转化生长因子β1影响三阴性乳腺癌顺铂耐药的机制研究  

Mechanism Study of Transforming Growth Factor β1 Affecting Cisplatin Resistance in Triple-Negative Breast Cancer

作　　者：朱雅婷[1] 许文婷[2] 尹秋玉 欧江华[2] 张晨光[2] ZHU Ya-ting;XU Wen-ting;YIN Qiu-yu;OU Jiang-hua;ZHANG Chen-guang(Department of Pharmaceutical,Xinjiang Medical University Cancer Hospital,Urumqi,Xinjiang,830011,China;Second Ward of Breast Surgery,Xinjiang Medical University Cancer Hospital,Urumqi,Xinjiang,830011,China;The Third Clinical School of Xinjiang Medical University,Urumqi,Xinjiang,830011,China)

机构地区：[1]新疆医科大学附属肿瘤医院药学部,新疆乌鲁木齐830011 [2]新疆医科大学附属肿瘤医院乳腺外科(二病区),新疆乌鲁木齐830011 [3]新疆医科大学第三临床医学院,新疆乌鲁木齐830011

出　　处：《现代生物医学进展》2023年第7期1236-1242,共7页Progress in Modern Biomedicine

基　　金：新疆维吾尔自治区高校科研计划重点项目(XJEDU2020I014);新疆维吾尔自治区药学会科研基金项目(YXH202101)。

摘　　要：目的:建立三阴性乳腺癌MDA-MB-231/顺铂(DDP)耐药细胞株,探讨转化生长因子β1(TGF-β1)调控三阴性乳腺癌DDP耐药的机制。方法:采用小剂量间歇诱导法建立MDA-MB-231耐药细胞株(MDA-MB-231/DDP),在MDA-MB-231/DDP中构建TGF-β1沉默细胞并分为TGF-β1沉默组(sh-TGF-β1)、阴性对照组以及对照组,实时定量聚合酶链反应(RT-qPCR)检测TGF-β1含量。另取MDA-MB-231细胞和MDA-MB-231/DDP细胞分为MDA-MB-231组(正常培养MDA-MB-231敏感细胞)、MDA-MB-231-DDP组(正常培养MDA-MB-231 DDP耐药细胞)、TGFβ1-shRNA组(MDA-MB-231 DDP细胞转染TGFβ1-shRNA慢病毒载体)和MDA-MB-231-DDP+3-MA组(MDA-MB-231 DDP细胞给予5mM 3-MA处理2 h)。细胞计数试剂盒(CCK-8)法检测耐药株的半数抑制浓度(IC50),并计算耐药指数及逆转耐药指数,RT-qPCR检测TGF-β1含量,蛋白印迹法(Western blot)检测TGF-β1、自噬相关蛋白LC3-I、LC3-II表达量,激光共聚焦显微镜观察自噬流的变化,应用SPSS 20.0软件进行统计分析。结果:成功建立DDP耐药细胞株MDA-MB-231/DDP,耐药指数为5.231;MDA-MB-231/DDP细胞的TGF-β1 m RNA表达和蛋白表达较MDA-MB-231细胞显著上调(P<0.05)。DDP耐药细胞MDA-MB-231/DDP中自噬相关蛋白LC3-II/LC3-I表达较MDA-MB-231细胞显著升高(P<0.05);应用自噬抑制剂3-甲基腺嘌呤(3-MA)后MDA-MB-231/DDP细胞自噬相关蛋白LC3-II/LC3-I表达显著下降(P<0.05);沉默MDA-MB-231/DDP细胞的TGF-β1基因后,DDP耐药细胞株的耐药指数从5.231下降到3.404,同时自噬相关蛋白LC3-II/LC3-I表达降低(P<0.05),且激光共聚焦显微镜观察到黄色和红色斑点的显著减少,表明自噬受到抑制。结论:TGF-β1与三阴性乳腺癌DDP耐药有关,其机制可能是增加自噬引起MDA-MB-231细胞DDP耐药。通过沉默TGF-β1可降低自噬水平,恢复三阴性乳腺癌细胞对DDP的敏感性。Objective:To establish MDA-MB-231/cisplatin(DDP)resistant cell lines of triple-negative breast cancer,and to explore the mechanism of transforming growth factorβ1(TGF-β1)regulating DDP resistance of triple-negative breast cancer.Methods:MDA-MB-231 resistant cell lines(MDA-MB-231/DDP)were established by low-dose intermittent induction method.TGF-β1 silencing cells were constructed in MDA-MB-231/DDP and they were divided into TGF-β1 silencing group(sh-TGF-β1),negative control group and control group.Real-time quantitative polymerase chain reaction(RT-qPCR)was used to detect TGF-β1 content.MDA-MB-231 cells and MDA-MB-231/DDP cells were divided into MDA-MB-231 group(MDA-MB-231 sensitive cells cultured normally)and MDA-MB-231-DDP group(MDA-MB-231 sensitive cells cultured normally DDP resistant cells),TGFβ1-shRNA group(MDA-MB-231 DDP cells transfected with TGFβ1-shRNA lentiviral vector)and MDA-MB-231-DDP combined with 3-MA group(MDA-MB-231 DDP cells were treated with 5 mM 3-MA for 2 h).Half inhibitory concentration(IC50)of drug-resistant strains was detected by cell counting kit(CCK-8),and drug resistance index and reverse resistance index were calculated.TGF-β1 content was detected by RT-qPCR,and expression levels of TGF-β1 and autophagy related proteins LC3-I and LC3-II were detected by Western blot.The changes of autophagy flow were observed by laser confocal microscopy.SPSS 20.0 software was used for statistical analysis.Results:The DDP resistant cell line MDA-MB-231/DDP was established successfully,and the resistance index was 5.231.The mRNA expression and protein expression of TGF-β1 in the MDA-MB-231/DDP cells were significantly up-regulated compared with MDA-MB-231 cells(P<0.05).The expression of autophagy related protein LC3-II/LC3-I in the MDA-MB-231/DDP resistant cells were significantly higher than those in the MDA-MB-231 cells(P<0.05).The expression of autophagy related protein LC3-II/LC3-I in the MDA-MB-231/DDP cells was significantly decreased after the application of autophagy inhibitor 3-met

关 键 词：转化生长因子Β1 自噬 三阴性乳腺癌 顺铂 耐药 

分 类 号：R737.9[医药卫生—肿瘤]