The Expression Of TRIM24 In Bladder Cancer Tissue And Its Effect On Biological Beheaviors Of Bladder Cancer Cell

Objective: Bladder cancer is the second most common genitourinary tumor worldwide.About 90% of bladder cancers are urothelial cell carcinomas which arise from bladder epithelial cells.Invasive bladder tumors are more aggressive,presenting with penetration of the basement membrane or invasion into muscle.Clinically,radical cystectomy remains the most common treatment for patients with invasive cancer.Despite advances in surgical technique,the 5-year cancer-speciic survival remains about 50%,which has not been significantly improved during the last decades.Therefore,exploring potential molecular targets which determine biological nature and behavior of bladder would be of great importance to optimize individual therapy.TRIM24,also named transcription intermediary factor 1-alpha(TIF1α),was identified as a co-regulator of retinoid signaling.Recently,TRIM24 has been found to be overexpressed in several human cancers.TRIM24 was found to be involved in acute promyelocytic leukaemia,papillary thyroid carcinoma and myeloproliferative syndrome.TRIM24 could bind chromatin to activate genes transcription which were associated with cellular proliferation and tumor development.TRIM24 could promote progression of prostate cancer and negatively correlated with survival of breast cancer patients.TRIM24 overexpression was also observed in non-small cell lung cancer and its overexpression promoted chemoresistance of glioma.The above reports suggest TRIM24 as a potential regulator during tumor progression.To data,expression pattern of TRIM24 in human bladder cancers has not been explored and its biological roles remain obscure.In the present study,we examined the protein expression of TRIM24 in 95 bladder cancer tissue specimens using immunohistochemistry and analyzed its correlation with clinicopathological factors.We also overexpressed and knocked down TRIM24 and explored its effect on biological behavior of bladder cancer cell lines.Methods: 1.Patients and specimens : The study protocol was approved by the institutional reviewer board of China Medical University.95 cases of primary bladder cancer underwent surgical resection and their corresponding paracancer tissues were collected from the Fourth Affiliated Hospital of China Medical University between Jun 2010 and Nov 2011.The histological diagnosis and tumor grade were evaluated for sections stained with hematoxylin and eosin according to the World Health Organization(WHO)classification guidelines.Tumors were classified into Ta,T1,T2,T3,T4 according to WHO guidelines(2007).2.Immunohistochemistry: Tumor pecimens were fixed with 10 % neutral formalin and embedded in paraffin,and 5-μm-thick sections were made.Immunostaining was performed using the avidin–biotin– peroxidase complex method(Ultrasensitive?,Mai Xin,Fuzhou,China).The sections were deparaffinized in xylene,graded alcohol,and then boiled in 0.01 M citrate buffer(p H 6.0)for 2 min in an autoclave.Hydrogen peroxide was applied to block endogenous peroxide activity.Sections were incubated with normal goat serum.Then,tissue sections were incubated with TRIM24 rabbit polyclonal antibody(1:600 dilution;Proteintech,USA).Rabbit immunoglobulin was used as a negative control.Staining for both antibodies was performed at room temperature for 2 h.Biotinylated goat antirabbit serum Ig G was used as a secondary antibody.After washing,the sections were incubated with streptavidin – biotin conjugated with horseradish peroxidase.After that,secion was developed with 3,3 ′-diaminobenzidine tetrahydrochloride(DAB).Counterstaining with hematoxylin was performed.The sections were dehydrated in ethanol before mounting.Two independent blinded pathologists examined all tumor slides randomly.Five views were examined per slide.Immunostaining of TRIM24 was scored on a semiquantitative scale by evaluating in representative tumor areas and staining intensity.Nuclear immunostaining in tumor cells was considered to be positive.The staining intensity was categorized as follows: 0,negative;1,weak;2,strong.The percentage of stained tumor cells was scored as 0,0 %;1,1-25 %;2,26-50 %;3,51-75 %,and 4,76-100%.The scores of each tumor sample were multiplied to give a final score of 0 to 8,and the tumor samples with a final score <4 were regarded as negative or weak staining,tumor sampleswith a final score of 