Influence Of Tandem Gene Survivin And CDK1shRNAs Expression Vector On Nasopharyngeal Carcinoma Cell Line CNE-2Growth And Apoptosis

Objective: To construct the vectors to knock down the expression of Survivin and/or CDK1by shRNA interference and study the effect to the biological behavior of nasopharyngeal carcinoma cell line CNE-2.Methods:Interference sequences to Survivin and CDK1were designed and synthesized on the date of GeneBank and the design philosophy of RNA interference. Each sequence was linked with plasmids and transfected to the CNE-2cell line by Lipofectamine2000. The recombinant plasmids SurvivinshRNA and CDK1shRNA were selected then the tandem plasmid pU6-SurvivinshRNA-CDK1shRNA was constructed. The tandem plasmid was identified by restriction enzyme digestion and DNA sequencing and then was transfected to CNE-2cell line by Lipofectamine2000. The changes of the expression of Survivin and CDK1caused by the plasmids were detected by RT-PCR and Western blot. The cell proliferation were determined by MTT. The apoptosis were determined by flow cytometry.Results:(1) Restriction enzyme cleave identification and sequencing proved that those recombinant plasmids of pU6-SurvivinshRNA, pU6-CDK1shRNA and pU6-SurvivinshRNA-CDK1shRNA were successfully constructed.(2) The result of RT-qPCR proved that:The mRNA expression of Survivin in the pU6-SurvivinshRNA and pU6-SurvivinshRNA-CDK1shRNA interference group can be locked down effectively52.7%and82.4%and the mRNA expression of Survivin in the pU6-CDK1shRNAis not locked down; The mRNA expression of CDK1in the pU6-CDK1shRNA and pU6-SurvivinShRNA-CDK1shRNA interference group can be locked down effectively56.7%and85.0%and the mRNA expression of CDK1in the pU6-SurvivinshRNA is not locked down.(3) The target protein expression levels of Survivin and CDK1were declined in the pU6-SurvivinshRNA, pU6-CDK1shRNA and pU6-SurvivinshRNA-CDK1shRNA interference group.(4)The cancer cell growth rate was decreased by MTT in the pU6-SurvivinShRNA, pU6-CDK1shRNA and pU6-SurvivinshRNA-CDK1shRNA interference group and the rate of inhibition were25.60%,15.62%and32.54%.(5) The rate of apoptosis were increased by flow cytometry in the pU6-SurvivinshRNA, pU6-CDK1shRNA and pU6-SurvivinshRNA-CDK1shRNA interference group and the rate of apoptosis were10.27%,8.00%and14.87%.Conclusions:(1) Those recombinant plasmids of pU6-SurvivinShRNA, pU6-CDK1shRNA and pU6-SurvivinshRNA-CDK1shRNA were successfully constructed.(2) The nasopharyngeal carcinoma cell proliferation activity could be reduced and the rate of apoptosis could be increased by the recombinant vectors of pU6-SurvivinshRNA, pU6-CDK1shRNA and pU6-SurvivinshRNA-CDK1shRNA·(3) Survivin and CDK1can be selected as the target genes for nasopharyngeal carcinoma therapy. The tandem gene Survivin and CDK1shRNAs expression vector can enhances the interference effect of nasopharyngeal carcinoma.