Study On The Expression And Relationship Of ATP5B,Cav-1 And CK18 In Breast Cancer Tissues And MCF-7 Cells

Breast cancer is the most common cancer disease among women worldwide,and it is also one of the major causes of female cancer death.Therefore,it is particularly important to clarify the internal mechanism of breast cancer occurrence and development for its diagnosis and treatment.ATP5 B is generally located in the intracellular mitochondria,while in breast cancer cells,prostate cancer cells and other tumor cells,ATP5 B is more ectopic expressed in the cell membrane.Cav-1 is the main component of the small fovea and has a variety of biological functions.The previous experiment of our research found that Cav-1 is involved in the migration and invasion of breast cancer MDA-MB-231 cells regulated by ATP5 B.CK18(Cytokeratin 18),as a type of intermediate filament,which not only participates in the assembly of cytoskeleton,but also participates in the intracellular signal transduction pathways and other biological processes.This study aims to clarify the relationship between ATP5 B,Cav-1 and CK18 in breast cancer MCF-7 cells and breast cancer tissues: through the promotion and inhibition model of ATP5 B,Cav-1 sh RNA,immunohistochemistry and other methods to explore whether the expression of CK18 is regulated by ATP5B/Cav-1 and thus participates in the migration and invasion of tumor cells regulated by ATP5B/Cav-1.Methods:1.The effect of cholesterol stimulation on ATP5 B membrane ectopia was detected in MCF-7 cells,the ATP5 B membrane ectopia was inhibited in MCF-7 cells when incubated with piceatannol,and the relationship between ATP5 B and CK18 was detected by western blot.2.Knock down Cav-1 by sh RNA,the knock down effect of Cav-1 in MCF-7 cells was identified by western blot and PCR.Western blot and CO-IP were used to detect the regulatory effect of ATP5 B / Cav-1 on CK18.3.The expressions of ATP5 B,Cav-1 and CK18 in breast cancer tissues were detected by immunohistochemistry,and the relationship between ATP5 B,Cav-1 and CK18 was analyzed by statistical methods.Results:1.The final concentration of cholesterol action is 20ug/ml.The effect of different time of cholesterol action on the ectopic expression of plasma membrane ATP5 B was detected by western blot.It was found that ATP5 B membrane ectopic expression was most significant when cholesterol stimulated for 12 h,so 12 h was determined as the best action time for follow-up experiments.The concentration of piceatannol is 10 u M.Western blot showed that cholesterol promoted the ectopic expression of ATP5 B,and the expression of CK18 was up-regulated,while piceatannol inhibited the ectopic expression of ATP5 B and down-regulated the CK18 expression.2.Using sh RNA technology to inhibit the expression of Cav-1 in MCF-7 cells,PCR and western blot results showed that,compared with the scramble group,the expression of Cav-1 in the sh RNA group was down-regulated at both the m RNA level and the protein level.At the same time,compared with the scramble group,the expression of CK18 protein in the sh RNA group was down-regulated,while after cholesterol stimulation,the protein expression levels of Cav-1 and CK18 in the sh RNA group did not change significantly compared with the scramble group.3.The expression of ATP5 B,Cav-1 and CK18 in breast cancer tissues was detected by immunohistochemistry: ATP5 B was positively expressed on the cell membrane in14 cases of breast cancer tissues,and the positive rate of low expression was 50.00%,and the positive rate of high expression was 50.00 %;in 17 cases of breast cancer tissues,the positive expression rate of Cav-1 was 88.24%;in 29 cases of breast cancer tissues,CK18 was positively expressed,of which the positive rate of low expression was 37.93%and the positive rate of high expression was 62.07%.The relationship between ATP5 B,Cav-1 and CK18 and clinicopathological characteristics was analyzed by statistical methods.It was found that the expression of ATP5 B and Cav-1 was not significantly correlated with the patient’s age,tumor size,tumor metastasis,and TNM staging;the patient’s tumor size is related to the increased expression of CK18(P<0.05),while the patient’s age,tumor metastasis and TNM stage were not significantly correlated with CK18 expression.Through statistical analysis of the clinical correlation between Cav-1 and CK18,it is found that the two are correlated.Conclusions:Through the above experiments,the following conclusions are drawn that the expression of ATP5 B,Cav-1 and CK18 are correlated in MCF-7 cells,the expression of CK18 can be affected by ectopia ATP5 B and Cav-1 regulates the expression of CK18.CK18 expression increased when ATP5 B membrane ectopic overexpression was stimulated by cholesterol,and CK18 expression decreased after inhibition of expression;CK18 expression decreased when Cav-1 sh RNA was used to knock down Cav-1,and this performance cannot be reversed by cholesterol.ATP5 B and Cav-1 form aggregates on the surface of MCF-7 cells and perform biological functions.In vivo experiments verify that ATP5 B is ectopically expressed on the surface of breast cancer cells,and the expressions of ATP5 B,Cav-1 and CK18 are correlated.