The Expression Of Genes Related To Gallbiadder Carcinoma And The Apoptosis Induced By Antisense Oligonucleotides Targeting Survivin And Bcl-2 On Gallbladder Carcinoma Cell

Background: Gallbladder carcinoma is the most common malignant tumor of bile duct,accounting for 25% of carcinoma of bile duct. It carries a poor prognosis and the improvement in survival rates over the past few decades has been disappointing.To search a new method for diagnosis and treatment is an important task for medical workers.Regulation of apoptosis ,or programmed cell death,is crucial to the preservation of homeostasis and morphogenesis of human tissue. Disturbance of this process by aberrantly extending cell viability or favouring accumulation of transforming mutation is thought to contribute to carcinogenesis.Survivin is a new member of the inhibitor of apoptosis proteins family, Bcl-2 is the earliestly detected protein inhibiting apoptosis ,they have been described to be selectively expressed in the most common human neoplasms and to be associated with clinical tumour progression . Moreover , they appear to be involved in tumour cell resistance to some anticancer agents and ionizing radiation.Caspase family is a group of proteases,which play active roles in the course of apoptosis,among them,Caspase-3 is the most important excuter ofapoptosis.On the basis of these findings, Survivin and Bcl-2 have been proposed as promising target for anticancer interventions.Antisense technique ,one of the gene therapy,is based on the priciple of base pair when antisense oligodeoxynucleotides incubated with tumour cells , sequence specific complementary to the target sequence would block transcription and expression of the relevant gene . Because of its effectiveness , safety , simplicity , antisense technique is a promising approach in the treatment of gallbladder carcinoma.In this study,we studyed the expression of Survivin,Bcl-2 and Caspase-3 ,their mutual correlations and correlations with the gallbladder carcinoma patient's characteristics were analyzed.Then we designed antisense oligodeoxynucleotides targeting Survivin and Bcl-2 ,which were intended to try to inhibit the growth and proliferation of gallbladder cells and induce apoptosis.Our research was composed of three parts.Part one:The expression of genes related to apoptosis in gallbladder carcinomaObjective: There are three gene families closely related to apoptosis , they are IAP family,Bcl-2 family and Caspase family.In order to evaluate the significance of Survivin,Bcl-2 and Caspase-3 which are the most typical ones in the three families,we studyed the expression of the three genes and their correlations with clinicopathological features in gallbladder carcinoma and their mutual correlations.Methods: The expression of Survivin ,Bcl-2 and Caspase-3 protein in tissues from 35 cases of gallbladder carcinoma, 15cases of gallbladder adenoma and 12 cases of chronic cholecystitis were studied by SABC immunohistochemical assay, and their correlations withclinicopathlogical features in gallbladder carcinoma were analyzed.The correlation analyses between the expression of the three genes were made.Results:l.The positive expression rate of Survivin protein was 71.8%(28/39) in gallbladder carcinoma , but there was no expression in benign gallbladder disease.2. The positive expression rate of Bcl-2 protein was 38.5%(15/39) in gallbladder carcinoma, 93.3% (14/15)in gallbladder adenoma and 8.3% (1/12) in chronic cholecystitis.The expression of Bcl-2 protein in gallbladder adenoma was higher than that in carcinoma (p<0.01), and the expression of Bcl-2 protein in gallbladder carcinoma was higher than that in chronic cholecystitis (p<0.01).3. The positive expression rate of Caspase-3 protein was 43.6% (17/39) in gallbladder carcinoma , 100% (15/15) in gallbladder adenoma , and 100% (12/12) in chronic cholecystitis. The expression of Caspase-3 protein in gallbladder carcinoma was significantly lower than that in adenoma (p<0.01) and chronic cholecystitis (p<0.01).4.No correlations were found between the expression of Survivin and age,sex of the patients or the volume of the tumor and the status of metastasis.5. No correlations were found between the expression of Bcl-2and age,sex of the patients and the volume of the tumor ,but there were significant differences between the expression of Bcl-2 and the differentiation and the Nevin stage of the tumor (P<0.01) .6.No correlations were found between the expression of Caspase-3 and age,sex of the patients and the volume of the tumor ,but there were significant differences between the expression of Caspase-3 and the differentiation and the Nevin stage of the tumor (P<0.01) .7. No correlation was found between the expression of Survivin and that of Bcl-2 or that of Caspase-3.8.There was positive correlation between the expression of Bcl-2 and that of Caspase-3.Conclusions:1. There were high expression of Survivin and Bcl-2 in gallbladder carcinoma ,while the expression of Caspase-3 was decreased.The expression of Bcl-2 and Caspase-3 were closely related to the differentiation and the stage of gallbladder