The Ectopic High Expression Of ATP5B Promotes The Migration And Invasion Of PC-3M Cells In Prostate Cancer By Activating ZNF703/MMP2 Pathway

BackgroundProstate cancer is the most common form of cancer in adult men.In our previous work,we through differential screening of phage peptide library,in human normal prostate epithelial cells,human prostate cancer PC-3 cells and human prostate cancer PC-3M cells(a highly metastatic cell line of PC-3)obtained a short peptide B04 that could specifically bind to the cell membrane of human prostate cancer PC-3M cells.Then,by reverse fishing and mass spectrometry,we identified ATP5 B as a specific binding protein to peptide B04.In response to this phenomenon,we successfully constructed a model of ectopic high expression of ATP5 B on the cell membrane,ectopic low expression of ATP5 B on the cell membrane and closed ectopic expression of ATP5 B on the cell membrane in PC-3M cells.Further studies found that ectopic high expression of ATP5 B on the cell membrane could promote the migration and invasion of PC-3M cells.The migration and invasion ability of PC-3M cells were inhibited by ectopic low expression of ATP5 B and ectopic expression blockade of ATP5 B.In order to explore the mechanism of ATP5 B overexpression promoting the migration and invasion of prostate cancer PC-3M cells,we used PC-3M cells as the control group and PC-3M cells with ectopic high ATP5 B expression as the experimental group.Among the proteomics results,we successfully screened the protein associated with the migration and invasion of PC-3M cells,ZNF703,and its downstream molecule,MMP2,by analyzing the differential proteins.Thus,based on the analysis of the proteomic results,it is suggested that our cell membrane ectopically expressed ATP5 B may affect the migration and invasion of prostate cancer PC-3M cells through the ZNF703/MMP2 pathway.ObjectiveTo investigate whether ATP5 B can affect the migration and invasion of prostate cancer PC-3M cells through ZNF703/MMP2 pathway,and to provide theoretical and experimental basis for the discovery,clinical diagnosis and treatment of prostate cancer.Methods1.The ZNF703/MMP2 pathway was activated by the ectopic expression of ATP5B(1)Western Blot assay was used to detect the ectopic expression of ATP5 B,ZNF703 and MMP2 in the cell membrane in the control group,ATP5 B ectopic high expression group,ATP5 B ectopic low expression group and ATP5 B ectopic closed expression group,respectively.(2)Gelatin enzyme assay was used to detect MMP2 enzyme activity in control group,ATP5 B group with ectopic high expression,ATP5 B group with ectopic low expression and ATP5 B group with ectopic closed expression.(3)ZNF703 was silenced in PC-3M cells with ATP5 B ectopic high expression.The lentivirus infection efficiency was observed by fluorescence microscopy.The m RNA and protein expression of ZNF703 were detected by RT-q PCR and Western Blot,respectively,to identify the transfection efficiency.(4)Western Blot assay was used to detect the ectopically expressed ATP5 B,ZNF703 and MMP2 proteins in the cell membrane in NC group,Scramble group and Sh ZNF703 group,respectively.The enzyme activity of MMP2 was detected in Scramble group and Sh ZNF703 group by gelatin enzyme profile experiment.2.The ectopic high expression of ATP5 B promotes the migration and invasion of PC-3M cells in prostate cancer and is correlated with the activation of ZNF703/MMP2(1)The effect of ectopic expression of ATP5 B in the migration and invasion ability of PC-3M was examined using wound healing and Transwell migration and invasion assays in the ectopic high expression of ATP5 B,ectopic low expression of ATP5 B and ectopic closed expression of ATP5 B groups.(2)In Scramble and Sh ZNF703 groups,protein expression of ATP5 B,ZNF703and MMP2 ectopically expressed in the cell membrane was detected using Western Blot assay;gelatinase profiling assay was performed to detect the enzymatic activity of MMP2.3.Correlation analysis in TCGA database and the ectopic expression of ATP5 B,ZNF703 and MMP2 in human prostate cancer and surrounding tissues and their relationship with Gleason grade(1)UCSC XENA website was used to analyze the correlation between ATP5 B,ZNF703 and MMP2 in prostate cancer patients’ tumor tissues in TCGA database.