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Objective To explore the effect and mechanism of fragile site WWOX gene on regulating proliferation of gallbladder cancer cells in vitro. Methods The pcDNA3.0 - WWOX recombinant plasmid which was previous successfully built was transfected to GBC-SD cells and empty carrier by liposome medium. Liposome and GBC-SD were served as the negative control and the blank control,respectively. After 48 hours transfection, inverted microscope was used to observe the changes of gallbladder cancer cells' morphology,MTT and BrdU were used to detect the proliferation level of gallbladder cancer cells,and flow cytometry instrument was used to detect the change of the cell proliferation cycle. Results The results of inverted microscope shown: the number of GBC-SD cells in pcDNA3.0-WWOX group decreased significantly,the suspension cells and cell debris increased,while cells in the vector control,NC and Mock groups were in normal proliferation state. MTT test showed the proliferation levels of GBC-SD cells in pcDNA3.0-WWOX group was lower than those in the control group in 24 h,48 h,72 h,96 h and 120 h,and the differences were statistically significant(P 0.05). Conclusion The overexpression of WWOX gene in vitro could effectively inhibit the proliferation activity of gallbladder cancer cells. WWOX might participate in the development of the malignant biological behavior of gallbladder cancer cells. It is expected to become a new potential target for the gene therapy to gallbladder cancer.
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Objective Via targeted inhibition of oncogene Bmi-1 expression by RNAi interfering technology in vitro, to observe its effect on the proliferation and cell cycle of gallbladder cancer cells. Methods Four miRNABmi-1 recombinant plasmids were constructed according to different Bmi-1 sites. RT-PCR and Western blot were used to mRNA and protein expression of Bmi-1 in gallbladder cancer cells were measured by RT-PCR and Western blot. mRNA and protein expression of Bmi-1 in gallbladder cancer cells. The most effective interfering plasmids in the miRNABmi-1 groups were transfected into GBC-SD cells. Cell proliferation and cell cycle were analyzed 48 h after transfection by BrdU and flow cytometry. Results Bmi-1mRNA expression in miRNAbmi1-1,-3 and-4 was significantly lower than the control group (P<0.05);and Bmi-1 protein expression in miRNAbmi1-2,-3 and-4 was significantly lower than the control group (P<0.05). The recombinant plasmid in miRNAbmi1-4, with the strongest inhibitive effect of Bmi-1mRNA and protein expression, was transfected into GBC-SD cells,then the cell proliferation rate (46.63 ± 5.31) was significantly lower in mRNABmi1-4 group than the control groups (P<0.05);G0/G1 phase cells increased (72.20 ± 1.71) and G2/M and S phase cells decreased (18.30 ± 7.21, 9.50 ± 6.01) in miRNABmi1-4 group. Both were significantly different from the control groups (P<0.05). Conclusions Targeting and silencing Bmi-1 expression can effectively inhibit the proliferation of GBC-SD cells and restrain the cell cycle atin G0/G1 phase. Bmi-1 gene may be a novel target for geneic therapy of gallbladder carcinoma.
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Objective To investingate the effect of survivin shRNA on chemotherapy resistance in GBC-SD cells. Methods They were divided into three groups of GBC-SD, GBC-SD/EGFP and GBC-SD/survivin. MTT assay was used to detect cell viability in the 3groups. mRNA and protein of survivin were tested by RT-PCR and Western blot. Then the cells were treated with proper construction DDP. The cell survival rate and IC_(50) were determined with MTT, cell apoptosis detected by FACS and the alteration of nucleus observed by TUNEL. Meanwhile, caspase-3 activity was determined using the colorimetric method. Results Cell viability was reduced remarkably in GBC-SD/survivin and survivin expression was decreased obviously. After being treated with DDP, cell survival rate and IC_(50) were decreased obviously in GBC-SD/survivin, apoptotic rate elevated remarkably compared with other groups. There were brownly nucleuses in three groups. Caspase-3 activity increased first and then decreased, but it exceeded in GBC-SD/survivin than that in other two groups. Conclusion The survivin shRNA can down-regulate the expression of survivin in GBC-SD cells remarkably and improve the sensibility to chemotherapy.
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AIM To investigate the inhibition and apoptosis mechanism of GBC-SD cells induced by rubescensine B. METHODS Using MTT, convert microscopy, electron microscopy, flow cytometry, an immunohistochemical assay, and spectrofluorometry demonstrate the presence and pathogenesis of apoptosis after treated by rubescensine B. RESULTS After exposure to Rubecensine B GBC-SD cells were induced to apoptosis in dose-dependent manner, and the level of Bcl-2,p53,C-myc,Fas/APO-1 were decreased within 24 hours, reversely the activity of Caspase-3 was enhanced with the appearance of apoptosis. CONCLUSION Rubecensine B can induce GBC-SD cells apoptosis related to Bcl-2,p53,Fas/APO-1 and C-myc.
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Objective To investigate the effect of cyclooxygenase-2 inhibitor Celebrex on the growth of(gallbladder) carcinoma GBC-SD cell line.Methods Cell growth suppression was counted by MTT method.Apoptotic index(AI) was evaluated by TUNEL staining.The apoptotic rate was counted by flow cytometry(FCM),fluorescence microscopy(FM) and transmission electron microscopy(TEM).Results Celebrex(inhibiting) the growth of GBC-SD cell line was dose-depend.The growth inhibition rate with 40?mol/L,80?mol/L,120?mol/L and 160?mol/L was 18.77%,25.32%,46.58% and 52.19%(respectively),(P