Preliminary Study Of Biological And Suppressing Effects Of Lnazyme Target EBV-LMP1Gene On B95-8Cells

Nasopharyngeal cancer (Nasopharyngeal Carcinoma, NPC) is one ofthe common malignant tumors in southern China.The number of newpatients ranks first in the world annually. Now that the EB virus (Epstein-Barr virus, EBV) are closely related with nasopharyngeal carcinoma,whichencod latent membrane protein1(latent membrane protein1, LMP1) is theonly clear evidence of oncogene gene. Gene therapy target EBV-LMP1became widely research hotspot. Based on DNAzyme, introduced one ormore locked nucleic acid (LNA) monomers and formed the new specificcleavage of RNA antisense oligonucleotide,which called Locked nucleicribosome (LNAzyme). LNA is an oligonucleotide derivative with a doublering structure, so that the introduction of LNA makes LNAzyme cuttingefficiency more significantly improved than the corresponding DZ,especially10to23types DNAzyme.In this study our group based on the10to23type DNAzyme(DZ167)which screened from pre-study target EBV-LMP1gene, DZ167modifiedby LNA and synthetic LANzyme(LNA167). EBV-LMP1positive B95-8cells as an object, for comparing the inhibition efficiency of DZ167and LNA167target LMP1and observing their biological effect on B95-8cell.First explore the application prospect of LNAzyme asEBV-LMP1inhibitors in NPC gene therapy. Objective: Based on the preliminary screening DZ167,synthesizingLANzyme(LNA167) against the EBV-LMP1gene and comparinginhibitory effect of DZ167and LAN167target EBV-LMP1gene in B95-8cells.Methods: Taking EBV-LMPl gene as target point and B95-8asprototype, and synthesizing DZ167.Marking the5’and3’terminal modifiedby thio-modification or two LNA monomers in terminal2bases, anddesigning disordered DNAzyme as control which has no catalytic activitycenter, all sequences5’end labeled with FITC.Transfecting them intoB95-8cells by lipotectamine,called disordered DZ167group、S-DZ167group、LNA167group respectively. Taking untransfected B95-8cell as blank group.24hours after transfection,observing the distribution offluorescence in living cells under the fluorescence microscope and testingtransfection efficiency by fluorescent flow cytometry.Transfection12h,24h,48h，72h and96h, using Real-Time PCR evaluation the stability andsuppression time-effect relationship for LMP1between S-DZ167andLNA167.48h after transfection using the Western blot method to detectinhibition expression of EBV-LMP1protein by LNA167, S-DZ167inB95-8cells.Results: After transfection, green fluorescence were showed in allgroups under fluorescence microscopy, and they were mainly distributed inthe cytoplasm and cytomembrane. Disorderly DZ167group, S-DZ167group, LNA167group transfection efficiency was75.5%,76.7%and77.6%respectively.The stability and suppression effect relationship for LMP1shown: afterTransfection12h,24h,48h,72h and96h,compared with non-transfectedgroup, LNA167group and S-DZ167group, both can maintain a certaininhibitory effect, inhibition rate were23.73%，33.93%，44.89%，27.45%，12.24%；45.76%，51.79%，71.43%，54.90%，22.45%，respectively. At each timepoint,the difference was significant (P <0.05) compared withnon-transfected group.The inhibition effect reached a peak at48h, and thendecreased gradually; whereas transfection96h compared with the S-DZ167group, the LNA167groups had no significant different(P>0.05), but LNA167group inhibitory effect and stability was still higher than S-DZ167group.Western blot analysis showed that: compared with non-transfectedgroup, inhibition rate of disorderly DZ167group on EBV-LMP1proteinwas5.89%, which had no significant different (P>0.05); inhibits rates ofLNA167, S-DZ167were49%,75.02%, LNA167and S-DZ167can inhibitEBV-LMP1protein in different degrees, but inhibitory effect of LNA167was significantly higher than S-DZ167, the difference was statisticallysignificant (P <0.05).Conclusion: Compared with the S-DZ167, LNA167can efficientlyinhibit the EBV-LMP1gene expression by LNA modified; LNA167,S-DZ167inhibition of EBV-LMP1expression show a certain time-effectrelationship,48h inhibition effect reached a peak, and then graduallydecline, but LNA167inhibitory effect was still higher than S-DZ167. Objective: To discuses the cytotoxicity, proliferation, oncogenicity and apoptosis on B95-8cells after transfection.Methods: Transfecting disordered DZ167、S-DZ509and LNA167into EBV-LMP1positive B95-8cells and negative CNE-2cells,respectively. Taking untransfected B95-8and CNE-2cells as blankcontrols.24、48、72hours after transfection, evaluate the cytotoxicity ofdifferent concentrations of S-DZ167, LNA167on CNE-2cells;24、48、72hours detected B95-8cells proliferation by CCK8;Observing colonyformation of B95-8cells in soft agar14days after transfection; Detectingapoptosis by Flow cytometry(FCM)48hours after transfection.Results: Cytotoxicity results showed that: the different concentrationsof LNA167and S-DZ167had show no effect on CNE-2cell proliferation,compared with non-transfected group at the same time points,which alsohas no significant changes in cell morphology before and after transfection.CCK8cell proliferation showed:24h after B95-8cells transfection,the inhibition rates of S-DZ167and LNA167group were23.52%,29.41%,compared with non-transfected group, which has significant difference (P<0.05). Transfection48h, each group reached the highest inhibitionefficiency, inhibition rates of S-DZ167and LNA167were,26.57%,62.74%,LNA167significantly higher than S-DZ167group, the difference wassignificant (P <0.05). From48h to72h, the inhibition efficiency on B95-8was decreased, compared with the control group, the difference wassignificant (P <0.05), the inhibitory effect LNA167group> DZ167group. Soft agar colony formation: observing the clones in each group, during4～5days the non-transfected and disorderly DZ167began to formcolonies, but LNA167and S-DZ167group showed no colony formation.Training to14days,the number of formed clones of LNA167, S-DZ167,disorderly DZ167and non-transfected group were15±1.67、32±2.31、62±5.13、58±2.36, clone formation rates were3%、6.4%、12.4%、11.6%,respectively. LNA167、S-DZ167group compared with the non-transfectedgroup had significant difference (P <0.05), between the two groups weresignificant difference (P <0.05).Targeting LMP1by LNAzyme,theoncogenicity of B95-8cell significant decreased.Though FCM，48hours after transfection,comparing to blank controlgroup，the cell apoptosis rate of disordered group was0.4%,which has nosignificant difference(P>0.05).The cell apoptosis rate of S-DZ167、LNA167groups were about19.55%、24.16%，the effect on apoptosis：disordered DZ167<S-DZ167<LNA167.Conclusion: LNA167, S-DZ167had show no significant toxicity oncells.Compared with the S-DZ167, LNA167can more effectively inhibitthe proliferation of EBV-LMP1cells and decreased oncogenicity, inducedapoptosis on B95-8cells.