Experimental Study On The Estrogen Promotes Mammary Epithelial Cell Stemness And Epithelial-mesenchymal Transition Via Regulation The Calpain-ERK Signaling Pathway

Objective:The main purpose of this study was to investigate the effects of E2-induced stemness and epithelial-mesenchymal transition of MCF-10A of human mammary epithelial cells,and the mediating role of calpain and its downstream pathways in this process.Methods:1.Cell transformation:MCF-10A of mammary epithelial cells were used as the research model.The cells were divided into three groups:DMSO（blank control,Cont）,E2 experimental group（E2-transformed cells,E2-T）and E2+Calpeptin experimental group（E2-C）,were respectively treated by DMSO（0.1%）、E2（50nmol/L）and E2（50nmol/L）+Calpeptin（1μmol/L）for15 generations.Experimental was divided into three groups to conduct the following experiments:（1）morphological observation;（2）cell proliferation inhibition rate was detected by MTT assay;（3）the cloning rate of cells was detected by colony formation assay.2.E2-induced MCF-10A cell stem cell characteristics,epithelial-mesenchymal transition and Calpain inhibitor Calpeptin in the intervention of E2-induced related effects:The experiment was divided into 3 groups,blank control group（DMSO）,E2-transformed cell group and E2+Calpeptin group to conduct the following experiments:（1）The cell mammosphere numbers was detected by Mammosphere assays;（2）The migration and invasion rates of cells were detected by wound healing experiment and transwell experiment respectively（3）Stemness related m RNA CD44,CD24,ALDH1A1,Nanog and OCT4 were detect;ed by q RT-PCR.（4）Western blot was used to detect the expressions of CD44,Nanog and OCT4 stemness related proteins,FN,E-cad and Vim EMT related proteins and p-ERK proteins.3.Nude mice subcutaneously into tumor experiment:the nude mice were randomly divided into 3 groups（each group n=3）:the blank control group,E2-transformed cells group and E2+Calpeptin experimental group.blank control group respectively（DMSO）,E2-transformed cell group and E2+Calpeptin experimental cells were inoculated in nude mouse breast pad,record each group of nude mice tumor volume,tumor weight and weight curve.4.Effect of ERK inhibitor U0126 on stemness and epithelial-mesenchymal transition of E2-transformed cells:blank control group（DMSO）+DMSO（0.1%）group,E2-transformed cells group+（0.1%）DMSO and E2 transformed+U0126(10μmol·L^-1)as the experimental group to conduct the following experiments:colony formation assay,Mammosphere assays,wound healing experiment,transwell assays and the expression of CD44,Nanog and OCT4 stemness related proteins,FN,e-cad and Vim epithelial-mesenchymal transition related proteins were detected by Western blot.Results:1.E2-induced MCF-10A cell transformation and Calpeptin’s intervention on E2-induced MCF-10A cell transformation:（1）The blank control group showed irregular polygon.Compared with the blank control group（DMSO）,the cell bodies of E2-transformed cells were larger and showed long spindle shape with mesenchymal cell characteristics.Compared with E2 transformed cells,E2+Calpeptin cells showed irregular polygon and small cell body;（2）Compared with the blank control group（DMSO）,the proliferation ability of E2-transformed cells was significantly enhanced（P<0.01）;Compared with E2-transformed cells,the proliferation ability of E2+Calpeptin group was significantly reduced（P<0.01）;（3）compared with the blank control group（DMSO）,the cloning ability of E2-transformed cells was significantly enhanced（P<0.01）;Compared with E2 transformed cells,the cloning ability of E2+Calpeptin group was significantly reduced（P<0.01）.2.E2-induced stemness and epithelial-mesenchymal transtion of MCF-10A cells and intervention of Calpeptin:（1）Compared with the blank control group（DMSO）,the self-renewal ability,migration rate and invasion rate of E2-transformed cells were significantly enhanced（P<0.01）;compared with E2-transformed cells,the self-renewal ability,migration rate and invasion rate of E2+Calpeptin experimental group were significantly reduced（P<0.05）;（2）Compared with the blank control group（DMSO）,the stemness m RNA expressions of CD44,ALDH1A1,Nanog and OCT4 in E2-transformed cells were up-regulated（P<0.01）,and the stemness m RNA expressions of CD24 were down-regulated（P<0.05）,the expressions of CD44,Nanog and OCT4 stemness-related proteins in E2-transformed cells were up-regulated（P<0.01）,the expressions of FN and Vim proteins were up-regulated（P<0.01,P<0.05）,and the expressions of E-cad proteins were down-regulated（P<0.01）;Compared with the E2-transformed cell group,the stemness m RNA expressions of CD44,ALDHA1,Nanog and OCT4 in the E2+calpeptin experimental group were down-regulated（P<0.05）,and the stemness m RNA expressions of CD24 were up-regulated（P<0.01）;CD44,Nanog and OCT4 were down-regulated（P<0.01）,FN and Vim were down-regulated（P<0.01）,and E-cad were up-regulated（P<0.01）.（4）Compared with blank control group（DMSO）,E2-induced MCF-10A cell transformation resulted in high expression of p-ERK（P<0.01）;Compared with E2-transformed cells,the expression of p-ERK protein in E2+Calpeptin group was significantly down-regulated（P<0.01）.3.Subcutaneous tumor formation in nude mice:（1）Compared with the blank control group（DMSO）,the tumor volume and tumor weight were significantly increased in the E2 transformed cell group（P>0.05）,and the weight of nude mice decreased with the increase of tumor volume（P<0.05）.（2）Compared with the E2 transformed cell group,there was no tumor formation in the E2+Calpeptin group,and the nude mice gained weight（P<0.05）.4.Inhibition of ERK inhibitor U0126 on stemness and epithelial-mesenchymal transition of E2-transformed cells:（1）compared with the E2-transformed cells+DMSO（0.1%）group,the E2-transformed cells+U0126（10μmol/L）group could significantly inhibit the proliferation,self-renewal,migration and invasion of E2-transformed cells（P<0.01）;Down-regulated CD44,Nanog,Oct4 dry-related proteins（P<0.05）,down-regulated the expression of FN,Vim proteins（P<0.05）,up-regulated the expression of E-cad proteins（P<0.05）.Conclusion:E2 can promote stem cell characteristics and epithelial-mesenchymal transition of mammary epithelial cells,and promote cell proliferation,clone formation,self-renewal,migration and invasion.The enhancement of stem cell characteristics and epithelial-mesenchymal transition of mammary epithelial cells promoted by E2 may be related to the Calpain-ERK signaling pathway.