4-8 were finally determined as TRIM24 overexpression.3.Cell culture and transfection: BIU-87 and 5637 cell lines were obtained from American Type Culture Collection(Manassas,VA,USA).The cells were cultured in 1640(Invitrogen, Carlsbad,CA,USA)containing 10% fetal calf serum(Invitrogen),100 IU/ml penicillin(Sigma,St.Louis,MO,USA),and 100 μg/ml streptomycin(Sigma).Cells were grown on sterilized culture dishes and were passaged every 2 days with 0.25% trypsin(Invitrogen).The plasmid of TRIM24 was purchased from Origene.Plasmid was transfected into cells using Attractene Transfection(Qiagen,Hilden,Germany).Empty vector was used as a negative control.Cells were harvested 48 hours later.On-Target Plus SMARTpool si RNA for TRIM24 and ON-TARGETplus Non-targeting si RNA #2 were purchased from Dharmacon(Lafayette,CO,USA).The cells were transfected with si RNA using the Dharma FECT 1(Thermo Fisher Scientific)according to the manufacturer)Scientific 4.Western blot analysis:Total proteins from cells were extracted in lysis buffer(Pierce,Rockford,IL)and quantified using the Bradford method.Samples of 50 mg of protein were separated by SDS-PAGE.Proteins were transferred to polyvinylidene fluoride membranes(Millipore,Billerica,MA,USA)and incubated overnight at 4°C with antibody against TRIM24(1:1000,Proteintech,USA)Cyclin D1,Cyclin E,p-IκBα,IκBα,p-AKT,AKT(1:1000;Cell signaling,Boston,MA,USA)and GAPDH(1:2000;Santa Cruz,USA).After incubation with peroxidase-coupled anti-rabbit/mouse Ig G(Santa Cruz)at 37°C for 2 hours,bounded proteins were visualized using ECL(Pierce,USA)and detected using DNR Bio Imaging System(Jerusalem,Israel).5.CCK8 assays:Cells were plated in 96-well plates in medium containing 10% FBS at approximately 1000 cells per well 24 h after transfection.For quantitation of cell viability,cultures were stained in cck8 assays 12、24、36、48、60 hours later.In brief,20 μl of CCK8 solution was added to each well and incubated for 4 hours.Then each solution was measured spectrophotometrically at 490 nm.6.Colony formation assay:Cells were transfected for 48 h and then plated into three 6-cm cell culture dishes(1000 per dish)and incubated for 12 days.Plates were washed with PBS and stained with Giemsa.The number of colonies with more than 50 cells was counted.The colonies were manually counted using a microscope.7.Matrigel invasion assay: Cell invasion assay was performed using a 24-well Transwell chamber with a pore size of 8 μm(Costar,Cambridge,MA).The inserts were coated with 20 μl Matrigel(dilution 1:3,BD Bioscience,San Jose,CA,USA).Forty-eight hours after the transfection,cells were trypsinized and 1×105 cells in 100 μl of serum-free medium were transferred to the upper Matrigel chamber and incubated for 16 hours.Medium supplemented with 15% FBS was added to the lower chamber as the chemoattractant.After incubation,the non-invaded cells on the upper membrane surface were removed with a cotton tip,and the cells that passed through the filter were fixed with 4% paraformaldehyde and stained with hematoxylin.8.Statistical analysis:SPSS version 11.5 for Windows was used for all statistical analyses.A c2 test was used to examine possible correlations between TRIM24 expression and clinicopathologic factors.All p values are based on a two-sided statistical analysis,and we defined p<0.05 as statistical significance.Results: 1.The expression of TRIM24 in bladder cancer and the association with the clinical stage.⑴ TRIM24 protein is overexpressed in bladder cancer: We investigated TRIM24 protein levels in a panel of 95 primary bladder cancer samples as well as their corresponding paracancer tissues by immunohistochemistry.We found that the TRIM24 protein was localized in the nuclear compartment of cancer cells.Strong nuclear staining was found in 39 out of 95(41.1%)bladder cancer tissues.⑵ The expression of TRIM24 in bladder carcinoma was significantly associated with invasion depth and tumor grade: Then we analyzed the relationship between TRIM24 overexpression and clinical parameters.The expression of TRIM24 in bladder carcinoma was significantly associated with,invasion depth(P=0.012)and tumor grade(P =0.028),but it had no association with age,gender and nodal status.2.The regulation and potential mechanism of TRIM24 in bladder cancer.⑴ Expression pattern of TRIM24 in cell line of normal bladder epithelium and bladder cancer cell lines: Using western blot,we found that strong expression in 5637 cell line and negative TRIM24 expression in T24,BIU-87 and SV-HUC-1 cell lines.