Silencing AKAP13 Enhances The Chemosensitivity Of Bladder Cancer Cells To Cisplatin

Objective:A wide array of studies found that the recognization and identification of biomarkers that can predict the response of bladder cancer to cisplatin is of great significance for the selection of personalized treatment.Previous studies have shown that AKAP13 and its splicing isoforms play an important role in the mechanism of drug resistance of some tumors,but up to now,there is no literature report on whether there is a correlation between AKAP13 and drug resistance of bladder cancer.The purpose of this study is to explore the effect of AKAP13 on the chemosensitivity of bladder cancer to cisplatin and whether it can be used as a biomarker to predict the response of bladder cancer to cisplatin.Methods:The expression of mRNA levels of AKAP13 in bladder cancer cells and normal bladder epithelial cell was detected by qRT-PCR,and then the protein levels of AKAP13 in bladder cancer and adjacent tissues were measured by immunohistochemistry.Based on the expression levels of AKAP13 in bladder cancer cell lines,two cell lines were chosen for drug resistance assay after siRNA transfection.Cell lines transfected with siRNA were then used to conduct CCK-8 assay including the proliferation ability of tumor cells,the IC50 value of tumor cells to cisplatin and drug resistance of tumor cells to cisplatin at IC50 concentration.In order to further verify the CCK-8 results,the cell cycle kit was used to detect the effect of AKAP13 silencing on the cell cycle distribution of bladder cancer cells incubated with or without cisplatin.Similarly,the effect of AKAP13 silencing on the apoptosis of bladder cancer cells with or without cisplatin was detected by cell apoptosis kit.Finally,the relevant variations of AKAP13 in the process of chemotherapy resistance of bladder cancer were verified by analyzing the Chip datasets of GEO.Results:1.The expression level of AKAP13 in bladder cancer cells was lower than that in normal bladder epithelial cell,which was consistent with the detection levels of AKAP13 protein in bladder cancer tissues compared with that in paracancer tissues.2.After verifying interference efficiency of siRNA,CCK-8 assay showed that IC50 values of 5637 and J82 cells to cisplatin were 1.14±0.04?g/ml and 0.97± 0.075?g/ml respectively in siCtrl group,while in siAKAP13 group,IC50 values were 0.51 ±0.077 ?g/ml and 0.55±0.028 ?g/ml respectively,the difference is statistically significant.Although AKAP13 silencing has no effect on cell proliferation without cisplatin,it can significantly reduce the proliferation of tumor cells under cisplatin.3.Cell cycle assay results indicated AKAP13 silencing had no effect on cell cycle distribution,while under cisplatin incubation,significant arrest of S-phase and G2/M-phase was observed in 5637 and J82 cell lines respectively.4.Cell apoptosis assay demonstrated that AKAP13 interference significantly increased the apoptotic rate of 5637,but had no effect on J82.When tumor cells incubated with cisplatin after siRNA tranfection,the apoptotic rates of 5637 and J82 were 32.75±2.28%?11.36 ± 1.46%respectively in siAKAP 13 group,but in siCtrl group,they were 25.47±3.12%?6.50±1.05%respectively.Significant difference was observed between two groups5.The datasets of GEO showed that the expression of AKAP 13 in bladder cancer tissues increased after neoadjuvant chemotherapy,and no matter before or after neoadjuvant chemotherapy,the expression levels of AKAP 13 were not related to the prognosis of patients.Conclusions:While the expression levels of AKAP 13 in tumor were lower than that in normal cells and tissues,moreover,AKAP 13 silencing had no effect on cell proliferation and cell cycle without cipatin,it is crucial for bladder cancer to tolerate the cytotoxicity of cisplatin.Through CCK-8 and apoptosis assay,we found that cisplatin inhibited the proliferation of bladder cancer cells and increased the apoptosis rate,and cell cycle assay showed that S-phase arrest of 5637 and G2/M-phase arrest of J82 were significantly higher than that of the control group.Although the effect of AKAP13 interference on the apoptosis rate of bladder cancer cells is still controversial,there is no doubt about its effect on cisplatin induced apoptosis.Finally,The results of microarray datasets showed that the expression of AKAP13 increased in the course of chemotherapy resistance of bladder cancer.In conclusion,the silencing of AKAP13 significantly enhanced the chemosensitivity of bladder cancer cells to cisplatin,and whether AKAP13 can be used as a biomarker to predict response of bladder cancer to cisplatin still needs further experimental verification.