The Effects On The Malignant Biological Behaviors Of Testicular Cancer Cells Of BOK And Its Possible Mechanism

Background:Testicular cancer(TC)is the most common malignancy tumor in young men aged 15-34.Its 5-year survival rate is over 95%.It is currently considered to be a"curable"malignant tumor disease.Testicular tumors are divided into germ cell tumors and non-germ cell tumors,of which germ cell tumors(GCT)are mostly,more than 95%.The cause of testicular tumors is still unclear,and the risk factors include:cryptorchidism or testicular failure(testicular hypoplasia syndrome),Klinefelter syndrome,family genetic factors,contralateral testicular tumors and infertility.Recent studies have found that B-cell lymphoma 2 ovarian killer(BOK)acts as a pro-apoptotic protein and acts as a tumor suppressor in malignant tumors.Up to now,there are few reports on the study of BOK protein in testicular cancer,and its specific biological effects are still unclear.Objective:In this study,immunohistochemistry was used to detect the expression of BOK protein in 10 cases of testicular cancer and adjacent tissues.The role and potential mechanism of BOK protein in testicular cancer cell lines were further studied by BOK overexpression and knockdown.Methods:1.This study collected surgical resection of the Department of Urology,Shanghai Changzheng Hospital from 2014 to 2016.The pathological experts confirmed 10cases of testicular cancer and corresponding adjacent tissues.The specimens were fixed by embedding and confirmed by pathologist after HE staining.2.Immunohistochemical staining method was used to detect the protein expression of BOK antibody in testicular cancer and corresponding adjacent tissues.3.Cell culture and transfection:Two TC cell lines(TCam-2 and I-10)and normal testis Hs1.Tes were purchased from the American Type Culture Collection,containing 10%fetal bovine serum,100 U/ml penicillin sodium.100 mg/ml streptomycin was cultured in DMEM and F-12K medium.When the cells grow to 50-70%confluence,the cells are transfected with a DNA transfection reagent.4.Western blot analysis:Total protein was extracted using sodium dodecyl sulfate polyacrylamide gel electrophoresis and then transferred to a nitrocellulose membrane.The blotting membrane was incubated with a 1:500 dilution of BOK antibody or a caspase-3/cleaved caspase-3 antibody.After washing,the membrane was incubated with a horseradish peroxidase-conjugated goat anti-rabbit 1:2000 dilution.The assay was performed using an EasySee Western blotting kit(TransGen Biotech,Beijing,China).5.CCK8 analysis:Cells were digested 24 hours after transfection,counted,seeded in 96-well plates,cultured for 0,12,24,36 and 48 hours,and finally evaluated using a cell counting kit.6.Transwell invasion assay:A 8-?m pore size permeable isolation chamber with an artificial basement membrane was used for in vitro invasion experiments.The cells are digested and counted.100?L of a total of 1×10 ~5 cells were plated in the upper chamber,and the culture medium supplemented with 500 mL of 10%FBS was used as chemical attractant to cover the bottom chamber.4%paraformaldehyde was used to fix the non-migrating cells on the bottom and stained with 0.1%crystal violet for 15 minutes.Cells were counted under a microscope at x400 magnification.7.TUNEL assay:TCam-2and I-10 cells were cultured in 12-well plates for 24 hours,washed with cold PBS and fixed with 4%paraformaldehyde.The assay was performed using the one-step TUNEL Apoptosis Assay Kit.Apoptotic cells in the nucleus show green fluorescence.8.Statistical analysis:Statistical analysis was performed using SPSS 19.0 statistical software.All data are expressed as mean±standard error of the mean(SEM).Assuming the two-sided independent variance,the mean between the groups was statistically analyzed using a two-tailed t test and analysis of variance.P<0.05 was considered statistically significant.Results:1.Compared with adjacent tissues,BOK was mainly expressed in normal cells and significantly down-regulated in testicular cancer tissues.The expression of BOK mRNA and protein was verified by RT-PCR and Western blot,respectively.The results were consistent with the results of immunohistochemical staining(p<0.001).2.BOK overexpression inhibits TC cell proliferation and invasion,and BOK knockdown promotes TC cell proliferation and invasion.We first examined the expression levels of BoK in two TC cell lines and normal testicular cells by Western blot.Western blot confirmed the validity of the BOK overexpression plasmid.CCK8 assays and in vitro invasion assays were performed to detect cell proliferation and invasion.The results showed that BOK overexpression inhibited proliferation and invasion of TCam-2 and I-10 cells.siRNA was transfected in TCam-2 and I-10 cells and subjected to Western blotting to confirm the effect of siRNA.The expression of BOK was significantly reduced after siRNA transfection.Cell proliferation and invasion are enhanced after BOK knockdown.These data support the role of BOK in tumor suppression in TC.3.BOK overexpression promotes apoptosis in testicular cancer cells.We performed a Western blot to detect caspase-3/cleaved caspase-3,and TUNEL detected DNA cleavage in TCam-2 and I-10cells overexpressing BOK.The results showed that cleaved caspase-3 occurred when TCam-2 and I-10 cells overexpressed BOK.BOK overexpression significantly increased TUNEL positive cells.These data indicate that BOK inhibits TC cell proliferation by inducing apoptosis.Conclusion:1.Compared with adjacent tissues,BOK is frequently down-regulated in testicular cancer tissues.2.BOK overexpression inhibits proliferation and invasion of testicular cancer cells.In contrast,BOK knockdown promotes proliferation and invasion of testicular cancer cells.3.BOK overexpression promotes apoptosis in testicular cancer cells.