信号转导和转录激活因子3/CC趋化因子配体2信号通路对乳腺癌细胞活力、迁移和侵袭的影响实验研究

Effect of signal transducer and activator of transcription 3/C-C motif chemokine ligand 2 signaling pathway on viability,migration and invasion of breast cancer cells

目的:探讨信号转导和转录激活因子3(STAT3)/CC趋化因子配体2(CCL2)信号通路对乳腺癌细胞活力、迁移和侵袭的影响。方法:体外培养人乳腺癌细胞株(HCC1937、MCF-7、MDA-MB-231、ZR-75-1)和人正常乳腺上皮细胞(MCF-10A)。采用蛋白印迹法检测HCC1937、MCF-7、MDA-MB-231、ZR-75-1和MCF-10A细胞磷酸化STAT3(p-STAT3)、STAT3、CCL2蛋白表达。选取p-STAT3/STAT3蛋白比值、CCL2蛋白表达最高的人乳腺癌细胞进行后续实验。将MCF-7细胞分为对照组、STAT3抑制剂(WP1066)Ⅰ组(2μmol/L WP1066)、Ⅱ组(4μmol/L WP1066)、Ⅲ组(8μmol/L WP1066)。分别采用细胞计数试剂盒-8、划痕实验、Transwell实验检测MCF-7细胞活力、迁移率及侵袭能力。采用蛋白印迹法检测MCF-7细胞p-STAT3、STAT3、CCL2、基质金属蛋白酶(MMP)-2、MMP-9蛋白表达。结果:与MCF-10A细胞比较,HCC1937、MCF-7、MDA-MB-231、ZR-75-1细胞p-STAT3/STAT3蛋白比值、CCL2蛋白表达升高(均P<0.05)。其中MCF-7细胞p-STAT3/STAT3蛋白比值、CCL2蛋白表达最高,因此选用MCF-7细胞进行后续实验。与对照组比较,STAT3抑制剂Ⅰ、Ⅱ、Ⅲ组MCF-7细胞活力、迁移率、侵袭数目依次降低(均P<0.05)。与对照组比较,STAT3抑制剂Ⅰ、Ⅱ、Ⅲ组MCF-7细胞p-STAT3/STAT3蛋白比值、CCL2、MMP-2及MMP-9蛋白表达依次降低(均P<0.05)。结论:乳腺癌细胞中STAT3/CCL2呈高表达,抑制STAT3/CCL2信号通路能够降低乳腺癌细胞活力,减少迁移和侵袭。

Objective:To investigate the effect of signal transducer and activator of transcription 3(STAT 3)/CC motif chemokine ligand 2(CCL2)signaling pathway on viability.migration and invasion of breast cancer cells.Methods:Human breast cancer cll lines(HCC1937,MCF-7,MDA-MB 231,ZR-71-1)and human normal breast epithelial cells(MCF-10A)were cultured in vitro.The expressions of p-STAT3,STAT3 and CCL2 in HCC1937,MCF-7,MDA-MB-231,ZR-75-1 and MCF-10A cells were detected by Western blot.Human breast cancer cells with the highest p-STAT3/STAT3 protein ratio and CCL2 protein expression were selected for follow up experiments.MCF-7 cells were divided into control group,STAT3 inhibitor(WP1066)group Ⅰ(2 pmol/L WP1066),group Ⅱ(4μmol/L WP1066)and group Ⅲ(8μmol/L WP1066).Cell count kit 8,scratch test and Transwell test were used to detect MCF-7 cell viability,mobility and invasion ability,respectively.The protein expressions of p STAT3,STAT3,CCL2,matrix metalloprotei-nase(MMP)-2 and MMP-9 in MCF-7 cells were detected by Western blot.Results:Compared with MCF-10A cells,the expressions of p-STAT3/STAT3 protein ratio and CCL2 protein were increased in HCC1937,MCF-7,MDA-MB-231 and ZR-75-1 cells(all P<0.05).Among them,the p-STAT3/STAT3 protein ratio and CCL2 protein expression were the highest in MCF-7 cells,so MCF-7 cells were selected for follow uP experiments.Compared with the control group,MCF-7 cells viability,mobility and invasion number in STAT3 inhibitor groups Ⅰ,Ⅱ and Ⅲ were decreased successively(all P<0.05).Compared with the control group,the p STAT3/STAT3 protein ratio,CCL2,MMP-2 and MMP-9 protein expressions of MCF-7 cells in STAT3 inhibitor groups Ⅰ,Ⅱ and Ⅲ were decreased successively(all P<0.05).Conclusion:STAT3/CCL2 is highly expressed in breast cancer cells,and inhibition of STAT3/CCL2 signaling pathway can reduce the via bility,migration and invasion of breast cancer cells.