Role Of BAG2 During The Apoptosis Induced By Proteasome Inhibitor In Bladder Cancer Cells

Objective: Bladder cancer is the most common malignant tumor in the urogenital system,and the incidence of bladder cancer in our China has been increasing year by year.The BAG gene family is a family of anti-apoptotic genes found in recent years,It has proved that the proteins of this family are involved in various processes such as tumor occurrence,cell apoptosis,nerve differentiation and cell cycle.Ubiquitin-proteasome system is one of the important protein degradation pathways in biological organisms,it can selectively degrade certain proteins within cells,so that it can regulate cell apoptosis,cell cycle,cell proliferation and other physiological processes.As a new anti-tumor research direction,this system has obvious clinical significance.This research is aimed to investigate the expression of BAG2 gene,the effect of BAG2 gene and proteasome inhibitor MG132 on cell proliferation and apoptosis and the role of BAG2 during the apoptosis induced by proteasome inhibitor MG132 in bladder cancer cells.The aims of this research are to find new tumor biomarker and new therapeutic targets for bladder cancer and provide theoretical basis for the clinical application of new anti-tumor drug-proteasome inhibitor MG132.Methods:1.The expression level of the BAG2 gene in two bladder cancer cell lines(5637,T24)and normal human bladder epithelial cells(SV-HUC-1)were detected by q RT-PCR and Western blot;Flow cytometry,Western blot and q RT-PCR were used to detect the efficiency of si RNA-BAG2 transfection;CCK8 assay was used to test the survival rate of bladder cancer cells,Cellular apoptosis was assayed by flow cytometry;The relationship between BAG2 gene expression and prognosis of bladder cancer patients was analyzed by bioinformatics method.2.5637 bladder cancer cells were treated with MG132 at various concentrations(0,1,2,3,4,6,8 and 10μmol/L)and various time(0,1,2,4,8,12,24 and 48h),the survival rate of 5637 bladder cancer cells was detected by CCK8 assay.The optimal concentration and time of drug were selected out;Cellular apoptosis was assayed by flow cytometry,Western blot was used to analyze the expression of caspase-3 protein.3.Western blot and q RT-PCR were used to detect the effect of MG132 on the expression of BAG2 gene in 5637 bladder cancer cells;5637 bladder cancer cells were divided into four groups(control group,si RNA-BAG2 group,siRNA-BAG2 plus MG132 group and MG132 group),the survival rate of 5637 bladder cancer cells was detected by CCK8 assay,Cellular apoptosis was assayed by flow cytometry.Results:1.The results of q RT-PCR and Western blot showed that the expression levels of BAG2 genes in bladder cancer cells(5637 and T24)were significantly higher than those in normal bladder epithelial cells(P < 0.05),and the results of q RT-PCR and Western blot were consistent;Comparing with the control group,silencing BAG2 gene expression could significantly decreased the cell proliferation activityand increased the cells total apoptosis rate in 5637 and T24 in bladder cancer cells(P < 0.05).2.Proteasome inhibitor MG132 had significant inhibitory effect on the proliferation activity of 5637 bladder cancer cells(P < 0.05),the effect was in a time and dose dependent manner.The results of flow cytometry showed that MG132 could improve the total apoptosis rate of 5637 bladder cancer cells(P < 0.05).The results of Western blot indicated that MG132 could significantly decrease the expression of Procaspase-3 protein and increase the expression of Cleaved caspase-3 protein(P < 0.05).3.Proteasome inhibitor MG132 could significantly improve the m RNA and protein expression of BAG2 gene(P<0.05);The results of CCK8 assay showed that the cells proliferation activity of the si RNA+MG132 group was decreased in different degrees comparing with control group,si RNA-BAG2 group,and MG132 group in 5637 bladder cancer cells,and flow cytometry showed that the cells total apoptosis rate of the si RNA+MG132 group was increased(P < 0.05).Conclusion:1.The BAG2 gene was highly expressed in both 5637 and T24 bladder cancer cells;Silencing BAG2 gene expression could significantly decreased the cell proliferation activity and increased the cells total apoptosis rate in 5637 and T24 bladder cancer cells;The 5-year survival rates of bladder cancer patients with high expression of BAG2 gene were significantly lower than those with low expression;These results suggest that BAG2 gene have the function of anti-apoptosis in bladder cancer cells,the expression level of it can be used as a prognostic factor in patients of bladder cancer,and it may be a new molecular target for the treatment of bladder cancer.2.Proteasome inhibitor MG132 could significantly inhibit the proliferation activity of bladder cancer cells and induce apoptosis in 5637 bladder cancer cells.These results suggest that MG132 is expected to provide a new drug treatment for bladder cancer.3.Proteasome inhibitor MG132 could improve the expression of BAG2 gene in 5637 bladder cancer cells;Silencing the expression of BAG2 gene could improve the killing activity of bladder cancer cells by proteasome inhibitor MG132;These results suggest that BAG2 gene may be resistant to the apoptosis induced by proteasome inhibitor MG132 in bladder cancer cells in some ways,si RNA-BAG2 can be used in combination with proteasome inhibitor MG132 for the treatment of bladder cancer in the future.