Construction Of Stathmin SiRNA Plasmid Expression Vector And The Effcts Of Apoptosis And Cell Cycle In Npc 5-8F Cell By Silencing Expression Of Stathmin Gene

Objective :Constructing stathmin specific SiRNA plasmid expression vector and investigating the effects of expression of stathmin by SiRNA plasmid expression vector,and also investigating the effects of apoptosis and cell cycle in nasopharyngeal carcinoma 5-8F cell line by silencing stathmin gene.Methods: Synthesize stathmin specific interfering expression DNA fragments, obtain doubled DNA fragment by annealing and combine the fragment with linear plasmid pGenesil1.1. The recombinant plasmid vectors were transferred and screened in Bacillus coli JM109. Then, the recombinant vectors were amplified and identified by di-enzyme digestion and DNA sequencing. There are 3groups of cells including 1)Control group (Liposome group); 2) Negative control group (transfecte pGenesil1.1-HK);3)STM group (transfecte pGenesil1.1-STM). The identified plasmid vector was transfected into nasopharyngeal carcinoma 5-8F cell line, and then extract total RNA and total protein in nasopharyngeal carcinoma cell after 48 hour, RT-PCR and Western blot were adopted to analyze the expression of stathmin at the mRNA and protein level, moreover collect nasopharyngeal carcinoma cell after 48h, then detect the cell cycle and apoptosis by flow cytometry.Results: Di-enzyme digestion analysis and DNA sequencing showed that the basic sequence and direction of stathmin specific DNA fragment, which was inserted into SiRNA plasmid expression, was correct and reduced the expression of stathmin effectively in nasopharyngeal carcinoma cell line 5-8F. Flow cytometry test show that in the STM group ,compared with Liposome group and Negative control group, the proportiona of G2 phase (21.27±0.76%)and apoptosis %index(9.20±1.03%)both increased significantly(P<0.05).Conclusion:we obtained the stathmin specific recombinant siRNA plasmid vector ,and the constructed stathmin specific recombinant siRNA plasmid expression vector could silence the expression of stathmin ,induce cell apoptosis and arrest the cells in G2 phase, this provides basic theory for gene therapy of the nasopharyngeal carcinoma.