The Functional/Mechanism Studies Of Ectopic ATP5B In Metastasis Ability Of Human Prostate Cancer Cells

Prostate cancer is the most common malignancy of the male genitourinary system.The treatment is more difficult after metastasis,and the prognosis is poor.Therefore,it is important to study the mechanism of prostate cancer metastasis and to identify potential therapeutic targets for high metastatic potential prostate cancer.In the preliminary work of our group,we developed a phage-displayed 7-mer peptide library to screen the target peptides that were specifically bound to PC-3M cells with subtractive panning from normal prostate cells and PC-3 prostate cancer cells.A novel short peptide(B04)was found to have high affinity to highly metastatic PC-3M cells.And we found that B04 can inhibit the proliferation and metastasis of highly metastatic PC-3M cells.Then,ATP5 B which located on the PC-3M cell surface was identified as a binding protein of B04.ATP5 B is usually expressed in the mitochondrial inner membrane,it can also be located in the cytomembrane of some types of cells.But its function is not clear yet.The function of ATP5 B expressed on the surface of PC-3M membrane is to be identified.Therefore,in this study,we aimed to explore the effect and its mechanism of ectopic ATP5 B on the invasion and metastasis of human prostate cancer cell line PC-3M.Results:1.Expression and localization of ectopic ATP5 B in prostate cancer and adjacent tissuesThe expression of ATP5 B protein in 98 cases of prostate cancer tissue microarray was detected by immunohistochemical staining.The prostate tissue chip consisted of 58 cases of prostate cancer(including 2 cases of ductal adenocarcinoma),37 cases of adjacent tissues and 3 cases of normal prostate tissue,(1)The expression of ATP5 B in tumor cell membrane can be seen in prostate cancer tissue,and the positive rate of ectopic expression of cell membrane was significantly increased in infiltrating proliferative prostate cancer(p <0.05).(2)The positive rate of ATP5 B in the cell membrane was significantly higher in Gleason?8(P <0.05).2.Effects of ectopic ATP5 B on the invasion and metastasis of prostate cancer cells(1)Establishing ectopic ATP5 B overexpression and inhibition of expression modelThe expression of ATP5 B in PC-3M cell membrane was detected by Western blot,quantitative real-time PCR and ATP level detection from the aspects of protein,gene and function level.The results show below:(1)When treated with cholesterol,ATP5 B in mitochondria protein decreased,the ATP5 B in membrane protein increased,and the total protein ATP5 B did not change significantly.The mitochondrial ATP5 B of PC-3M did not change significantly,the membrane protein ATP5 B and total protein ATP5 B decreased when treated with piceatannol.(2)The relative expression of ATP5 B m RNA has no significant change after treated with cholesterol,and the relative expression of ATP5 B m RNA was decreased after treatment with piceatannol.(3)Cholesterol loadinng promotes PC-3M extracellular ATP production,while piceatannol inhibit extracellular ATP production.These results suggest that cholesterol promotes ATP5 B ectopia from mitochondrial to cell membrane and increases the expression of ATP5 B in the cell membrane.Piceatannol can inhibit the expression of ATP5 B in the cell membrane.(2)In vitro and in vivo study on the invasion and metastasis of prostate cancer cell line PC-3M effected by ectopic ATP5BThe effect of ectopic ATP5 B on invasion and metastasis of PC-3M in vitro were tested by cell wound healing test,Transwell chamber migration test,Transwell chamber invasion test.The results showed that the migration and invasion of PC-3M were enhanced with the increased expression of cell membrane ATP5B(p <0.05).After treated with cell membrane ATP5 B inhibitor piceatannol,the ability of invasion was significantly decreased(p <0.05).Subsequently,the changes of PC-3M transfer function in vivo were measured in human prostate cancer Chicken embryo Chorioallantoic membrane metastasis model.The results showed that when inhibit PC-3M cell membrane ATP5 B,the transfer function was significantly decreased(p <0.05).The above results suggest that cell membrane ATP5 B promotes prostate cancer invasion and metastasis.(3)The mechanism studies of ectopic ATP5 B in metastasis ability of human prostate cancer cellsThe changes of c-Myc,VEGFA protein and m RNA levels were detected by Western blotting and PCR.The results showed that changes in ATP5 B of the cell membrane caused changes in c-Myc and VEGFA.And after changes the ectopic expression of ATP5 B in the PC-3M cell membrane,the ability of induce tubular formation of HUVEC cells also changes.It is speculated that ATP5 B promotes the invasion and metastasis of prostate cancer may via activating c-Myc proto-oncogenes and up-regulating VEGFA.The above experimental results show that ATP5 B is highly expressed in high-grade prostate cancer and infiltrating prostate cancer tissue.Cell membrane ATP5 B overexpression and inhibitory expression models can be constructed by treating PC-3M cells with cholesterol and piceatannol.Ectopic ATP5 B promotes invasion and metastasis of prostate cancer in vitro and in vivo,ATP5 B promotes the invasion and metastasis of prostate cancer may via activating c-Myc proto-oncogenes and up-regulating VEGFA.The above results also further indicate that the ectopic ATP5 B can promote the invasion and metastasis of prostate cancer,which is expected to become the detection of metastatic prostate cancer indicators or therapeutic targets.