M1巨噬细胞培养液对ER阳性乳腺癌细胞上皮-间质转换的影响及Calpain的介导作用

Effects of M1 macrophage culture medium on epithelial-mesenchymal transition of ER-positive breast cancer cells and the mediating effect of Calpain

目的 探讨M1巨噬细胞对乳腺癌细胞上皮细胞-间充质转换(EMT)的影响及钙蛋白酶(Calpain)在其中的介导作用。方法 以人乳腺癌细胞系雌激素受体阳性(ER+)细胞MCF-7、T47D及雌激素受体阴性(ER-)细胞MDA-MB-231为模型细胞,实验分为MCF-7细胞及T47D细胞的RPMI-1640组(RPMI-1640纯培养基培养)、M1巨噬细胞条件培养液培养组(M1-CM组)、M1-CM+Calp组[M1-CM联合Calpain抑制剂Calpeptin(50μmoL/L)处理]、M1-CM+CI-Ⅲ组[M1-CM联合Calpain抑制剂Calpain inhibitorⅢ(50μmoL/L)处理],MDA-MB-231细胞的RPMI-1640组、M1-CM组;采用划痕实验和侵袭实验检测各组中MCF-7细胞、T47D细胞、MDA-MB-231细胞的迁移能力及MCF-7细胞的侵袭能力,采用qRT-PCR检测M0巨噬细胞、M1巨噬细胞趋化因子受体7CCR7、趋化因子配体10(CXCL10)及白细胞介素-12(IL-12) mRNA表达水平,采用Western blot实验检测MCF-7细胞、T47D细胞及MDA-MB-231细胞中上皮-钙黏素(E-cad)、纤连蛋白(FN)、波形蛋白(Vim)蛋白水平表达。结果 与RPMI-1640组相比,M1-CM组ER+细胞MCF-7及T47D迁移能力增强(P<0.05),MCF-7细胞侵袭能力增强(P<0.05);ER+细胞MCF-7及T47D E-cad蛋白表达水平下调,而FN、Vim表达上调(P<0.05),而ER-细胞MDA-MB-231中只有E-cad表达上调(P<0.05);与M1-CM组相比,ER+细胞MCF-7及T47D的M1-CM+Calp组、M1-CM+CI-Ⅲ组中M1-CM诱导的ER+乳腺癌细胞EMT效应被逆转(P<0.05)。结论 M1巨噬细胞条件培养液可以促进ER+细胞人乳腺细胞的EMT,但对ER-细胞EMT无明显影响,其促进ER+乳腺癌细胞EMT信号机制可能与Calpain通路有关。

Objective To investigate the effect of M1 macrophages on epithelial-mesenchymal transition(EMT)of breast cancer cells and the mediated role of Calpain.Methods Estrogen receptor(ER+)positive human breast cancer cells MCF-7,T47D,and ER-negative human breast cancer cells MDA-MD-231 were used as model cells.In the experiment,MCF-7 and T47D cells were divided into RPMI-1640 group(RPMI-1640 pure medium culture),M1-CM group(M1 macrophage conditioned medium culture group),M1-CM+Calp group[M1-CM combined with Calpain inhibitor Calpeptin(50μmoL/L)],and M1-CM+CI-Ⅲgroup[M1-CM combined with Calpain inhibitor Calpain inhibitorⅢ(50μmoL/L)];MDA-MB-231 cells were divided into RPMI-1640 group and M1-CM group.The cell migration ability of MCF-7 cells,T47D cells,and MDA-MB-231cells and the cell invasion ability of MCF-7 cells in each group were detected by Wound-healing assay and Transwell assay.The mRNA expression levels of CC-chemokine receptor 7(CCR7),CXC motif chemokine ligand 10(CXCL10)and interleukin-12(IL-12)in M0 macrophages and M1 macrophages were detected by qRT-PCR.The protein levels of epithelial cadherin(E-cad),fibritin(FN),and vimentin(Vim)in MCF-7 cells,T47D cells,and MDA-MB-231cells were detected by Western blot.Results Compared with the RPMI-1640 group,the migration ability of ER+cells MCF-7 and T47D in the M1-CM group was enhanced(P<0.05),and the invasive ability of MCF-7 cells was enhanced(P<0.05).The expression levels of MCF-7 and E-cad in ER+cells decreased,while the expressions of FN and Vim increased(P<0.05),and only E-cad expression increased in ER-cells MDA-MB-231(P<0.05).Compared with the M1-CM group,the EMT effect of ER+breast cancer cells induced by M1-CM in the M1-CM+Calp group and M1-CM+CI-Ⅲgroup of ER positive cells MCF-7 and T47D was reversed(P<0.05).Conclusion M1 macrophages conditioned medium can promote EMT of ER+cells and human breast cells,but has no obvious effect on EMT of ER-cells.Its mechanism of promoting EMT of ER+breast cancer cells might be related to Calpain signaling.