Preparation Of Short Peptide B04 Liposome And Its In Vitro Inhibition Of ATP5B Ectopic Expression In Mitochondria Of Prostate Cancer Cells

In the preliminary work of the research,we obtained the short peptide B04 specifically bound to human high metastatic prostate cancer cell line PC-3M by phage display technology,and then obtained the target protein ATP5 B specifically bound to B04 on the cell membrane of PC-3M by reverse fishing and screening.ATP5 B is usually expressed in the mitochondrial endometrium,and can be ectopic in the cell membrane in some cells.Its function and ectopic route are still unclear,and the ectopic expression of ATP5 B is related to the metastasis ability of tumor.Therefore,in order to illustrate specific source and effectively inhibit tumor cell membrane surface ATP5 B ATP5B ectopic expression,this research intends to adopt short peptide antibody specificity ATP5 B B04,cells by liposome uptake unique mechanism,establish a short peptide B04 combined with mitochondrial ATP5 B not only with the combined method of cell membrane ATP5 B,specificity of mitochondria ATP5 B,closed by protein imprinting method to observe the cell membrane surface ATP5 B ectopic expression is effectively decreased,thus inhibiting ATP5 B ectopic expression.Methods:1.Prepare short peptide B04 and verify whether it has the same function as short peptide which is located on the phage surface.A batch of short peptide B04 was synthesized according to the short peptide sequence,and the prepared B04 was co-incubated with PC-3M cells.The binding ability of B04 to ATP5 B was detected by cell immunofluorescence chemistry and Western blot.2.Preparation of liposomes coated with short peptide B04 and establishment of HPLC detection method for B04(1)the liposomes wrapped in B04 were prepared by inverse phase evaporation method,and the standard curve of short peptide B04 was established by HPLC.(2)the demulsification solution of liposomes was detected by HPLC,and the encapsulation rate was calculated.3.To detect the inhibitory ability of B04 liposome on mitochondrial ATP synthase ectopic and PC-3M transfer(1)the effects of B04 liposome on ATP5 B energy metabolism on mitochondria in cells were detected by ATP biological detection kit.(2)the effects of cholesterol stimulation on the ATP5 B content in the cell membrane and cytoplasm of PC-3M cells were detected by Western blot.(3)inhibition of ATP5 B ectopic ability of mitochondria by short peptide B04 was detected by Western blot.(4)the transwell chamber was used to detect the influence of B04 liposome on the migration and invasion ability of PC-3M cells.Results:1.To verify the effect of synthetic short peptide B04 on ATP5 B in PC-3M cellsCell fluorescence detection showed that after PC-3M cells were sealed with200ug/mL B04 solution,the fluorescence intensity and positive rate of ATP5 B were significantly reduced compared with the control group.Western blot analysis showed that the protein content of ATP5 B was significantly reduced after the use of 200ug/mL B04 solution compared with that of the unsealed group.The results showed that the single chain of B04 short peptide had the same sealing function as that of B04 expressed on phage surface.2.Synthesize B04 liposome and verify its performanceThe peak area of free B04 and the peak area of emulsion breaking B04 were taken into the standard curve formula,the corresponding concentration was calculated,and the encapsulation rate formula was used to obtain the encapsulation rate of B04 liposomes prepared by reverse evaporation method was 44.1%.3.Effect of B04 liposome on ATP5 B ectopic expression in PC-3M cell membrane of prostate cancer cellsWe found that compared with the no-load group,ATP5 B protein on the cell membrane surface of PC-3M increased significantly in the cholesterol-loaded group by Western blot after 6 hours of cholesterol loading,and the expression of ATP5 B in the cytoplasm could not be detected regardless of whether cholesterol stimulation was used.By cholesterol loading for 6h,Western blot detection showed that the PC-3M group(25-100ug/mL,4h)with added B04 liposome had a significant decrease in ATP5 B compared with the control group,while the total ATP5 B had no significant change.4.Effects of B04 liposome on the migration and invasion ability of PC-3M in prostate cancer cellsIn both Transwell chamber migration and invasion experiments,the number of cells in PC-3M group with B04 liposome was significantly decreased compared with the control group,which proved that the migration ability of tumor cells was positively correlated with the ATP5 B heterotopic expression on the cell membrane surface of PC-3M.Conclusion:1.The synthetic short peptide B04 can block ATP5 B on the cell membrane surface of pc-3m prostate cancer cells.2.The B04 liposome inhibited the ATP5 B expression on the pc-3m cell membrane surface of prostate cancer cells.3.B04 liposome has an inhibitory effect on the migration and invasion of pc-3m in prostate cancer cells.