EIF3B Regulates The Enrichment Of MiR-100-5p In Prostate Cancer PC-3-Derived Exosomes And Its Influence On Macrophage Phagocytosis

Objective: To study the effect of RNA-binding protein EIF3 B in prostate cancer cells PC-3 on the expression level of mi R-100-5p in exosomes,and then to explore the effect of exosomal mi R-100-5p on macrophage phagocytosis.Methods: 1.Exosomes were identified by transmission electron microscopy and nanoparticle size analysis,and exosome surface marker proteins were identified by Western Blot;2.The expression of mi R-100-5p in PC-3 and RWPE-1 exosomes was detected by q PCR.PC-3-derived-exosomes was labeled with PKH67,and then induce macrophages.The expression of miR-100-5p in PC-3-exo-M was detected by using q PCR;3.Human peripheral blood mononuclear cells were extracted by immunomagnetic beads method,and then induced into macrophages in vitro;4.The phagocytosis ability of PC-3-exo-M and macrophages was assesed using phagocytosis kit after transfection with mi R-100-5p inhibitor;5.Biyuntian reactive oxygen species ROS detection kit was used to detect ROS content of macrophages in the above group;6.QPCR and Western Blot were used to detect the expression level of target gene NOX4 after mi R-100-5p inhibition,Double luciferase report experiment verified the binding of mi R-100-5p to NOX4;7.Biotin-labeled mi R-100-5p probe was used to pull down the mi R-100-5p binding protein,and the expression of EIF3 B in samples and exosomes was detected by Western Blot;8.QPCR was used to detect the m RNA level of EIF3 B in PC-3 and RWPE-1 cells;9.PC-3 cells were transfected with EIF3 B si RNA,and the knockdown effect of EIF3 B was detected by Western Blot.QPCR was used to evaluate the effects of EIF3 B on the expression of mi R-100-5p in PC-3-exosomes.Results:1.The exosomes photographed by transmission electron microscopy are concave cup-shaped,and the size of the exosomes is about 100 nm by nanoparticle tracking analysis.WB identification of exosomes expressing CD63 and Tsg101proteins;2.The expression of mi R-100-5p in prostate cancer cellderived-exosomes(PC-3-exo)was significantly higher than that in normal prostate epithelial cell derived exosomes(RWPE-1-exo),prostate cancer cells PC-3-derived exosomes were absorbed in macrophages,the expression of mi R-100-5p in PC-3-exo-M was significantly increased compared with M0;3.The monocytes were successfully extracted and induced into macrophages in vitro;4.Exosomes derived from prostate cancer cells PC-3 inhibited the phagocytosis of macrophages.With inhibition of mi R-100-5p,the phagocytic ability of macrophages was enhanced;5.Compared with M0,the level of ROS in PC-3-exo-M decreased,and the content of reactive oxygen species in PC-3-exo-M increased after inhibiting the expression of mi R-100-5p;6.NOX4 is the target gene of mi R-100-5p,down regulates the expression of mi R-100-5p,and the level of NOX4 protein increases;7.Mi R-100-5p can bind to EIF3 B protein;8.The EIF3 B m RNA in PC-3 cells was higher than that in normal prostate epithelial cells RWPE-1;9.Inhibition of EIF3 B in PC-3 cells down regulated the expression of mi R-100-5p in PC-3-derived-exosomes.Conclusion: Conclusion: 1.The exosomes of prostate cancer cells PC-3 are the important sources of mi R-100-5p in macrophages,and the process of mi R-100-5p entering PC-3 exosomes is mainly regulated by EIF3 B protein.2.mi R-100-5p in PC-3-exo inhibits the phagocytic function of macrophages,and its mechanism is to reduce ROS production of macrophages by inhibiting the expression of NOX4.