Experimental Study Of The Inhibition By RNAi-mediated Survivin Gene Silencing On The Nude Mice Xenograft Tumor Of Nasopharyngeal Carcinoma Cell CNE-2

Part one Construction and identification of Survivin-shRNA expression vectorObjective To construct and identity Survivin-shRNA expression vector in order to make foundation of gene therapy for further exploration NPC of RNA interference.Materials and Methods According to survivin cDNA gene sequence in the gene bank (NM₀01168), a pair of 65 nt oligonucleotides which containing the sites of restriction endonuclease at both ends, were designed and synthesized by Reynolds design principles.Oligonucleotides were annealed and ligated with linedrized RNAi-Ready pSIREN-DNR-DsRed-Express by T4DNA ligase. Transfected into escherichia coli strains DH-5a the recombinants were finally sequenced and identified by restriction endonuclease digestion.Results pSIREN-survivin/shRNA was successfully constructed and identified by double endonuclease digestion. Sequence analysis of inserted fragment revealed the same sequence as synthesized shRNA Oligonucleotides.Conclusion Survivin-shRNA expression vector has been successfully constructed, make the foundation of research using liposome packaging transfecting NPC tissue cell for the next step. Part two Experimental studies on the effect of survivin expression suppressed by small interference RNA which induces apoptosis in nasopharyngeal carcinoma cell line CNE-2Objective To evaluate the effect of survivin siRNA silencing survivin gene on the apoptosis rate and proliferation of nasopharyngeal carcinoma cells.Materials and Methods The plasmid pSIREN-siRNA/survivin was constructed and transfected into NPC cells. The expression level of survivin mRNA and protein of were detected by RT-PCR and western blot after the transfection. Apoptosis rate and cell cycle were measured by flow cytometry. Cellular proliferation was measured by MTT.Results shRNA was transfected into NPC cells effectively and its rate was (46.9±1.55)% observed with fluorescence microscopy. Survivin mRNA and protein were inhibited after transfection for 24 hours and the inhibitory rates were (41.58±0.63)%,(68.29±0.52)%.The inhibitory rates increased by (63.64±0.96) %,(70.83±0.48)% respectively after transfection for 48 hours. Amount of S phase cells decreased, while G2/M phase increased through flow cytometry analysis. The apoptosis rates of positive groups transfected after 24h were (36.24±0.78)%and (50.37±0.85)% of that after 48h respectively, significantly higher than the negative and the untreated groups. MTT test showed that NPC cells proliferation were inhibited after transfection of plasmid pSIREN-shRNA/survivin.Conclusion Small interference RNA silencing survivin gene can effectively inhibit NPC cellular mRNA and protein expression and its proliferation and induce the cell's apoptosis. Part three Experimental study of the inhibition by RNAi-mediated survivin gene silencing on the nude mice xenograft tumor of nasopharyngeal carcinoma cell CNE-2Objective To probes into the influence about tumor growth of nasopharyngeal carcinoma CNE2 by short hairpin RNA targeting survivin gene in nude mice.Materials and Methods Construct a tumor-burdened model of nasopharyngeal carcinoma, then suppress the expression of survivin in nasopharyngeal carcinoma through multi-point injection and transfect on nude mice by liposome. MRNA and protein levels of survivin were detected by semi-quantitative reverse transcriptase polymerase chain reaction (RT-PCR)and immunohistochemistry.Determination of tumor growth in volume.Cell apoptosis was detected By transmission electron microscopy techniques liver and kidney function was tested.Results The expression of survivin mRNA and protein can be effectively and stably suppressed by survivin gene sequence-specific shRNA of NPC xenografts in nude mice. tumor growth can be significantly inhibited and apoptosis was promoted, and the liver and kidney function in nude mice had no effect on short-term.Conclusions The expression of survivin gene can be inhibited by RNA interference NPC xenografts in nude mice.and the proliferation of nasopharyngeal carcinoma cells can be reduced and cell apoptosis was promoted cancer tumor growth was reduced, as a novel therapeutic tool for nasopharyngeal carcinoma.