十溴联苯醚暴露通过胞外囊泡装载miR-221促进三阴性乳腺癌细胞转移

Decarbromodiphenyl ether exposure promotes migration of triple-negative breast cancer cells through miR-221 in extracellular vesicles

目的:探讨环境污染物十溴联苯醚(BDE-209)暴露促进三阴性乳腺癌(TNBC)恶性进展的机制。方法:将TBNC细胞分为空白对照组和BDE-209暴露组。超速离心法提取胞外囊泡,分别应用扫描电子显微镜和蛋白质印迹法对提取的胞外囊泡进行鉴定,采用纳米颗粒跟踪技术检测胞外囊泡生成量,细胞划痕实验和Transwell小室实验检测与胞外囊泡共培养后TNBC细胞的迁移能力和扩散能力,定量逆转录PCR(qRT-PCR)检测囊泡中的miR-221表达水平,蛋白质印迹法检测共培养后TNBC细胞中基质金属蛋白酶9(MMP9)的表达水平。结果:与空白对照组比较,2.00、20.00和200.00 ng/mL BDE-209暴露可显著增加胞外囊泡的生成(均P<0.05),其中200.00 ng/mL BDE-209暴露诱导释放的胞外囊泡共培养的细胞迁移率较空白对照组提高86%,穿膜细胞数提高至1.32倍,miR-221表达水平提高至2.71倍,MMP9表达水平提高至1.62倍(均P<0.05)。转染抗miR-221抗体能降低BDE-209暴露诱导生成的胞外囊泡内miR-221表达水平,使BDE-209促进MDA-MB-231细胞迁移的能力被显著抑制。结论:BDE-209暴露可通过增加胞外囊泡的生成及装载miR-221增强TNBC细胞的迁移能力,促进TNBC细胞转移。

Objective:To investigate the effect of decarbromodiphenyl ether(BDE-209)exposure on the migration ability of triple-negative breast cancer(TNBC)cells and to explore the underlying mechanism.Methods:Human TNBC MDA-MB-231 cells were divided into blank control group and BDE-209 exposure groups(treated with 0.02,0.20,2.00,20.00 and 200.00 ng/mL BDE-209 in high glucose DMEM).Extracellular vehicles(EVs)secreted by MDA-MB-231 cells were isolated by differential ultracentrifugation.Transmission electron microscopy(SEM),nanoparticle tracking analysis(NTA)and Western blotting were performed to characterize the EVs.The effect of the EVs induced by BDE-209 exposure(EVs-BDE-209)on the migration and invasion of MDA-MB-231 cells was detected by wound-healing assay and Transwell test.qRT-PCR was used to measure the miR-221 level in EVs-BDE-209.The expression of MMP9 in MDA-MB-231 cells was determined by Western blotting.Results:Compared with the blank control,BDE-209 exposure increased the tumor cell-derived EVs in dose-dependent manner.The MDA-MB-231 cells co-cultured with EVs released by 200.00 ng/mL BDE-209 exposure showed an 86%increase in cell migration rate,a 1.32-fold higher number of membrane-penetrating cells,a 2.71-fold higher expression level of miR-221,and a 1.62-fold higher expression level of MMP9 compared with the blank control group(all P<0.05).While transfection with anti-miR-221 antibody to decrease miR-221 level in EVs significantly reversed the increased invasion ability of the MDA-MB-231 cells treated with EVs-BDE-209.Conclusion:BDE-209 exposure may promote metastasis potential of MDA-MB-231 cells via EVs-BDE-209 transmitted miR-221.