SiRNA Against Survivin in Nasopharyngeal Carcinoma Cells Growth Inhibition Investigation
|Course||Department of Otolaryngology Head and Neck Surgery|
|Keywords||Survivin eukaryotic expression vector Lipofectamine2000Survivin siRNA Nasopharyngeal carcinoma cells|
The first partObjective:Through the construction of targeting Survivin shRNA eukaryotic expression vector method to explore the technology of RNAi in nasopharyngeal carcinoma cell growth inhibition, for nasopharyngeal carcinoma gene therapy provides a new experimental basis.Method:The expression of apoptosis inhibiting gene Survivin as the target, to construct the expression of small interfering RNA (siRNA) eukaryotic expression vector, by liposome transfection in nasopharyngeal carcinoma cell line CNE-2, by RT-PCR Survivin mRNA expression in nasopharyngeal carcinoma cells inhibit, preliminary identification of interference effect.Results:The recombinant vector pGPU6/GFP/Neo-shRNA1,2,3,4, N double digestion and genesequencing to detect, confirm that the base sequence of the template sequence, gene mutation, the vector was successfully constructed.The recombinant vector was transfected the Lipofectamine2000by liposome-mediated nasopharyngeal carcinoma cells, cell apoptosis occurs in varying degrees, the transfection efficiency of about55%.RT-PCR method for preliminary identification of its moderating effect of Survivin/GAPDH expression relative to changes in survivin mRNA, mRNA expression of each group is quite different,24h before transfection, measured mRNA relative to a certain decline, about in about50%; but with time, the interference effect of recombinant plasmid was gradually weakened.Conclusion:This experiment by building targeted Survivin shRNA eukaryotic expression vector,liposome-mediated transfection of nasopharyngeal carcinoma cell line CNE-2cells, the Survivin gene expression levels in the inhibited to some extent. RNAi in a certain degree of inhibition of nasopharyngeal carcinoma cell gene therapy for nasopharyngeal carcinoma provides a new experimental data. The second partObjective:Chemically synthesized small interfering RNA (small interfering RNA, siRNA) approach to investigate the RNAi technology to inhibit the growth of nasopharyngeal carcinomacells for nasopharyngeal carcinoma gene therapy to provide a new experimental basis.Method:SiRNA design principles for the survivin gene sequence-specific siRNA, liposome-mediated transfection of CNE-2cells.The RT-PCR and Western blotting detection of Survivin mRNA and protein expression in the disturbance, and filter out the interferenceeffect of siRNA sequences for cell growth inhibition test (CCK-8experiments) detectedon cell growth inhibition.Results:Transfection siRNA24h,48h and72h, using RT-PCR and Western blotting, in CNE-2Z cells survivin expression was significantly decreased, and filter out the best inhibitory effect on the1st sequence of CCK-8experiments, the results shown to significantly inhibit cell growth proliferation.Conclusion:Targeting Survivin siRNA transfection of human nasopharyngeal carcinoma CNE-2cells to inhibit the expression levels of survivin gene in the cells, and cell growth and proliferation of biological behavior inhibition.Application by chemical synthesis of siRNA, RNAi technology nasopharyngeal carcinoma cell growth was significantly inhibited for nasopharyngeal carcinoma gene therapy offers a new experimental data.