(2)Immunohistochemical staining was used to analyze the ectopic expression of ATP5 B,ZNF703 and MMP2 in prostate cancer and surrounding tissues with different Gleason grades.Results1.The ZNF703/MMP2 pathway was activated by the ectopic expression of ATP5B(1)The results of Western Blot assay showed that the protein expression of the cell membrane ectopically ATP5 B,ZNF703 and MMP2 was significantly higher in the ATP5 B ectopic high expression group compared with the control group,while the protein expression of the cell membrane ectopically ATP5 B,ZNF703 and MMP2 in ATP5 B ectopic low expression and ATP5 B ectopic expression closed group was significantly decreased.(2)Gelatin zymography showed that the enzymatic activity of MMP2 was significantly higher in the ATP5 B ectopic high expression group compared with the control group,while the enzymatic activity of MMP2 was significantly lower in the ATP5 B ectopic low expression and ATP5 B ectopic expression closed groups.(3)Immunofluorescence showed that the lentivirus infection efficiency was more than 90%.The results of Western Blot and RT-q PCR showed that,compared with the NC group,the m RNA and protein expression of ZNF703 cells in Scramble group were significantly increased.Compared with the Scramble group,the m RNA and protein expressions of ZNF703 cells in the Sh ZNF703 group were significantly reduced,indicating successful construction of a PC-3M cell line with high ATP5 B ectopic expression of ZNF703.(4)The results of Western Blot showed that,compared with the NC group,the Scramble group significantly increased the protein expressions of ATP5 B,ZNF703 and MMP2.Compared with Scramble group,ATP5 B protein expression in cell membrane ectopically expressed in Sh ZNF703 group did not change,while ZNF703 and MMP2 protein expressions were significantly decreased.Compared with Scramble group,the enzyme activity of MMP2 in Sh ZNF703 group was significantly reduced.2.The changes of migration and invasion of PC-3M cells in prostate cancer were correlated with the activation of ZNF703/MMP2(1)The results of wound healing and Transwell migration and invasion assays showed that the migration and invasion ability of cells in the ectopic high ATP5 B expression group was significantly higher compared with the control group,while the migration and invasion ability of cells in the ectopic low ATP5 B expression and ectopic closed ATP5 B expression groups was significantly lower.(2)The results of cell wound healing and Transwell migration and invasion assays showed that the migration and invasion ability of cells in the Sh ZNF703 group was significantly reduced compared with the Scramble group.3.Correlation analysis in TCGA database and the ectopic expression of ATP5 B,ZNF703 and MMP2 in human prostate cancer and surrounding tissues and their relationship with Gleason grade(1)According to the transcriptome data of prostate cancer patients in TCGA database,ATP5 B was positively correlated with ZNF703 and MMP2.(2)The results of immunohistochemical staining showed that in human prostate cancer tissues,ATP5 B was mainly expressed in the cell membrane,ZNF703 in the nucleus and MMP2 in the cytoplasm,all of which were stained brownish-yellow;meanwhile,the expression of ATP5 B,ZNF703 and MMP2 was significantly higher in prostate cancer tissues compared with surrounding tissues and showed a significant positive correlation with Gleason grade.The ectopic expression of ATP5 B,ZNF703 and MMP2 in prostate cancer tissues was significantly higher and positively correlated with Gleason grade.Conclusions1.The ZNF703/MMP2 pathway was activated by the ectopic expression of ATP5 B.2.The changes of migration and invasion of PC-3M cells in prostate cancer were correlated with the activation of ZNF703/MMP2.3.In TCGA database,the expression of ATP5 B was positively correlated with ZNF703 and MMP2 in human prostate cancer patients.the ectopic expression of ATP5 B,ZNF703 and MMP2 are highly expressed in human prostate cancer and surrounding tissues,and are positively correlated with Gleason grade.