(2)Testing efficiency of TRIM24 plasmid transfection in BIU-87 cell line: We performed q RT-PCR to test the TRIM24 m RNA expression level in BIU-87 cell line transfected TRIM24 overexpressed compared with empty vector.We found that TRIM24 plasmid transfection significantly increased TRIM24 m RNA expression level compared with empty vector.Similarly,we performed Western blot to test the TRIM24 protein expression level in BIU-87 cell line transfected TRIM24 plasmid compared with empty vector.We found that TRIM24 plasmid transfection significantly increased TRIM24 protein expression level compared with empty vector.⑶ TRIM24 overexpression increased proliferation rate and colony formation ability of BIU-87 cell line: We performed CCK-8 assay and colony formation assay to test the effect of TRIM24 on bladder cancer growth.We found that TRIM24 plasmid transfection significantly increased cell proliferation rate and colony formation ability compared with empty vector.(4)TRIM24 overexpression increased invading ability of BIU-87 cells: Matrigel invasion assay was performed to examine its role on invading ability.TRIM24 overexpression significantly enhanced invading cell line number compared with empty vector.(5)The potential mechanism of TRIM24 facilitated proliferation and invasion of BIU-87 cell line: We performed Western blot to test protein level of AKT、IκBα、Cyclin D1 and Cyclin E,and the phosphorylation level of AKT and IκBα,in BIU-87 cell line transfected TRIM24 plasmid compared with empty vector.We found that TRIM24 transfection led to increase of Cyclin D1 and Cyclin E levels.These results suggested that TRIM24 regulated cell proliferation possibly through regulation of Cyclin proteins.Furthermore,to explored potential mechanism of TRIM24 mediated biological effects,we analyzed several signaling pathways and found that TRIM24 could regulate NF-κB and AKT pathways.Transfection of TRIM24 plasmid upregulated the level of p-IκBα and p-AKT in BIU-87 cells,NF-κB inhibitor BAY 11-7082 can reversed the effect of TRIM24 on p-IκBα and cyclin D1.3.The role of TRIM24 as a potential target to therapy bladder cancer.(1)Testing efficiency of TRIM24 si RNA transfection in 5637 cell line: We performed q RT-PCR to test the TRIM24 m RNA expression level in 5637 cell line transfected TRIM24 si RNA compared with empty vector.We found that TRIM24 si RNA transfection significantly decreased TRIM24 m RNA expression level compared with empty vector.Similarly,We performed Western blot to test the TRIM24 protein expression level in 5637 cell line transfected TRIM24 si RNA compared with empty vector.We found that TRIM24 si RNA transfection significantly decreased TRIM24 protein expression level compared with empty vector.(2)TRIM24 si RNA depressed proliferation rate and colony formation ability of 5637 cell line: We performed CCK-8 assay and colony formation assay to test the effect of TRIM24 on bladder cancer growth.We found that TRIM24 si RNA transfection significantly decreased cell proliferation rate and colony formation ability compared with empty vector.(3)TRIM24 si RNA decreased invading ability of 5637 cells: Matrigel invasion assay was performed to examine its role on invading ability.TRIM24 silencing significantly decreased invading cell line number compared with empty vector.(4)The potential mechanism of TRIM24 si RNA inhibited proliferation and invasion of 5637 cell line: We performed Western blot to test protein level of AKT、IκBα、Cyclin D1 and Cyclin E,and the phosphorylation level of AKT and IκBα,in 5637 cell line of TRIM24 si RNA compared with empty vector.We found that TRIM24 si RNA transfection led to decrease of Cyclin D1 and Cyclin E levels.These results suggested that TRIM24 regulated cell proliferation possibly through regulation of Cyclin proteins.Furthermore,to explore potential mechanism of TRIM24 mediated biological effects,we analyzed several signaling pathways and found that TRIM24 could regulate NF-κB and AKT pathways.Transfection of TRIM24 si RNA downregulated the level of p-IκBα and p-AKT in 5637 cells.Conclusion: 1.TRIM24 is overexpressed in bladder cancer tissues and correlated with its invasive depth and grade.2.TRIM24 can drive proliferation and invasion through regulation of NF-κB and AKT signaling in bladder cancer,3.TRIM24 may be a potential treatment target in urinary bladder carcinoma.