鱼藤素通过FZD7调控Wnt通路抑制三阴性乳腺癌细胞侵袭、迁移、EMT的实验研究

目的:探讨鱼藤素对三阴性乳腺癌（TNBC）细胞增殖、侵袭、迁移和上皮-间质转化（EMT）的抑制作用及可能的机制。方法:体外培养人TNBC细胞系（MDA-MB-231和BT-20），加入鱼藤素（5、10、20 μmol/L）诱导后，采用CCK-8法检测MDA-MB-231和BT-20细胞的存活率;克隆形成实验检测细胞克隆形成能力;流式细胞术分析细胞凋亡情况;Transwell实验检测细胞迁移和侵袭能力;Western Blot检测细胞中卷曲同源物7（FZD7）、Ki-67、E-钙黏蛋白（E-cadherin）、波形蛋白（Vimentin）、β-连结蛋白（β-catenin）、c-myc蛋白表达。利用FZD7过表达载体质粒（pcDNA-FZD7）转染建立FZD7过表达细胞，进一步研究FZD7过表达对鱼藤素抗TNBC作用的影响;裸鼠成瘤实验检测鱼藤素对MDA-MB-231细胞体内生长的影响。结果:与对照组（0 μmol/L）相比，鱼藤素（5、10、20 μmol/L）呈剂量依赖性的显著降低MDA-MB-231和BT-20细胞存活率，并显著增加MDA-MB-231和BT-20细胞的凋亡率（P＜0.05）。与对照组相比，鱼藤素可显著降低MDA-MB-231和BT-20细胞增殖活力、克隆形成能力、迁移与侵袭能力和细胞内Vimentin、FZD7、β-catenin、c-myc蛋白表达，升高细胞凋亡率和细胞内E-cadherin蛋白表达（P＜0.05）;过表达FZD7可逆转鱼藤素对TNBC细胞恶性表型的影响。体内实验中，与对照组相比，鱼藤素组裸鼠的肿瘤体积和肿瘤质量、肿瘤中FZD7蛋白表达和FZD7、Ki-67阳性表达显著降低（P＜0.05），与鱼藤素组和鱼藤素+pcDNA-NC组相比，鱼藤素+pcDNA-FZD7组肿瘤体积和肿瘤质量、肿瘤中FZD7蛋白表达和FZD7、Ki-67阳性表达显著升高（P＜0.05）。结论:鱼藤素可抑制TNBC细胞增殖、迁移、侵袭和EMT，并诱导细胞凋亡，其作用机制可能与下调FZD7的表达，抑制Wnt/β-catenin信号通路的活化有关。

Experimental study on deguelin inhibiting the invasion,migration and EMT of triple-negative breast cancer cells by regulating the Wnt pathway through FZD7

Objective:To investigate the inhibitory effects of deguelin on the proliferation,invasion,migration,and epithelial-mesenchymal transition (EMT) of triple-negative breast cancer (TNBC) cells and its possible mechanism.Methods:Human TNBC cell lines (MDA-MB-231 and BT-20) were cultured in vitro,after induction by adding deguelin (5,10,20 μmol/L),the CCK-8 method was used to detect the survival rates of MDA-MB-231 and BT-20 cells.Clone formation experiment was used to detect cell clone formation ability.Flow cytometry was used to analyze the cell apoptosis.Transwell experiment was used to detect cell migration and invasion abilities.Western Blot was used to detect the expression of Frizzled-7 (FZD7),Ki-67,E-cadherin,Vimentin,β-catenin,and c-myc in cells,FZD7 overexpression vector plasmid (pcDNA-FZD7) transfection was used to establish the FZD7 overexpression cells,and the effect of FZD7 overexpression on the anti-TNBC effect of deguelin was further studied.The tumorigenesis experiment in nude mice was used to test the effect of deguelin on the growth of MDA-MB-231 cells in vivo.Results:Compared with the control group (0 μmol/L),deguelin (5,10,20 μmol/L) significantly reduced the survival rates of MDA-MB-231 and BT-20 cells in a dose-dependent manner,and significantly increased the apoptosis rates of MDA-MB-231 and BT-20 cells (P＜0.05).Compared with the control group,deguelin could significantly reduce the proliferation viability,clone formation,migration and invasion abilities of MDA-MB-231 and BT-20 cells,and the expression of Vimentin,FZD7,β-catenin,and c-myc proteins in the cells,and significantly increased the cell apoptosis rate and expression of E-cadherin protein in the cells (P＜0.05).Overexpression of FZD7 could reverse the effects of deguelin on the malignant phenotype of TNBC cells.In the in vivo experiment,compared with the control group,the tumor volume and tumor weight,expression of FZD7 protein and positive expression of FZD7 and Ki-67 in the tumor of the deguelin group were significantly reduced (P＜0.05),and compared with the deguelin group and the deguelin+pcDNA-NC group,the tumor volume and tumor weight,expression of FZD7 protein and positive expression of FZD7 and Ki-67 in the deguelin+pcDNA-FZD7 group were significantly increased (P＜0.05).Conclusion:Deguelin can inhibit the proliferation,migration,invasion,and EMT of TNBC cells,and induce cell apoptosis.Its mechanism may be related to down-regulating the expression of FZD7 and inhibiting the activation of Wnt/β-catenin signaling pathway.