The Killing Effect Of HSVtk/GCV Suicide Gene System On Hepatoma Carcinoma Cells Driven By The Survivin Promoter

Objectives:1. Construct a kind of plasmid vector pcDNA3.1-pSurvivin-TK driven by survivin promoter.2. Whether herpes simplex virus thymidine kinase(HSVtk) suicide gene regulated by the survivin promoter could be targetedly expressed in hepatoma carcinoma cells was detected.3. Evaluate the efficiency of HSVtk/GCV system on hepatoma carcinoma cells under the control of the survivin promoter.Methods:1. HL-7702 normal liver cells, HEK293 T human embryonic kidney cells, HepG2 hepatoma carcinoma cells and HuH-7 hepatoma carcinoma cells were cultured in vitro.2. Genomic DNA was extracted from the HepG2 cells, PCR was applied to obtain survivin promoter sequence. The recombinant plasmid pcDNA3.1-pSurvivin-TK was constructed and sequenced. BLAST was used to detect the homology of DNA sequence.The plasmid was transformed into DH5α, HSVtk gene was amplified by bacterial PCR, and PCR products were detected by agarose electrophoresis.3. The plasmid pcDNA3.1-pSurvivin-TK was transfected into the HL-7702 cells,HEK293 T cells, HepG2 cells and HuH-7 cells respectively. The total mRNA and protein were extracted from the corresponding cells after 48 hours, the expressions of HSVtk mRNA and protein were analyzed by RT-PCR and Western blot.4. The HL-7702 cells, HepG2 cells and HuH-7 cells were transfected with the plasmidpcDNA3.1-pSurvivin-TK. 48 hours later, the cells were interfered with ganciclovir(GCV),cell viability was assayed by MTT.5. The plasmid pcDNA3.1-pSurvivin-TK was transfected into the HL-7702 cells,HepG2 cells and HuH-7 cells. 150 μg/ml of GCV was added to cell medium for 24 hours,Hoechst staining was used to detect the morphological change of nucleus; cell apoptosis and reactive oxygen species(ROS) levels were observed with flow cytometry; Caspase-3staining was used to detect activated caspase-3 in apoptotic cells by fluorescence microscopy.Results:1. Survivin promoter sequence was amplified by PCR, the recombinant plasmid pcDNA3.1-pSurvivin-TK driven by the survivin promoter was constructed successfully,the sequence of HSVtk fragment was identical to GenBank.2. RT-PCR and western blot results showed, HSVtk gene was effectively expressed in HepG2 and HuH-7 cells transfected with pcDNA3.1-pSurvivin-TK plasmid, whereas HSVtk gene expression was not detected in the HL-7702 and HEK293 T cells.3. MTT assay revealed that, with the increasing of GCV concentration, cell viability gradually decreased in the HepG2 and Hu H-7 cells treated with HSVtk/GCV suicide gene system, there were significantly statistical difference in comparison with the blank groups(P﹤0.05), while HSVtk gene showed no cytotoxicity in the HL-7702 cells.4. Hoechst staining revealed that nucleus of the HepG2 and Hu H-7 cells treated with HSVtk/GCV stained translucent, uneven color and nuclear chromatin pyknosis, while the HL-7702 cells remained uniform blue fluorescence.5. Flow cytometry showed, in the HepG2 and Hu H-7 cells treated with HSVtk/GCV suicide gene system, cell apoptosis rates and ROS levels were increased dramatically in comparison with the blank groups(P<0.05), however, in the HL-7702 cells, cell apoptosis rates and ROS levels had no significant change.6. Caspase-3 staining showed that activated caspase-3 increased significantly in theHepG2 and HuH-7 cells interfered with HSVtk/GCV suicide gene system, this confirmed the apparent cell apoptosis, whereas in the HL-7702 cells activated caspase-3 increase was not observed.Conclusions:1. A novel recombinant plasmid pcDNA3.1-pSurvivin-TK was constructed successfully.2. HSVtk gene was obviously expressed in the HepG2 and HuH-7 cells specifically,but not detected in the HL-7702 and HEK293 T cells.3. HSVtk/GCV suicide gene system showed effective killing effect on HepG2 and HuH-7 cells, while in the HL-7702 cells, no apparent impact was observed.