Experimental Study On The Eukaryotic Expression And Inducing Apoptosis Effect Of VP3 Gene And Improved TAT-VP3 Gene In Human Bladder Cancer Cells In Vitro

Objective To explore the expression and apoptosis inducing effect of chickenanemia virus(CAV) VP3 gene and improved TAT-VP3 gene in human bladder cancercell lines BIU-87.Methods The recombinant eukaryotic expression vector PcDNA3-VP3 andPcDNA3-TAT-VP3 were constructed. Then, the positive recombinant containing VP3gene and improved TAT-VP3 gene was transfected into human bladder cancer celllines BIU-87, using Lipofectamine ^TM2000 mediated transfection in vitro. After that,RT-PCR was performed to detect the expression condition of VP3 gene and improvedTAT-VP3 gene in vivo. HE staining, transmission electronmicroscope were used todetermine the apoptotic morphological changes. From 24 to 72 hours aftertransfection of PcDNA3-VP3 and PcDNA3-TAT-VP3 into BIU-87 cells, opticalmicroscopy, fluorescein stain of JC-1, Annexin V-EGFP/PI dual staining were used toverify apoptosis of tumor cells in different stages. The proliferation of BIU-87 cellswas detected by MTT method. Apoptotic rates were quantified by flowcytometry(FCM) using TUNAL method.Results We constructed the two recombinant plasmids successfully, which wascorroborated by analyse of gene sequence respectively. The results of RT-PCRsuggested that VP3 gene and improved TAT-VP3 gene was expressed in BIU-87 cells.The typical morphological changes of apoptosis such as cell shrinkage, chromatincondensation, nuclear fragmentation and apoptotic body were observed by both lightand electron microscope. The green fluorescence was observed in early apoptotic cellsby JC-1 method. Annexin V-EGFP/PI dual staining can differentiate early and lateapoptotic cells. The proliferation of BIU-87 cells which was detected by MTT method both decrease distinctively(P＜0.01). The apoptotic rates in PcDNA3-VP3 group aftertransfection for 24, 48, and 72 hours were (26.3±1.96)%, (48.83±1.03)%,(68.50±3.00)% and (14.3±0.59%), (30.50±0.77)%, (42.27±2.66)% for transfection ofPcDNA3-TAT-VP3 group. The apoptotic rates were both higher than that in controlgroup(P＜0.01).Conclusion The expression of chicken anemia virus VP3 gene can induce early andhigh efficiency apoptosis in human bladder cancer cell lines BIU-87. At the same time,the results of our experimental data also suggests that the improved TAT-VP3 fusionprotein is very potent in inducing apoptosis in human bladder cancer cell lines BIU-87,which will provide a foundation for the future work such as protein manufacturingand function researching of this fusion protein.