The Effect Of ATP5B Upregulation On MMP-2 In Caveolae Caused By Cholesterol Stimulation In Prostate Cancer Cells

Cholesterol levels on the cell membranes of normal cells are tightly regulated,but cholesterol metabolism is abnormal in many solid tumors,including prostate cancer.Cholesterol,as the main component of membrane lipids,accumulates in large quantities in membrane rafts and stabilizes membrane domains by interacting with other lipids and specific raft proteins.Cholesterol can promote ATP5 B ectopic on cell membrane,protein and observe prostate cancer cells(PC-3M)migration invasive ability increase,and MMP-2 can degradation of extracellular matrix of basic skeleton type Ⅳ collagen,destroy the tumor cell invasion and metastasis of histology barrier,the activation of MMP-2 also rely on the cell membrane.In addition,the research group obtained the ATP5B-Cav-1 protein complex in breast cancer cells MDA-MB-231 by immunoco-precipitation technology.Cav-1 was only located in the concave of membrane raft,and was responsible for its unique concave shape as a structural protein.Cav-1,as a marker of membrane raft,is considered to be an important protein in the development process of prostate cancer.Therefore,in order to further clarify the mechanism image of ATP5 B ectopic expression on the invasion ability of PC-3M tumor cells in prostate cancer cells,various promotion and inhibition models were adopted to explore the relationship between CAV-1,ATP5 B and MMP-2.Methods:1.Cytoplasmic membrane proteins were extracted and the binding of ATP5 B,Cav-1 and MMP-2 on PC-3M cell membrane surface was detected by immunoco-precipitation method.2.In PC-3M prostate cancer cells,plasma membrane proteins were extracted after cholesterol treatment for 0h,2h,6h,12 h and 24 h.Western blot was used to detect the expression levels of ATP5 B and MMP-2 proteins on the plasma membrane at different times of loading.3.The silencing effect of shRNA Cav-1 in prostate cancer was determined by PCR and Western blot from RNA and protein levels.The expressions of Cav-1 and MMP-2 were detected by Western blot.The cell supernatant was extracted and the synthesis of MMP-2 was detected by gelatinase.Transwell chamber was used to describe PC-3M invasion of prostate cancer cells.4.After the liposome B04 was co-incubated with the cells,cholesterol stimulation was added,and the plasma membrane expressions of ATP5 B,CAV-1 and MMP-2 were detected by Western blot.The secretion of MMP-2 in cell supernatant was detected by gelatinase assay.Results:1.In PC-3M,ATP5 B,Cav-1 and MMP-2 were co-located on the cell membrane of prostate cancer cellsImmunoprecipitation results showed that the MMP-2 antibody was immunoprecipitated by the cell membrane protein,and the expression of Cav-1 and ATP5 B were detected in the immunoprecipitation products.2.After cholesterol stimulation,the expression of ATP5 B,Cav-1 and MMP-2proteins on plasma membrane was increased,and it was time-dependentIn the cholesterol stimulation model,Western blot was used to detect the protein expressions of ATP5 B,Cav-1 and MMP-2 on the plasma membrane at different times of the load,and it was found that cholesterol could significantly promote the membrane ectopic level of ATP5 B,and the ATP5 B expression level was the highest at 12 h and slightly decreased at 24 h over time.Meanwhile,the expression levels of Cav-1 and MMP-2 on the membrane increased with the increase of cholesterol loading time,but the effect was not lasting,which was generally consistent with the change trend of ATP5 B protein expression.3.Cav-1 expression down-regulation induced MMP-2 synthesis and secretion down-regulation in PC-3M cellsShRNA was used to inhibit the expression of Cav-1 in PC-3M cells.PCR and Western blot revealed that the expression of shRNA was down-regulated at mRNAand protein levels compared with those of the scramble group.Western blot detected the down-regulation of MMP-2 expression in plasma membrane.The results of gelatinase assay showed that MMP-2 secretion decreased after Cav-1 was silenced,and invasion ability of PC-3M cells was weakened after Cav-1 expression was down-regulated.4.Effect of ATP5 B ectopic expression down-regulation on MMP-2 synthesis and secretionAfter the closure of cytoplasmic ATP5 B by liposome B04,cholesterol stimulated PC-3M cells and promoted the expression of ATP5 B,MMP-2 and Cav-1on the membrane surface.Western blot showed that after the closure of B04,the expression of ATP5 B on the plasma membrane decreased significantly,but the expressions of MMP-2 and Cav-1 were almost not affected.However,gelatinase results showed that MMP-2 secretion decreased.Conclusion:1.MMP-2 co-localized with ATP5 B and Cav-1 on the cell membrane of PC-3M prostate cancer cells.2.After cholesterol stimulation,the expression of ATP5 B,Cav-1 and MMP-2proteins on the plasma membrane was increased,and it was time-dependent.3.The down-regulation of ATP5 B expression in the membrane recess of prostate cancer cells inhibited the secretion of MMP-2,but did not affect MMP-2 protein expression.