Study On Specific Silence Of EBV Latent Gene EBNA2 Expression By SiRNA

Epstein-Barr virus (EBV) nuclear protein 2 (EBNA2) is one of the first two proteinsexpressed from the EBV genome and is encoded by BYRF1 gene.EBNA2 lackssequence-specific DNA-binding activity and recognizes specific promoters throughcellular proteins such as RBP-Jκ. EBNA2 is a phosphoprotein that localizes to variouscompartments of the nucleus, including the nucleoplasm, the chromatin fraction, and thenuclear matrix. The EBNA2 protein does not bear any significant sequence homologywith cellular proteins.EBNA2 carries the characteristic features common to alltranscription factors: A transactivation domain, nuclear localization motifs and a regionwhich mediates promoter contact.RNAi is a phenomenon of genetic interference mediated by double strand RNAwhich can degrade mRNA specifically and effectively and cause post-transcriptional genesilencing. Recent research showed 21-23nt siRNA (small interference RNA) can mediatespecific gene silencing in mammalian cells. Now siRNA has been a strong tool inknockdown of specific gene in mammalian cells.In our study we transfected chemically synthetic siRNA targeted EBNA2 gene intoGT38 cells with lipofectamine 2000 and observed inhibition of EBNA2 gene expressionby siRNA. We also studied whether siRNA can induce cell cycle change or apoptosis ofGT38 cells. Meanwhile we construct a recombinant retroviral vector pSUPER-EBNA2that target EBNA2 and select a stable virus-producing cell clone, to compare inhibitioneffects to target gene expression and influence on target cell between two methods.Part one Specific Silencing of EBNA2 Gene Expression by siRNA in GT38 CellsObjective To synthesize siRNA that targets EBNA2 coding gene and transfect EBVpositive GT38 cells. To investigate the specific silencing effect of siRNA to theexpression of EBNA2 and the influence on target cell due to EBNA2-specific silence onEBV positive cells.Methods The chemically synthetic siRNA was transfected into target cells GT38 which isⅢtype latency by lipofectamine 2000. The silencing effect on the expression of EBNA2was tested by RT-PCR and the apoptosis and cell cycle of GT38 was detected withHoechst 33258 stain, transmission electron microscope (TEM) and flow cytometry.Results①RT-PCR result showed that siRNA789, siRNA764 and siRNA257 markedlyinhibited the expression of EBNA2 mRNA in GT38 cells comparing with theuntransfected GT-38 (P＜0.01), and the effect of siRNA789 was most obvious.② Compared with the untransfected GT-38, levels of EBNA2 mRNA insiRNA789-trancfected GT-38 didn't decreased (P＞0.05) 24h post-transfection, whilemarkedly decreased 48 and 72h post-transfection (P＜0.01).③In siRNA789-trancfectedGT-38, levels of EBNA2 mRNA decreased along with the increasing siRNA789concentration during 0～50nM (P＜0.01), wheras the level between 50nM, 75nM and100nM was not significant (P＞0.05).④It was not showed any nucleus concentration, apoptosis bodies or apoptosis hump in experiment group GT38 comparing to the controlgroup cells. Flow cytometry analysis showed that siRNA transfection in GT-38 increasedthe proportion of cells at S phase, and didn't induce cell apoptosis.Conclusion Chemically synthetic siRNA targeted EBNA2 effectively silences theexpression of EBNA2 in EBV positive cell GT38. The inhibition rate of EBNA2 mRNAis associated with the transfection time and is proportioned to siRNA789 concentrationduring 0～50nM. The effect of inhibition is specific and relates to the siRNA sequence.siRNA transfection in GT-38 increases the proportion of cells at S phase, and do not effectcell apoptosis. It provides evidence for investigating the therapeutic effect of specialsilence of EBNA2 to EBV positive tumors.Part two Construction of pSUPER retro RNAi system of the specific silencing EBVlatent protein EBNA2Objective To construct a recombinant retroviral vector pSUPER-EBNA2 that targetsEBV nuclear antigen 2(EBNA2) and select a stable virus-producing cell clone.Methods The 60nt sequences encoded for transcribing EBNA2 shRNA were cloned intoa retrovirual vector pSUPER. retro with DNA recombinant technique. The recombinantvector was identified by the electrophoresis analysis of restriction enzyme digestion andDNA sequencing. The packaging cell PA317 was transfected with recombinant plasmidusing G418 medium and the titer was determined.Results The result of DNA sequencing demonstrated that 60bp sequences had beeninserted in the expected site and the insertion sequenve was exactly correct. When therecombinant vector was transfected into the packaging cell, green fluorescent protein(GFP) was expressed. The anti-G418 positive clones were also selected and the titer was2.5×10~5 CFU/ml.Conclusion We succeed in constructing a recombinant retroviral vector pSUPER-EBNA2and selecting a stable virus-producing packaging cell line, which establishes foundationfor approaching specific silence of target gene with retroviral vector further.