Exosome From CAF Facilitates Epithelial Mesenchymal Transition Of Prostate Cancer Cells

ObjectiveUtilizing exosome from cancer-associated fibroblasts(CAF)and normal fibroblasts(NF)separately to treat prostate cancer cells and discuss the influence of exosome and it's specific biological molecules from CAF in the epithelial mesenchymal transformation(EMT)of prostate cancer cells.Methods(1)CAF and NF were distinguished by Western Blot and immunofluorescence(IF).Prostate cancer cells were treated by the conditioned medium of CAF and NF.The influence of different on cancer cells was examined through Western Blot.(2)Ultracentrifugation was applied to extract the exosome from CAF and NF and transmission electron microscope was used to detect such exosome.(3)Exosome from CAF and NF was dyed through PKH-67.The absorption of exosome above by prostate cancer cells was observed via IF.(4)Transwell migration assay,Western Blot and IF were utilized to analyse the impact of migration and EMT of prostate cancer cells mediated by different exosome.(5)Relative published literatures and database were searched and found some miRNA which could potentially lead to the results above.MiRNAs were detected and quantified by RT-PCR and qPCR.CAF and NF were treated with miRNA inhibitor and then repeated the previous experiment.Experimental results was verified via Transwell migration assay,Western Blot and IF.Results(1)Conditioned medium of Prostate cancer cells was used as the control group,and NF and CAF conditioned medium were used as the treatment groups on Tramp-C1 and LNCaP cells.Western Blot showed that compared with the tumor cells conditioned medium and NF conditioned medium,conditioned medium of CAF could promote EMT of prostate cancer cells.(2)Exosome from NF and CAF were extracted by gradient ultracentrifugation,and were successfully verified by Western Blot and transmission electron microscopy.Exosome stained with PKH-67 could be absorbed by prostate cancer cells by IF.Tumor cells' migration ability was enhanced and processing EMT after intaking the exosome from CAF.(3)We identified 4 potential miRNAs which may contribute to the above effect through literatures and databases.They were miRNA-18 a,miRNA-21,miRNA-181 ba and miRNA-214.The expression of miRNA-18 a and miRNA-21 in NF-and CAF-derived exosomes were detected to be statistically significant by PCR,qRT-PCR.And the difference could be transferred into Tramp-C1 and LNCaP cells via exosomes.After treatmenting CAF with miRNA inhibitor and NC,miRNA-21 was confirmed the main miRNA which promoted EMT of prostate cancer cells.The migration ability and EMT process of tumor cells decreased when miRNA-21 was inhibited.(4)The promotion of EMT of prostate cancer cells by CAF was a complex process involving multiple components.Our study showed that exosome containing miRNA-21 participated in this procedure.While,there were other substances which could take part in the transition in CAF conditioned medium and the exact composition is not yet clear.ConclusionsCAF was able to promote prostate cancer cells to EMT by secreting exosome which contained miRNA-21.The experimental results vanished after miRNA-21 being inhibited.