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目的:探讨大黄素抑制乳腺癌细胞侵袭与迁移的潜在作用机制。方法:体外培养乳腺癌细胞系MDA-MB-231，按照是否使用大黄素分为空白组及大黄素组，挽救实验补充大黄素＋氯喹组、740Y-P组、大黄素＋740Y-P组及氯喹组。检测伤口愈合试验中的伤口闭合情况；Transwell迁移与侵袭试验下的细胞数量；蛋白免疫印迹分析下的上皮-间充质转化(EMT)相关蛋白(E-钙黏蛋白、N-钙黏蛋白、β-连环蛋白、Snail、Slug)、自噬相关蛋白[Beclin1、家蚕隔离体蛋白1(SQSTM1)、微管相关蛋白1轻链3(LC3)Ⅱ/LC3Ⅰ]及通路相关蛋白[糖原合酶激酶3β(GSK3β)、磷酸肌醇3-激酶(PI3K)、蛋白激酶B(AKT)、哺乳动物雷帕霉素靶蛋白(mTOR)、磷酸化糖原合酶激酶3β(p-GSK3β)、磷酸化磷酸肌醇3-激酶(p-PI3K)、磷酸化蛋白激酶B(p-AKT)、磷酸化哺乳动物雷帕霉素靶蛋白(p-mTOR)]。结果:伤口愈合试验处理48 h后，空白组[(83.20±5.29)%]、15 μmol/L浓度大黄素组[(70.24±3.52)%]、30 μmol/L浓度大黄素组[(61.77±3.40)%]及60 μmol/L浓度大黄素组[(35.95±2.94)%]MDA-MB-231细胞的伤口愈合率比较，差异有统计学意义( P<0.05)。Transwell小室实验处理48 h后，空白组[(531±54)个]、15 μmol/L浓度大黄素组[(465±59)个]、30 μmol/L浓度大黄素组[(383±29)个]及60 μmol/L浓度大黄素组[(240±33)个]MDA-MB-231细胞的细胞侵袭数量比较，差异有统计学意义( P<0.05)；空白组[(403±37)个]、15 μmol/L浓度大黄素组[(370±51)个]、30 μmol/L浓度大黄素组[(296±36)个]及60 μmol/L浓度大黄素组[(162±30)个]MDA-MB-231细胞的细胞迁移数量比较，差异有统计学意义( P<0.05)。经Western blot法验证，大黄素处理MDA-MB-231细胞48 h后，空白组、15 μmol/L浓度大黄素组、30 μmol/L浓度大黄素组及60 μmol/L浓度大黄素组的E-钙黏蛋白(0.75±0.23、1.15±0.11、1.35±0.05、1.48±0.31)、N-钙黏蛋白(1.56±0.09、1.48±0.08、1.25±0.16、1.19±0.05)、β-连环蛋白(1.23±0.16、0.92±0.14、0.71±0.08、0.41±0.05)、Snail(0.93±0.06、0.97±0.11、0.40±0.02、0.29±0.01)及Slug(0.43±0.08、0.29±0.01、0.19±0.02、0.05±0.01)蛋白表达量比较，差异有统计学意义( P<0.05)。空白组、经60 μmol/L大黄素处理24 h的大黄素组、经60 μmol/L大黄素处理36 h的大黄素组及经60 μmol/L大黄素处理48 h的大黄素组的SQSTM1(1.01±0.01、0.72±0.08、0.49±0.05、0.39±0.01)及LC3Ⅱ/LC3Ⅰ(1.03±0.04、1.39±0.10、1.71±0.08、2.75±0.15)表达量比较，差异有统计学意义( P<0.05)。空白组、大黄素组、氯喹组及大黄素＋氯喹组N-钙黏蛋白(0.03±0.01、0.15±0.01、0.03±0.01、0.47±0.01)、β-连环蛋白(0.85±0.08、0.29±0.01、0.79±0.02、0.65±0.05)、Snail(0.75±0.02、0.37±0.02、0.71±0.01、0.67±0.01)、Slug(0.14±0.01、0.07±0.01、0.21±0.01、0.06±0.01)表达量比较，差异有统计学意义( P<0.05)。空白组、15 μmol/L浓度大黄素组、30 μmol/L浓度大黄素组及60 μmol/L浓度大黄素组的p-GSK3β(0.80±0.03、0.50±0.07、0.21±0.01、0.07±0.02)、p-PI3K(0.58±0.04、0.39±0.04、0.21±0.04、0.12±0.02)、p-AKT(0.85±0.08、0.69±0.07、0.39±0.07、0.15±0.05)及p-mTOR(0.31±0.01、0.21±0.01、0.17±0.01、0.05±0.01)比较，差异有统计学意义( P<0.05)；空白组、大黄素组、740Y-P组及大黄素＋740Y-P组的p-GSK3β(1.00±0.03、0.97±0.07、0.41±0.01、0.57±0.02)、p-PI3K(0.21±0.04、0.64±0.04、0.41±0.05、0.12±0.02)、p-AKT(0.95±0.08、1.39±0.07、0.13±0.01、0.45±0.05)及p-mTOR(0.31±0.01、0.51±0.01、0.17±0.01、0.30±0.01)表达水平比较，差异有统计学意义( P<0.05)。 结论:大黄素通过自噬与EMT之间的串扰机制抑制乳腺癌细胞转移。

Objective:To explore the potential mechanism of emodin inhibiting invasion and migration of breast cancer cells.Methods:Breast cancer cell line MDA-MB-231 was cultured in vitro and divided into blank group and emodin group according to whether emodin was used or not, and rescue experiment supplemented emodin + chloroquine group, 740Y-P group, emodin + 740Y-P group and chloroquine group.Wound closure in wound healing tests, number of cells in Transwell migration and invasion tests; epithelial-mesenchymal transition(EMT) associated proteins(E-cadherin, N-cadherin, β-catenin, Snail, Slug) under protein immunoblot analysis, autophagy-related proteins[ Beclin1, sequestosome 1(SQSTM1), microtubule-associated protein light chain 3(LC3)Ⅱ/LC3Ⅰ]and pathway-related proteins[ glycogen synthase kinase 3β(GSK3 β), phosphoinositol 3-kinase(PI3K), protein kinase B(AKT), mammalian target of rapamycin(mTOR), phosphorylated glycogen synthase kinase 3β(p-GSK3β), phosphorylated phosphoinositol 3-kinase(p-PI3K), phosphorylated protein kinase B(p-AKT) and phosphorylated mammalian