Research On Targeting Radio-gene Therapy Through The Expression Of CD/UPRT.UL49 Mediated By E6.BARF1p In Nasopharyngeal Carcinoma

Nasopharyngeal carcinoma is a kind of malignant tumor treated mainly by radiotherapy, how to improve regional control rate and survival rate, reduce the radiation damage to normal tissue and improve the quality of life of patients has always been the focus of this study, therefore, this article is going to discuss the research on targeting radio-gene therapy through the expression of CD/UPRT.UL49 mediated by E6.BARFlp(referred to as BARFlp) in nasopharyngeal carcinoma.Objective To design the of BARF1 gene promoter sequence, verify whether this sequence has promotive activity by comparing the promoter activity in human nasopharyngeal epithelial cells NP69 and that in normal human nasopharyngeal carcinoma cells CNE-2, and confirm whether the promoter of BARF1 gene has relative specificity of tumor.Methods Combining results of promoter prediction software (TRANSFAC and MATINSPECTOR) and primer design software (Primer premier 5.0), we designed the sequence of BARF1 gene promoter, extracted the genomic DNA from B95-8 cell lines, amplified the designed BARF1 gene promoter by PCR, connected the amplified promoter to the luciferase reporter gene vector, then transfected this vector into NP69 cells and CNE-2 cells transiently through liposome way, and verified its promoter activity, also compared the promoter activity in NP69 cells with that in CNE-2 cells, to verify whether this promoter has tumor relative specificity, t-test of SPSS 10.0 was used to detect the difference between them.Results BARF1 gene promoter showed promoter activity in CNE-2 cells, but the activity is low, yet there is no promoter activity detected in NP69 cells, and the promoter activity showed in CNE-2 cells is much higher than that in human nasopharyngeal epithelial cells NP69, difference is significant(p<0.05).Conclusion The promoter sequence of BARF1 gene has promotive activity, and also has tumor relative specificity.Objective To construct nasopharyngeal carcinoma CNE-2 cell lines expressing stable suicide gene.Methods The plasmids of pcDNA3.1 (-) E6.BARF1p. CD/UPRT.UL49 was transfected into CNE-2 cells through lipofectamine, and the transfected CNE-2 cells were selected by G418 to get the cells expressing CD/UPRT.UL49 gene. The protein produced by the suicide gene was tested by Western-blotting in CNE-2 cells.Results Suicide genes was expressed stably in CNE-2 cells. Conclusions We constructed CNE-2 cell lines expressing stable suicide gene through lipofectamin transfection.Objective To observe killing effect towards CNE-2 cells through the expression of suicide gene and prodrug system of CD/UPRT.UL49/5-FC in vitro gene therapy.Methods Given different quantity of Y radiation combined with different quantity of prodrug 5-Fc to wild CNE-2 cells and CNE-2 cells transfected with CD/UPRT.UL49 respectively, after 48h, the killing effects was tested by MTT method; again given 2Gy radiation and 200μg/ml prodrug 5-Fc to wild CNE-2 cells and CNE-2 cells transfected with CD/UPRT.UL49 respectively, after 48h, the killing and bystander effects on CNE-2 cells transfected with CD/UPRT.UL49 was tested by flow cytometry (FCM). T test and ANOVA were used to detect the differences. P＜0.05 represents significant.Results The growth of CNE-2 cells expressing suicide genes was suppressed more or less, and such a suppression effect of CD/UPRT.UL49 was of dose dependence to the 5-FC. Though there were only 10 percent of the cells expressing CD/UPRT.UL49 gene, it was demonstrated that 37.46 percent of the cells were killed. Conclusion The suicide gene and prodrug system of CD/UPRT.UL49/5-FC has direct cytotoxic and bystander effects to CNE-2 cells.Objective To observe the enhancement of the sensitization of the NPC to radiation under the intervention of prodrugs by plasmid vector expressing CD/UPRT gene in situ and in vivo gene-radiotherapy.Method CNE-2 cells in log phase were inoculated subcutaneously in nude mice to construct a nude mouse model of NPC,and an in situ gene therapy was performed with plasmid packed by plasmid. When the size of tumors reaching 0.8-1.0cm, the nude mice were randomized to different grouped, the size of the tumor was recorded and the suppression of tumor was compared among different treating groups. Biopsy of the tumor were performed to find the difference between treatment groups.Then targeted RNA was isolated from tumor tissues and RT-PCR was performed to define the expression of suicide gene.Results In situ gene therapy experiment showed that compared with cotroll group,growth of transplant tumour of each treatment group was distinctly suppressed(P<0.01).Conclusions The suicide gene E6.BARF1p.CD/UPRT. UL49/prodrug system, is both radiosensitive and tumor specifically effective in anticancer therapy, and exert killing effect to CNE-2 cells under radiation. From this study, we established a new strategy for radio-gene therapy of NPC.