Regulation And Mechanism Of Estrogen On Stemness Of ER Positive Breast Cancer Cells

Objective To investigate the effect of estrogen(Estradiol,E2)on the stem cell characteristics and epithelial-mesenchymal transition(EMT)of estrogen receptor(ER)-positive breast cancer cell MCF-7 and PI3 K /AKT/mTOR signaling pathway mediation.Methods 1.Western blot was used to detect the expression of estrogen receptor alpha(Estrogen receptor Alpha,ERα)in normal breast epithelial cells MCF-10 A and breast cancer cell lines.2.Use Mammosphere formation experiment to enrich breast cancer stem cells(Breast cancer stem cells,BCSCs);use quantitative real-time PCR(qPCR)and Western blot to detect the expression of BCSCs-related stem genes and proteins.3.The CCK-8 method was used to screen the optimal concentration of E2 on MCF-7 cells;qPCR and Western blot were used to verify the effect of E2 on the expression of BCSCs-related stemness genes and proteins;the Mammosphere formation experiment was used to observe the effect of E2 on the enriched BCSCs;The effect of E2 on the ratio of acetaldehyde dehydrogenase 1(ALDH1+)in BCSCs was observed by flow cytometry.4.Scratch experiment and cell clone formation experiment were used to detect the effect of E2 on the migration and proliferation of MCF-7cells;qPCR and Western blot were used to detect the changes in EMT-related gene and protein expression of MCF-7 cells.5.Scratch test and cell clone formation test to detect the effect of estrogen antagonist Fulvestrant on the migration and proliferation of MCF-7 cells;Western blot was used to detect EMT-related proteins in the control group(CT),E2 treatment group,and E2+Fulvestrant treatment group,and the expression of Ras/Raf and PI3K/AKT pathway proteins.6.qPCR and Western blot were used to detect changes in the expression of genes and proteins of PI3K/AKT/mTOR signaling pathway in MCF-7 cells by E2.7.Mammosphere formation experiment to observe the effect of AKT inhibitor MK-2206 on the enriched BCSCs;Western blot detection of E2,MK-2206 and E2+MK-2206 treatment group compared to CT group stemness-related protein,PI3K/ The expression of AKT/mTOR signaling pathway related proteins and EMT related proteins.Results 1.ERα was high expressed in ER-positive breast cancer MCF-7 cells,and the difference statistical was significantly(P<0.01).2.Compared with breast cancer cells,the BCSCs related genes and proteins ABCG-2,nanog and Oct-4 were significantly up-regulated in MCF-7 cells(P<0.05).3.Based on CCK-8 experiments,the optimal concentration of E2 on MCF-7 cells was 10 n M.At the concentration,the expression of BCSCs related genes and proteins ABCG-2,nanog,and Oct-4 are significantly increased(P<0.05);An increased volume and number of microspheres proved E2 could promote the spheroidization ability of BCSCs in vitro;Flow cytometry showed that compared with the CT group,the proportion of ALDH1+ cells in the BCSCs was significant increase after treated with E2(P<0.01).4.Compared with the CT group,the MCF-7 cells migration and clone formation rate in the E2 treatment group were increased;The expression of EMT-related genes and proteins N-cadherin,Vimentin,Slug,Snail,ZEB1 were increased(P<0.05).5.After treated with Fulvestrant,the migration and clone formation rate of MCF-7 cells were decreased;The expression of EMT and PI3K/AKT/mTOR pathway related proteins Vimentin,Slug,Snail,Ras,Raf,PI3 K and AKT was significantly reduced in the E2+Fulvestrant treatment group(P<0.05).6.In the E2 treatment group,the expression of PI3K/AKT/mTOR pathway related genes PI3 K,AKT,mTOR were increased,and the protein expression of PI3 K,AKT,mTOR,p70S6 K,P-PI3 K,P-AKT,P-mTOR,and P-p70S6 K were significantly increased(P<0.05).7.AKT inhibitor MK-2206 can significantly inhibited the spheroidization ability of BCSCs,and decreased the expression of stemness proteins ABCG-2,nanog,and Oct-4;MK-2206 can also reduce the expression of AKT,mTOR,p70S6 K,P-AKT,P-mTOR,P-p70S6 K and N-cadherin,Vimentin,Slug,and Snail(P<0.05).Conclusion E2 can induce and promote BCSCs enrichment in MCF-7 cells.E2 promoted EMT of MCF-7 cells through Ras/Raf/ and PI3K/AKT signaling pathways.E2 activated of PI3K/AKT/mTOR signaling pathway in MCF-7 cells and enhances the expression of BCSCs.