target of rapamycin(p-mTOR)]were analyzed.Results:After 48 hours of treatment by wound healing test, the wound healing rate of MDA-MB-231 cells in blank group[(83.20±5.29) %], emodin group with 15 μmol/L concentration[(70.24±3.52) %], emodin group with 30 μmol/L concentration[(61.77±3.40) %]and emodin group with 60 μmol/L concentration[(35.95±2.94) %]were compared, the differences were statistically significant( P<0.05).After 48 h of Transwell laboratory treatment, comparison of cell invasion number of MDA-MB-231 cells in blank group(531±54), emodin group with 15 μmol/L concentration(465±59), emodin group with 30 μmol/L concentration(383±29) and emodin group with 60 μmol/L concentration(240±33), the differences were statistically significant( P<0.05).Cell migration number of MDA-MB-231 cells in blank group(403±37), emodin group with 15 μmol/L concentration(370±51), emodin group with 30 μmol/L concentration(296±36) and emodin group with 60 μmol/L concentration(162±30) were compared, the differences were statistically significant( P<0.05).Western blot verified that after emodin treated MDA-MB-231 cells for 48 h, E-cadherin(0.75±0.23, 1.15±0.11, 1.35±0.05, 1.48±0.31), N-cadherin(1.56±0.09, 1.48±0.08, 1.25±0.16, 1.19±0.05), β-catenin(1.23±0.16, 0.92±0.14, 0.71±0.08, 0.41±0.05), Snail(0.93±0.06, 0.97±0.11, 0.40±0.02, 0.29±0.01)and Slug(0.43±0.08, 0.29±0.01, 0.19±0.02, 0.05±0.01)protein expression levels in blank group, emodin group with 15μmol/L concentration, emodin group with 30 μmol/L concentration and emodin group with 60 μmol/L were compared, the differences were statistically significant( P<0.05).Blank group, treated with 60 μmol/L concentration emodin group for 24 h, treated with 60 μmol/L concentration emodin group for 36 h and treated with 60μmol/L concentration emodin group for 48 h, the expression levels of SQSTM1(1.01±0.01, 0.72±0.08, 0.49±0.05, 0.39±0.01) and LC3Ⅱ/LC3Ⅰ(1.03±0.04, 1.39±0.10, 1.71±0.08, 2.75±0.15) were compared, the differences were statistically significant( P<0.05).N-cadherin(0.03±0.01, 0.15±0.01, 0.03±0.01, 0.47±0.01), β-catenin(0.85±0.08, 0.29±0.01, 0.79±0.02, 0.65±0.05), Snail(0.75±0.02, 0.37±0.02, 0.71±0.01, 0.67±0.01), Slug(0.14±0.01, 0.07±0.01, 0.21±0.01, 0.06±0.01) in blank group, emodin group, chloroquine group and emodin + chloroquine group of expression level were compared, the differences were statistically significant( P<0.05).After 48 h of emodin treatment, in blank group, emodin group with 15 μmol/L, emodin group with 30 μmol/L concentration and emodin group with 60 μmol/L concentration, p-GSK3β(0.80±0.03, 0.50±0.07, 0.21±0.01, 0.07±0.02), p-PI3K(0.58±0.04, 0.39±0.04, 0.21±0.04, 0.12±0.02), p-AKT(0.85±0.08, 0.69±0.07, 0.39±0.07, 0.15±0.05) and p-mTOR(0.31±0.01, 0.21±0.01, 0.17±0.01, 0.05±0.01)were compared, the differences were statistically significant( P<0.05).In blank group, emodin group, 740Y-P group and emodin + 740Y-P group, p-GSK3β(1.00±0.03, 0.97±0.07, 0.41±0.01, 0.57±0.02), p-PI3K(0.21±0.04, 0.64±0.04, 0.41±0.05, 0.12±0.02), p-AKT(0.95±0.08, 1.39±0.07, 0.13±0.01, 0.45±0.05) and p-mTOR(0.31±0.01, 0.51±0.01, 0.17±0.01, 0.30±0.01)were compared, and the differences were statistically significant( P<0.05). Conclusions:Emodin inhibits breast cancer cell metastasis through a crosstalk mechanism between autophagy and EMT.