| 朱雅婷,许文婷,尹秋玉,欧江华,张晨光.转化生长因子β1影响三阴性乳腺癌顺铂耐药的机制研究[J].,2023,(7):1236-1242 |
| 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 |
| 投稿时间：2022-11-23 修订日期：2022-12-19 |
| DOI：10.13241/j.cnki.pmb.2023.07.007 |
| 中文关键词: 转化生长因子β1 自噬 三阴性乳腺癌 顺铂 耐药 |
| 英文关键词: Transforming growth factor β1 Autophagy Triple-negative breast cancer Cisplatin Drug resistance |
| 基金项目: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） |
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| 中文摘要: |
| 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 mRNA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。 |
| 英文摘要: |
| ABSTRACT Objective: To establish MDA-MB-231/cisplatin (DDP) resistant cell lines of triple-negative breast cancer, and to explore the mechanism of transforming growth factor β1 (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), TGF β1-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-methyladenine(3-MA)(P<0.05). After silencing TGF-β1 gene in the MDA- MB-231/DDP cells, the drug resistance index in the DDP resistant cell lines decreased from 5.231 to 3.404, while the expression of autophagy related protein LC3-II/LC3-I were decreased(P<0.05), and the yellow and red spots were significantly reduced by confocal laser microscopy, it showed that autophagy was inhibited. Conclusion: TGF-β1 is associated with DDP resistance in the triple-negative breast cancer, and the mechanism may be increased autophagy causing DDP resistance in the MDA-MB-231 cells. By silencing TGF-β1, autophagy levels are reduced and DDP sensitivity of triple-negative breast cancer cells is restored. |
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