The Latent Membrane Protein-1 Gene Silence By Combination Treatment Of Short Hairpin RNA And Deoxyribozyme In Nasopharyngeal Carcinoma

Nasopharyngeal carcinoma (NPC) is a common human malignancy in China. Latent membrane protein-1(LMP1) of Epstein-Barr virus (EBV) plays a significant role in pathogenesis of NPC and is regarded as the oncogene of NPC.RNA interference(RNAi) and deoxyribozyme(DNAzyme, DZ) are two potent molecular biological tools in degrading expression of aimed genes and have been applied in studies of anti-virus or tumor and gene function, extensively. In our researches, we constructed recombination plasmids expressing short hairpin RNA(shRNA) targeting EBV-LMP1 gene and investigated EBV-LMP1 gene silence by combination treatment of shRNA and deoxyribozyme in nasopharyngeal carcinoma cells.PartⅠInhibition of GFP Gene Expression in Nasopharyngeal Carcinoma Cells by shRNAObjective: To investigate the RNA interference phenomenon induced by shRNA in nasopharyngeal carcinoma cells by observing the inhibition effect and cytotoxicity of shRNA targeting intrinsic GFP.Methods: HNE1 cells transfected with pEGFP-N1 were followed by screening with G418 to obtain a stable nasopharyngeal carcinoma cell strain expressing GFP(HNE1-GFP) ; pshRNA-GFP were transfected into HNE1-GFP with various ratio levels. The fluorescence was observed by fluorescent microscope; The inhibition ratios and cytotoxicity of pshRNA-GFP were analyzed by software and MTT assay; The mRNA and protein level of GFP were detected by RT-PCR and western blot, respectively.Results: pshRNA-GFP suppressed the intrinsic expression of GFP with maximum ratios of 86.7%, without significant cytotoxicity. pshRNA-GFP 1:3/2:5 were of vivid inhibitory effect on GFP mRNA and protein expression.Conclusion: Knocking out the expression of aimed genes by shRNA was demonstrated in nasopharyngeal carcinoma cells.PartⅡFusion Protein Expression of EBV-LMP1 and GFP Gene in Nasopharyngeal CarcinomaObjective: To construct GFP fusion protein eukaryotic expression vectors and express LMP1-GFP fusion protein in HNE1 cells.Methods: pMD18-T-LMP1m was constructed based on LMP1 cDNA sequence gained through PT-PCR from total RNA of B95-8 cells. The eukaryotic expression vectors of pEGFP-N1-LMP1m, pEGFP-C1-LMP1m, pEGFP-N1-LMP1a and pEGFP-C1-LMP1c were constructed on the basis of pMD18-T-LMP1m, followed by being transfected into HNE1 cells with LipofectamineTM2000.The expression of LMP1-GFP fusion protein was observed by fluorescence inverted microscope and RT-PCR as well as western blot.Results: The recombination vectors of pEGFP-N1-LMP1m, pEGFP-C1-LMP1m, pEGFP-N1-LMP1a, pEGFP-C1-LMP1c were constructed and transfected into HNE1 cells successfully. The LMP1-GFP fusion protein is mainly located on cellular membrane and in cytoplasm. The fusion protein was detected by both RT-PCR and western blot.Conclusion: The expression of LMP1 fragments fusion protein is obviously observed in HNE1 cells, including LMP1 N terminal, C terminal and total LMP1-GFP fusion protein. A visible cellular system of LMP1 gene expression is successfully established in our research, which is available in screening procedure of short hairpin RNA targeting EBV-LMP1 gene in NPC cells. PartⅢThe Construction and Screening of pshRNA-LMP1 Expression Vectors in the Visible Cellular System of HNE1 CellsObjective: To construct pshRNA-LMP1 expression vectors; to estimate silencing efficiency of pshrna-LMP1 vectors in nasopharyngeal carcinoma cells.Methods: The pshRNA-LMP1 expression cassettes were designed complement to a 35bp long internal repeated sequence in EBV-LMP1 cDNA fragment. The pEGFP-N1-LMP1m and pshRNA-LMP1 vectors were co-transfected into HNE1 cells to estimate transfection efficiency. The efficiency and specificity of pshRNA-LMP1 were estimated by fluorescence observation and RT-PCR as well as western blot. The proliferation and apoptosis of HNE1 cells was detected by MTT and AO/EB assay respectively.Results: pshRNA-LMP1 expression vectors were obtained successfully and its co-transfection ratio with eukaryotic expression vectors of LMP1-GFP fusion protein in HNE1 cells was 1.0μg:0.5μg. pshRNA-LMP1-3519 was efficient to inhibit the expression of LMP1 gene and the growth of HNE1 cells without any cytotoxicity, but to promote the apoptosis of HNE1 cells.Conclusion: Our visible cellular system is of convenience to screening pshRNA-LMP1 vectors in nasopharyngeal carcinoma cells. pshRNA-LMP1-3519 is efficient in down regulating the expression of EBV-LMP1 gene and promoting the apoptosis of NPC cells.PartⅣThe Latent Membrane Protein-1 Gene Silence by Combination Treatment of pshRNA-LMP1 and DeoxyribozymeObjective: To evaluate the EBV-LMP1 gene silence by combination treatment of pshRNA-LMP1 and DNAzymes.Methods: pshRNA-LMP1-3519 combined with DZ167/509 were co-transfected into HNE1 cells with pEGFP-N1-LMP1m. FCM was used to assay inhibition ratio of fluorescent light, RT-PCR as well as western blot were used to detect the expression of EBV-LMP1 gene in HNE1 cells.Results: The inhibition ratio of fluorescent light was 75.15% in pshRNA-LMP1-3519 group compared to that of 86.31% in pshRNA-LMP1-3519 combined with DZ167 and 88.88% in pshRNA-LMP1-3519 combined with DZ509 groups. The fluorescence intensity in pshRNA-LMP1-3519 group and pshRNA-LMP1-3519 combined with DZ509 group were 12.99% and 5.60%, respectively.Conclusion: The visible cellular system is of power to estimate silencing efficiency of combination therapy in nasopharyngeal carcinoma cells. The combination group presented a more efficient gene silencing capacity compared to RNA interference group or DNAzymes group.