The Relationship And Mechanism Between MiR-26b/RAB31 Mediated Lysosome-Related Exosome Secretion And BPA Induced Proliferation And Migration Of MCF-7 Cells

BackgroundBisphenol A(BPA)is a widely used chemical raw material and a typical environmental endocrine disruptors(EEDs).Studies have shown that BPA exposure can promote the occurrence of various tumors and is closely related to the clinical outcome of patients.In recent years,as an environmental pollutant,the link between BPA exposure and breast cancer has been widely concerned.Studies have found that BPA exposure can promote abnormal proliferation and migration of breast cancer cells,but the mechanism is unknown.Exosomes,with a diameter of about 30-200 nm,widely exist in various body fluids such as peripheral blood,saliva,urine and lotion,and play an extremely important role in the stress response of human cells to external environmental stimuli.Studies have found that under the stress of chemical substances such as cigarette smoke extract,alcohol,polychlorinated biphenyls(PCBs)and hydroquinone(HQ),the secretion of exosome in cells was significantly enhanced and played an important role in the stress response of cells.At present,the role and mechanism of exosomes in the stress response to BPA are still unclear.It was reported that BPA exposure resulted in abnormal miRNA profile in breast cancer cells,among which,microRNA-26b(miR-26b)was down-regulated.The luciferase reporter assay showed that miR-26b,as a tumor suppressor gene,can directly target the expression of RAB31.RAB31 could promote the formation of intraluminal vesicles(ILVs)independently of the classical endosomal sorting complex required for transport(ESCRT)and tetraspanin pathways,and inhibit multivesicular bodies(MVBs)-lysosomal fusion,reduce the degradation of exosome precursors,thus enhance the secretion of exosomes.However,whether miR-26b/RAB31 mediated exosome secretion is involved in the toxic effects of BPA remains elusive.ObjectiveThe purpose of this study is to explore the relationship and molecular mechanism between the proliferation and migration of human breast cancer cells(MCF-7)induced by BPA and lysosome related exosome secretion mediated by miR-26b/RAB31 pathway.Methods1.CCK-8 method was used to determine the cytotoxicity of BPA and calculate IC50.2.Taking untreated MCF-7 cells as control group,qRT-PCR and Western blot were used to analyze the expression level of miR-26b and RAB31 in cells of different treatment groups.3.Lysotracker and Lysosensor staining were used to analyze the number and activity of lysosomes in different treatment groups.4.Exosomes of different treatment groups were extracted by ultrahigh speed centrifugation and characterized by transmission electron microscopy.5.CCK-8 method and cell scratch test were used to analyze the cell proliferation and migration ability of different treatment groups.6.The miR-26b mimic was used to up-regulate the expression of miR-26b in cells,qRT-PCR and Western blot were used to analyze the expression level of RAB31 in different treatment groups before and after transfection,the changes of exosome secretion,proliferation and migration of cells were analyzed.Result1.The IC50 value of MCF-7 cell exposure to BPA for 24h was 65.82 μM.2.The exposure of MCF-7 to 10 μM BPA resulted in a decreased miR-26b expression,the expression of RAB31 was up-regulated,and the activation of miR-26b/Rab-31 pathway,consequently,the number and activity of lysosomes decreased,the secretion of exosomes increased,cell proliferation and migration were enhanced obviously.3.when GW4869 was used to directly inhibit the exosome secretion of MCF-7 cells treated with BPA,their proliferation and migration were down-regulated.4.miR-26b mimic down-regulated the expression of RAB31,increased the number and activity of lysosomes in MCF-7 cells,exosome secretion was down-regulated,cell proliferation and migration decreased.Conclusion1.BPA exposure activates the miR-26b/RAB31 pathway in MCF-7 cells.2.BPA exposure reduces the number and activity of lysosomes through miR-26b/RAB31 pathway.3.BPA exposure promotes exosome secretion through lysosome-related pathways.4.Exosome secretion is associated with the proliferation and migration of cell treated with BPA.