Expression Of Livin And The Correlation Study Of Livin In Bladder Cancer

IntroduceInhibitor of Apoptosis Protein(IAP)can suppress apoptosis induced by a variety of stimulators in cell and is a important anti-apoptotic factor which keeps close relation to appearance and development of tumor.Cell apoptosis is the key mechanism which modulates the balance of organism and is rigidly controlled.The execution molecules of apoptosis belong to the caspase/CED3 family,which is produced as zymogens and activated by proteolytic processing.IAP blocks apoptosis by binding and inhibiting specific intracellular proteases,especially caspases3,7 and 9.IAP can also suppress apoptosis through caspase-independent mechanisms too.Bladder cancer is the most common urological tumor in China,which is the most urological tumor on the occurrence and death rate.The biology character of bladder cancer is outstanding recurrence.More than half of patients will recur in two years who underwent transurethral resection(TUR-B)or partial cystectomy.About 16%—25% of patients tend to develop more malignant tumor.Accurate judgement on malignance degree is very important in selecting treatment measure of bladder cancer.Livin is a novel member of IAP family and expresses in proliferating cells during fetal development predominantly.However,it is very low or absent in normal differentiated tissues.Livin has also been shown to be abundantly expressed in a variety of tumor tissues and cell lines.On the other hand,Livin was not detectable in most normal adult tissues.There are two Livin isoforms named as Livinαandβ.Until now,only Livinαis reported to be expressed in bladder cancer.However,the actual role of Livin in the development and progression of bladder cancer remains poorly understood,and the relationship between Livin expression and chemotherapy resistance in bladder cancer is still unclear.Considering the above,we carried through a series of experiments as follows to try to explore the anti-apoptotic functions of Livin on bladder cancer development and its role in chemotherapy resistance.Objective1.To evaluate the expression of Livin in bladder transitional cell carcinoma(BTCC) and to investigate its clinical and prognostic implications.2.To explore effect of gene Livin transfection on the proliferation and apoptosis in BTCC cells.3.To explore the inhibitory effect of RNAi on livin expression and to investigate the mechanism of bladder cancer cell's chemosensitivity resumed by siRNA.MethodsSubjectChief reagent:Homo bladder cancer cell line T24 comes from Chinese Type Culture Collection Center;Lipofectamine^TM2000 lipid tranfection kit,Trizol and pcDNA3.1(+)come from Invitrogen Company;Wizard purefection plasmid DNA purification system come from Promega Company;RPMI1640,G-418 come from GIBCO Company;polyclonal rabbit antibody against Livin come from Imgenex Company;goat anti-rabbit antibody tagged by FITC come from Santa Cruz Company;MMC come from Roche Company; MTT and AO come from Amresco Company;DMSO,PI and goat anti-rabbit antibody come from Sigma Company;Annexin V-FITC & PI Apoptosis Detection Kit come from Beijing Baosai Company;PCR primer,100bp DNA marker,RT-PCR kit, pMD19-T,JM109,Genefinder,Hindâ…¢and BamHI come from TaKaRa Company; PVDF member come from Bio-rad Company;siRNA is designed and chemosynthesized by Shanghai Genefarmer Company;fetal bovine serum come from TBD Company;Others come from Huamei Company.Chief equipment:Olympus light microscope,convert microscope,Incubation box,autoclave sterilizer,refrigerator,ultraviolet/visible light absorption meter(Du-640,Beckman), hypothermy supercatrifuge(GS-15 Rebeckman),absorption meter,homogenate machine,ultrasonic disintegrator,electrophoresis apparatus,auto running gel imaging analysector,cell culture board and so on. Experimental methodTissue Samples and Cell LinesBladder cancer tissues were obtained from 48 patients with superficial bladder transitional cell carcinoma,who underwent transurethral resection(TUR-B)or partial cystectomy.Tumor extension limited to the mucosa(pTa),or the lamina propria(pT1) of the bladder wall was defined as superficial according to the post-surgical pathology. Among these patients,31 were primarily diagnosed and 17 were relapsed cases.The pathological tumor stage was classified according to World Health Organization(WHO) criterion,which showed G₁ in 23 cases,G₂ in 17 cases and G₃ in 8 cases.Of the 48 cases,17 developed relapse.As a control,normal appearing bladder tissues were obtained in area apart from tumor(≥3cm)in 15 patients.No evidence of histological change in normal control bladder samples was histopathologically confirmed.Tissues specimens were immediately frozen in liquid nitrogen and stored at-80℃until use.Bladder cancer cell line T24 was purchased from the Shanghai institute of biochemistry and cell biology,Chinese Academy of Sciences.The cell line were grown