Effects And Mechanism Of Exosomes Secreted By Mesenchymal Stem Cells Treated With Doxorubicin On Breast Cancer Chemotherapy Resistance

Objective: We will explore the effect and mechanism of Doxorubicin treated mesenchymal stem cells exosomes(Dt-MSC-exo)secreted by doxorubicin-treated mesenchymal stem cells on breast cancer chemotherapy resistance,and find possible molecular mechanisms of drug resistance In order to obtain possible drug resistance targets and provide new options for accurate treatment of breast cancer.Methods:1.The culture supernatants of mesenchymal stem cells(MSCs)treated with doxorubicin(Doxorubicin,Dox)and physiological saline were collected to obtain MSCs conditioned medium and Dox-treated MSCs(Doxorubicin treated MSCs,Dox-treated-MSC)conditioned medium,and co-cultured with MDA-MB-231,MCF-7 cells,through CCK-8(CCK-8 Assay),LDH(Lactate dehydrogenase),flow cytometry detection of MSCs conditioned medium and Dox-treated-MSC conditioned medium Effects on MDA-MB-231 and MCF-7cells tolerant to Dox.2.Isolation,extraction and identification of mesenchymal stem cells exosomes(MSC-exo).Exosomes were extracted from MSCs conditioned medium and Dox-treated-MSC conditioned medium by ultracentrifugation,and the morphology of exosomes was identified by transmission electron microscope analysis.Surface protein markers.3.After co-cultivation of MSC-exo and Dt-MSC-exo with MDA-MB-231 and MCF-7 cells,CCK-8,LDH and flow cytometry experiments were used to detect MDA-MB-231 and MCF-7 cells are resistant to Dox.4.Using the GEO database,analyze and screen the differentially expressed m RNA between untreated MCF-7 cells and MCF-7 cells treated with MSC-exo to make a heat map.Then the experiment was divided into three groups:(1)control group,(2)MSC-exo group,(3)Dt-MSC-exo group,and Real-time quantitative quantitative PCR(q PCR)and WB detection were used to detect the m RNA and protein expression of differential genes in MDA-MB-231 and MCF-7 cells.5.After down-regulating the differentially expressed gene S100A6 in MDA-MB-231 and MCF-7 cells,co-culture with MSC-exo and Dt-MSC-exo,using q PCR and WB to detect each group of MDA-MB-231 and MCF-7S100A6 m RNA and protein expression changes in cells.Next,the effects of MSC-exo and Dt-MSC-exo on Dox tolerance of MDA-MB-231 and MCF-7cells were tested by CCK-8 and flow cytometry.Subsequently,the expression of S100A6 was down-regulated in MSCs,use q PCR and WB to detect the expression changes of S100A6 m RNA and protein in MSCs and Dox-treated MSCs,and collect the exosomes of MSCs and co-culture with MDA-MB-231,MCF-7 cells,q PCR was used to detect the expression of S100A6 m RNA in MDA-MB-231 and MCF-7 cells.6.candidate mi RNA(mi R-21-5p)was determined,and then q PCR was used to detect the expression of MSCs and MSC-exo and mi R-21-5p m RNA after Dox treatment.After transfection of mi R-21-5p agomir(downregulated mi R-21-5p)in MDA-MB-231 and MCF-7 cells,they were co-cultured with MSC-exo and Dt-MSC-exo.The expressions of mi R-21-5p and S100A6 m RNA in MDA-MB-231 and MCF-7 cells of each group were detected by q PCR,and Confirm the correlation between S100A6 and mi R-21-5p.Subsequently,the expression of mi R-21-5p was down-regulated in MSC-exo and Dt-MSC-exo,co-cultured with MDA-MB-231 and MCF-7 cells,and each group of MDA-MB-231 and MCF-7 cells were detected by q PCR S100A6 m RNA expression in CCK8 and flow cytometry were used to detect the effect of MSC-exo and Dt-MSC-exo on MDA-MB-231 and MCF-7 cells tolerant to Dox.Results:1.MSCs conditioned medium and Dox-treated-MSC conditioned medium can significantly enhance the resistance of MDA-MB-231 and MCF-7 cells to Dox,and compared with MSCs conditioned medium,Dox-treated-MSC conditions The culture solution led to greater resistance of MDA-MB-231 and MCF-7 cells to Dox.2.Isolate exosomes from MSCs conditioned medium and Dox-treated-MSC conditioned medium by ultracentrifugation.Transmission electron microscopy observed oval or goblet-shaped double-layered membrane vesicle bodies with a diameter ranging from 40 to 100 nm.WB results showed that the surface proteins CD9,CD63,CD81 and TSG101 of the exosomes were positive.3.MSC-exo and Dt-MSC-exo can significantly enhance the resistance of MDA-MB-231 and MCF-7 cells to Dox,and compared with MSC-exo,Dt-MSC-exo leads to MDA-MB-231,MCF-7 cells are more resistant to Dox.4.GEO database(GSE46950)predicts that screening MSC-exo and Dt-MSC-exo can significantly increase the expression of S100A6 in MDA-MB-231 and MCF-7 cells,and compared with MSC-exo,Dt-MSC-exo resulted in higher expression of S100A6 in MDA-MB-231 and MCF-7 cells than MSC-exo.5.Down-regulate the expression of S100A6 in MDA-MB-231,MCF-7cells,can significantly inhibit the promotion of Dt-MSC-exo on MDA-MB-231,MCF-7 cells tolerant to Dox,and eliminate MSC-exo and Dt-MSC-exo had significant differences in Dox resistance between MDA-MB-231 and MCF-7cells.However,the expression of S100A6 was down-regulated in MSCs,and MSC-exo and Dt-MSC-exo had no significant effect on the promotion of S100A6 in MDA-MB-231 and MCF-7 cells.6.MSC-exo and Dt-MSC-exo promote the expression of S100A6 in MDA-MB-231 and MCF-7 cells by transmitting mi R-21-5p,S100A6 has a positive correlation with mi R-21-5p.Down-regulating the expression of mi R-21-5p in MSC-exo can significantly inhibit the promotion of Dt-MSC-exo on Dox tolerance of MDA-MB-231 and MCF-7 cells;More importantly,it eliminates the increasing effect of S100A6 levels and eliminates the significant difference in Dox resistance between MDA-MB-231 and MCF-7 cells between MSC-exo and Dt-MSC-exo.Conclusion:1.MSC-exo treated with Dox can significantly enhance the resistance of MDA-MB-231 and MCF-7 cells to Dox.2.Dox-treated MSC-exo promotes the expression of S100A6 in MDA-MB-231 and MCF-7 cells by delivering mi R-21-5p,which may enhance the resistance of MDA-MB-231 and MCF-7 cells to Dox One of the mechanisms.