Ectopic ATP5B Promotes PC-3M Cell Metastasis In Prostate Cancer By Activating The ITGB1/FAK Pathway

ATP5B is a β subunit in the F1 structural domain of mitochondrial intima ATP synthase,and it is also one of the most important subunits of F1F0 ATP synthase,which plays a crucial role in tumor progression.In the preliminary work of the research group,in order to find a new target for the treatment of prostate cancer patients,we obtained a short peptide B04 that can specifically bind to the cell membrane protein of PC-3M by phage reduction screening of human normal prostate epithelial cells,Prostate cancer PC-3 cells and prostate cancer PC-3M cells.The target protein specifically binding to short peptide B04 was screened as ATP5 B by reverse fishing,affinity chromatography and mass spectrometry,and it was confirmed that cholesterol promoted the ectopic expression of cell membrane ATP5 B and promoted the proliferation,migration and invasion of prostate cancer PC-3M cells.Treatment with short peptide B04 could effectively inhibit its proliferation,migration and invasion,while treatment with Piceatannol could inhibit the ectopic expression of ATP5 B on the surface of cell membrane and inhibit the migration and invasion of cells.In order to explore the mechanism of ATP5 B promoting the migration and invasion of prostate cancer PC-3M cells,the research group performed proteomic analysis on the prostate cancer PC-3M cell membrane ATP5 B group and prostate cancer PC-3M cell membrane ATP5 B group.Proteomic KEGG enrichment analysis showed that LAMA4 and other differential proteins were enriched in the ECM-receptor interaction signaling pathway.LAMA4 is a component of the extracellular matrix（ECM）.The integrin family is the membrane surface receptor of laminin.The recognition binding of the integrin to ECM affects the downstream adherent spot kinase（FAK）and causes a cascade of signal amplification.Genomic data of prostate cancer patients from TCGA database were analyzed by UCSC XENA website,and the results suggested that ATP5 B was positively correlated with ITGB1 and FAK in prostate cancer.According to proteomic analysis,we hypothesized that ATP5 B expressed ectopically on the cell membrane may affect migration and invasion through ITGB1/FAK signaling pathway in PC-3M cells of prostate cancer.Objective:To investigate the effect of ectopic expression of ATP5 B on the migration and invasion of prostate cancer PC-3M cells through ITGB1/FAK pathway,so as to provide theoretical and experimental basis for ectopic expression of ATP5 B in cell membrane as a potential therapeutic target for the treatment of prostate cancer.Method:1.Proteomic analysis of differential proteins and KEGG enrichment of prostate cancer PC-3M cells with ectopic high expression of ATP5 B and ectopic expression of ATP5 B in cell membrane,retrieval of transcriptome data of tumor tissues of prostate cancer patients from TCGA database,and analysis of the correlation between ATP5 B and ITGB1 and FAK,so as to provide basis for follow-up experiments.2.The optimal blocking time of short peptide B04 on ectopic expression of ATP5 B in cell membrane was detected by cellular immunofluorescence assay.3.The expression of ATP5 B and ITGB1 in PC-3M cells was detected by Immunocytochemistry.The positional relationship between ectopic ATP5 B and itgb1 in PC-3M cells was determined by immunofluorescence double staining.4.Prostate cancer PC-3M cells were transfected with plasmid containing ITGB1gene（expressing GFP indicator protein）,and the stable knock down ITGB1 cell line was obtained through puromycin screening.The knock down efficiency was detected at the m RNA and protein levels by RT-q PCR and Western Blot.5.Western Blot was used to detect ATP5 B ectopic high expression group,ATP5 B ectopic low expression group,ATP5 B ectopic closed expression group,control group,Scramble group,sh ITGB1 group and sh ITGB1 + ATP5 B ectopic high expression group Protein expression of ATP5 B,ITGB1,FAK and p-FAK.6.ATP5 B ectopic high expression group,ATP5 B ectopic low expression group,ATP5 B ectopic closed expression group and control group,Scramble group,sh ITGB1 group and sh ITGB1 + ATP5 B ectopic high expression group were detected by scratch test,Transwell compartment migration and invasion test cell migration and invasion ability of PC-3M.Result:1.The results of proteomic KEGG enrichment analysis showed that the differential proteins such as LAMA4 were enriched in the ECM receiver interaction signal pathway.After the transcriptome data analysis of prostate cancer patients in TCGA database,the results also showed that ATP5 B was positively correlated with ITGB1 and FAK.The results suggested that ATP5 B was closely correlated with ITGB1 / FAK pathway.2.The optimal blocking time of short peptide B04 on ATP5 B was 12 h.3.ITGB1 was positive in PC-3M cells.Immunofluorescence double staining showed that ATP5 B and ITGB1 were Co-located.4.The results of RT-q PCR and Western blot showed that the PC-3M cell line with knock down ITGB1 was successfully obtained.The following is uniformly expressed in sh ITGB1 group.5.Western Blot results showed that the expressions of ATP5 B,ITGB1 and p-FAK in ATP5 B ectopic high expression group were higher than those in the control group,while the expressions of ATP5 B ectopic low expression group and ATP5 B ectopic closed expression group were lower than those in the control group.Compared with Scramble,ATP5 B expression remained unchanged in the sh ITGB1 group,and p-FAK expression was decreased in the sh ITGB1 + ATP5 B ectopic high expression group,while p-FAK expression was decreased in the sh ITGB1 + ATP5 B ectopic high expression group.6.The results of scratch test and Transwell chamber migration and invasion test showed that the migration and invasion ability of the ATP5 B ectopic high expression group was higher than that of the control group,while the ATP5 B ectopic low expression group and the ATP5 B ectopic closed expression group were lower than that of the control group.The migration and invasion ability of sh ITGB1 and sh ITGB1 + ATP5 B ectopic high expression group was lower than that of the Scramble group.Conclusion:1.Ectopic expression of ATP5 B and ITGB1 co-located in PC-3M cells.2.Ectopic high expression of ATP5 B on cell membrane can activate ITGB1/FAK pathway.3.The ectopic high expression of ATP5 B on cell membrane promotes the migration and invasion of PC-3M cells in prostate cancer by activating the ITGB1/FAK pathway.