in tissue culture flasks in RPMI1640 supplemented with 10%FBS at 37℃and 5% CO₂.ImmunofluorescenceBladder cancer tissues were embed by OCT,then slice up to 6μm,adhered to the cover glass;Then,it was fixed 20-30 min with paraformaldehyde,treated 10-15min with 0.2%Triton-X100,rabbit-anti human Livin antibody(diluted 1:400)was added as first antibody,incubated at 4℃overnight.Then,the sample was added with FITC(diluted 1:200)labeled second antibody,incubated at room temperature for 2 hours.The glass was washed again and sealed with 50%glycerin,fluorescence microscope was used for observation and photographing.Western BlotProteins were separated by denaturing sodium dodecyl sulfate polyacrylamide gel electrophoresis(SDS-PAGE)and Western blot was conducted as described.The protein concentration was mensurated with lowry analysis.The soluble supernatants were immunoprecipitated using a polyclonal rabbit antibody against Livin or againstβ-actin(as internal control).Values are presented as the ratio of Livinαoptical density to β-actin optical density.RT-PCRTotal RNA was extracted from cultured cells and tumor tissues using Trizol reagent.RT-PCR was operated according to the manufacture's protocol.Based on mRNA sequences of Livin,primer for RT-PCR was described as KIM.GAPDH was used as an internal control.The following program was used for RT reaction:20min at 50℃,5min at 99℃,5min at 5℃;for PCR reaction:2min at 94℃,denature for 30s at 94℃,annealing for 30s at 55℃,extending for 2min at 72℃,repeating 35 cycles, extending for 4min at 72℃ridyzation solution.Construction of pcDNA3.1(+)-Livin recombinant plasmid vectorThe construction of recombinant vectors was performed according to reference. First,full length cDNA of Livin was obtained by RT-PCR.Second,after TA cloning, DNA sequencing was performed for the clones validated by enzyme digestion and PCR. Third,full length cDNA of Livin in TA clone was acquired by digestion with Hindâ…¢and BamHI and inserted into Hindâ…¢and BamHI sites in pcDNA3.1(+).Thus,positive recombinant clones pcDNA3.1(+)-Livin were obtained.Transfection of Livinα-pcDNA3.1(+)into T24 cell lineLivinα-pcDNA3.1(+)was confirmed by sequencing analysis.To determine the appropriate G418 concentration for cell screening after transfection,T24 cells was passaged at a concentration of 1×10⁵/mL,with presence of G418 at concentration of 200,400,600,800,and 1000μg/ml,respectively.The concentration that was sufficient to prevent total cell growth in 7-8days was determined by cell screening.In this study, 400μg/mL of G418 was used for screening.Gene transfection was performed by cationic lipid mediated transfection according to Lipofectamin^TM2000's protocol.The medium was replaced with fresh one every 2-3days until G418 resistant clones emerged(about 3-4weeks later).Single resistant clone was picked and cultured using limiting dilution assay.The untransfected blank control and the control transfected by pcDNA 3.1(+)were used as control.RNA interference:siRNA transfection was performed by cationic lipid mediated transfection according to Lipofectamin^TM2000's protocol.The final concentration of siRNA was 50nmol/l,100nmol/l and 200nmol/l respectively.Negative controls and positive controls were caught through at one time.6 hours after siRNA transfection,the efficiency of transfection was detected by fluorescence microscope.Colony-forming assayAttached cells in exponential proliferation stage were subjected to digestion,and resuspended in RPMI1640 containing 10%FBS for single cell suspension,and seeded at 100 cells/10mL culture medium in each 10mL dish.The cells were distributed uniformly in the dishes for 2-3weeks static culture.The clones containing more than 50 cells were counted under microscope.The rate of colony forming(%)=(average colony number/planted single cell number)×100%.MTT assayBladder cancer cells before and after transfection were detached using 0.25% trypsin.The cell suspension was distributed into 96 well plates at a concentration of 1.0×10⁵ cells/well,followed by addition of 0.2mL/well of RPMI1640 containing 10% FBS.The culture medium of each well was replaced with 0.2mL of RPMI1640 without FBS after 24h,then added MMC and made its final concentration at 48μmol/L.The sample was divided into 9 groups with 4 wells in each.After 24h,each well was added 20μL of MTT solution(5mg/mL in culture medium),then incubated for 4h at 37℃. The supernate was removed,0.15ml of dimethylsulfoxide(DMSO)was added into each well.After being shaken for 10min,the light absorption of each well was measured at 490nm on ELISA machine referring to absorbency(A49o).Finally,means of results were used to calculate the rate of the cell proliferation.The proliferative rate =ExperimentA₄₉₀/ ControlA₄₉₀×100%.Acridine Orange stainingThe apoptotic morphology of bladder cancer cells before and after transfection was stained by acridine orange and observed