carcinoma.2. Survivin,Bcl-2 and Caspase-3 all played an important role in the pathway of carcinogensis of gallbladder carcinoma.Part two:The apoptosis induced seperately and combinedly by antisenseoligonucleotide targeting survivin and bcl —2 on gall bladdercancer cell line GBC-SD in vitroObjective: It has been verified that lose control of Survivin and Bcl-2 activity was correlated with the carcinogensis and development of human malignant tumor.The carcinogenesis rate may be reduced by inhibiting abnormal activity of Survivin and Bcl-2 in cells.We want to study the apoptosis induced by Survivin and Bcl-2 antisense oligonucleotides (AsODN) on gallbladder carcinoma,and further offered new theory reference and idea for the prevention and therapy of gallbladder carcinoma.Men tods: The gallbladder carcinoma cell line GBC-SD was treatedwith Survivin and Bcl-2 antisense oligonucleotide separately and combinedly.The inhibition of gallbladder carcinoma cell proliferation was estimated by growth curve,MTT assay; The expression of Survivin mRNA,Bcl-2 mRNA and Survivin protein ,Bcl-2 protein were determined by reverse transcription polymerase chain reaction (RT-PCR) and immunohistochemistry SABC method.The cell cycle and apoptosis index(AI) were analyzed by flow cytometry(FCM). Results:1. After treated with Survivin and Bcl-2 AsODN separately and combinedly for 24h,the proliferation of GBC-SD cells was inhibited and the inhibition showed a time-dependent manner.With the prolong of time,the inhibition enhanced gradually. The highest growth inhibitory rate were 54.3±2.89%, 47.6±4.19%, 76.5±5.52 % respectively when GBC-SD cells were treated for 48hc2. After transfected with Survivin and Bcl-2 AsODN for 24h,the expression of Survivin and Bcl-2 protein were reduced in GBC-SD cells,and the labeling indices(LI) were decreased from 63.74%±11.56% to 39.27%±7.62% (P<0.01) and from 52.64%±9.87% to 30.15%±8.61% (P <0.01) respectively.3. By semi-quantitive analysis, the level of Survivin mRNA was down-regulated by 50.4 % ±2.3 % compared with that of control group,and Bcl-2 mRNA by 56.3 % ±1.5 % correspondingly.4. It was found that cell cycle was blocked significantly by G1 phase after action of Survivin AsODN and combined AsODNs for 48h,the percentage of cells in G0/G1 phase was increased significantly and that was decreased in S phase. There was the similar tendency after the transfection of Bcl-2 AsODN. The apoptosis index(AI) of the three AsODN groups were 11.38%±3.91 %,28.45%± 6.34%and 9.26%±4.15% respectively and each was markedly higher than control group ( P<0.01 )。5. Survivin and Bcl-2 AsODN were found to induce pronounced morphological changes characteristic of apoptosis and extensive DNA fragment in the GBC-SD cell line 24h after treatment.Conclusions:1. Either Survivin , Bcl-2 AsODN or combining the two AsODNs could inhibit the proliferation of gallbladder carcinoma cell line GBC-SD effectively and showed a time-dependent manner.2. Survivin AsODN could down-regulate the expression of Survivin mRNA and Survivin protein; Bcl-2 AsODN could down-regulate the expression of Bcl-2 mRNA and Bcl-2 protein.3. Survivin AsODN and combining Survivin ,Bcl-2 AsODN could inhibit the proliferation by influencing the distribution of cell cycle of gallbladder carcinoma cell line GBC-SD.They could increase the percentage of the cells in G0/G1 phase and reduce the percentage of the cells in S phase.Bcl-2 AsODN has the similar effect.4. It is more markedly in inducing apoptosis of GBC-SD cell line to combine Survivin and Bcl-2 AsODN.Part three:Therapeutic effect of combining antisense oligonucleotide targeting Survivin and Bcl-2 on human gallbladder carcinoma in vivoObjective: To evaluate the inhibitory effect of combining antisense oligonucleotide targeting Survivin and Bcl-2 on growth of human gallbladder carcinoma in nude mice .Methods: Human gallbladder carcinoma transplanted subcutaneouslyin nude mice was established.A total of 12 mice were divided into 3 groups,namely,control group,Survivin and Bcl-2 sODN group,Survivin and Bcl-2 AsODN group.Different treatment were given respectively for 12 days.The weight and volume of the subcutaneous tumors were measured;Histological changes were observed by HE staining;The expression of Survivin and Bcl-2 protein were examined by immunohistochemistry SABC method.Results: The mean volume and weight of subcutaneous tumors of AsODN group were lower than that of control group and sODN group(P< 0.01).The inhibitory rate of volume and weight of AsODN group were 55.53% and 56.24%, while that of sODN group were only 9.9% and 8.47%. A lot of apoptotic cells were seen in tissue section by HE staining,and the expression of Survivin and Bcl-2 protein were lower than that of control group and sODN group(P< 0.05), no difference was found between control group and sODN group(P>0.05).Conclusions: Combining antisense oligonucleotide targeting Survivin and Bcl-2 could inhibit the growth of subcutaneous tumors in nude mice,the reason might be that they could down-regulate the expression of Survivin and Bcl-2 and induce apoptosis of gallbladder carcinoma cell.