噬相关基因ATG7 自噬相关蛋白4A 三阴性乳腺癌 

分 类 号：R737.9[医药卫生—肿瘤]