under a fluorescent microscope BX51 as described.Flow cytometry analysisApoptotic rate was assessed by FCM after stained with PI and annexin-V/FITC. Bladder cancer cells were first stained with 1μg/mL PI for 30min at 4℃,followed by annexin-V/FITC staining as described previously.Apoptotic rate was determined by FCM and 10000 cells were scored for each sample.Caspase-3 activity assayCaspase-3 activity was measured using Caspase-3/CPP32 Colorimetfic Assay Kit. Bladder cancer cells were plated into a 96 well plate(5×10⁶ cells/well).Resuspend cells in 50μL of chilled cell lysis buffer and incubate cells on ice for 10min.Centrifuge for 1 min at 10000g.Transfer supernatant to a fresh tube and put on ice.Dilute 200μg protein to 50μL cell lysis buffer were used.Add 50μL of 2×reaction buffer to each sample.Add 5μL of the 4mM DEVD-pNA substrate(200μM final concentration)and incubate at 37℃for 2h.Finally,read samples at 405nm in a microtier plate reader.Statistical analysisX² analysis was used to determine the significance of the Livin expression in bladder cancer tissues.SPSS 12.0 was used for Statistic analyses.All determinations were made in at least four independent experiments,and the results were expressed as means±SD.A P-value of less than 0.05 was considered statistically significant in all analyses.Experimental resultLivin expression and the reeurrenee of bladder cancerLivin expression was not detected in 15 non tumor bladder tissues,whereas bladder tumor tissues showed a certain positive expression of Livinα,but not Livinβ. Total 19 of 48 bladder cancer samples were found to expressed Livinα,and the positive rate was 39.13%(9/23)in G₁,41.18%(7/17)in G₂,and 37.50%(3/8)in G₃ tumors.The differences of Livinαexpression among G₁,G₂ and G₃ tumors did not show statistical significance(P＞0.05).The expression rate of Livinαin relapse tumors was 58.82%(10/17),while it was 29.03%(9/31)in primary tumors.There was a significant difference between these two group of patients(P＜0.05).After 2-year follow up,the relapse rate in the patients who expressed Livinαwas 68.42%(13/19),which was significantly higher than that in patient without Livinαexpression(37.93%,11/29, P＜0.05).Livinαtransfection in bladder cancer cellsProtein and mRNA expression of either Livinαorβwere not detected in T24 bladder cancer cell.It showed that expression of Livinαwas positive in these transfected cell lines,while it was negative in the cells transfected by mock pcDNA3.1(+)or untransfected controls.Colony forming assayThe colony forming rate of T24 cell transfected by Livinα-pcDNA3.1(+)were 72.5±6.9,which were significantly higher than those of mock transfected cells (43.3±4.6),or those of untransfected cells(45.8±5.6)(P＜0.01).Efficiency of transfection observationIt was observed by fluorescence microscope after siRNA interference for 6h. When concentration of siRNA were 50nmol/l,100nmol/l and 200nmol/l,efficiency of transfection were 67.5%,86.7%and 73.6%,respectively.So 100nmol/l siRNA was selected to carry though the next experiment.MTT assayThe proliferate rates of T24 cell transfected by Livinα-pcDNA3.1(+)were 67.3±3.5%.By contrast,mock transfected T24 cell was shown with proliferate rate of 31.6±5.1%%,and 28.7±5.2%in those of untrasfected cells.The proliferation rate of bladder cancer cells transfected by Livinα-pcDNA3.1(+)was dramatically increased, when compared with those of the controls or those transfected by siRNA(P＜0.01).Acridine Orange stainingThe cells with stable Livinαexpression were found to be resistant to MMC treatment,whereas the cells without expression of Livinαrapidly underwent apoptosis, as determined by nuclear morphological evaluation using Acridine orange staining.A direct correlation was observed between the rate of apoptosis and expression of Livinα(data not shown).Flow cytometry analysisAfter MMC treatment for 24h,a significantly low rate of apoptosis was found in the cells transfected with Livinα-pcDNA3.1(+),when compared with control cells transfected with pcDNA3.1(+)or those untransfected or those transfected by siRNA.Caspase-3 activity assayAfter 24h treatment with MMC,caspase-3 activity in T24 cell transfected by Livinα-pcDNA3.1(+)were found to be considerably decreased(0.1695±0.0105),as compared to their controls transfected by siRNA(0.4678±0.0474)and untransfected controls(0.4955±0.0283)(P＜0.01).ConclusionsThe expression of Livin was associated with the relapse of BTCC,Livin may serve as a marker for diagnoses of BTCC and play an essential role in the prognostic of BTCC relapses.Livin expression in T24 cell line may promote the proliferation of the tumor cells.Livin can resist the cells apoptosis induced by chemotherapy,and give them more anti-apoptosis ability.Livin may serve as a promising marker to identify the relapse risk in bladder cancer,and targeting Livin could offer a therapeutic benefit in apoptosis-inducing treatment.