Biological Function Of NF-κB Silencing In Nasopharyngeal Carcinoma5-8F Cell

Objective: To clone the eukaryotic expression vectors of small interferingRNA (siRNA) against NF-κB p65gene and to evaluate their silencing effects inthe nasopharyngeal carcinoma5-8F cells,it may be provide basis for the study ofeffects of NF-κ B in nasopharyngeal carcinoma.Methods:(1)According to NF-κB p65gene sequence of GenBank, we usedRNAi designer software to design the siRNA specific target sequence (siNF-κB)and the synthetic negative sequence. Justice chain interference sequence synthesisand antisense strand annealing synthetic double stranded DNA fragments werecomposed. The annealed DNA fragments were subcloned into pGenesil-1.2plasmid expression vector to construct the specific pGenesil-1.2-NF-κB andgeneral negative pGenesil-1.2-HK expression vector.(2)Then we transformed therecombinant plasmid expression vectors into JM109strains for ampilificationcultured and used the method of enzyme digestion and sequencing to analyze andidentify the recombinant plasmid expression vectors.(3)The recombinant plasmidexpression vectors(pGenesil-1.2-NF-κB、pGenesil-1.2-HK) were transfected to thenasopharyngeal carcinoma5-8F cells with lipofectamine, then observed theirexpressions in eukaryotic cells by fluorescence microscope.(4)The experiment was set the control group(non transfection vector in5-8F cells), negative control group(to express pGenesil-1.2-HK plasmid5-8F cells), experimental group(pGenesil-1.2-NF-κ B expression vector into5-8F cells).The expression ofNF-κB p65protein was detected by Western blotting methods after transfection48h. Analysis the affect of NF-κB gene silencing to the proliferation inhibitionand the cell cycle by MTT and flow cytometry in5-8F cells.Results:(1) Restriction enzyme digestion and sequencing results showed thatthe NF-κB p65specific interfering DNA fragments inserted siRNA plasmidexpression vector were in the right sequence and direction, and was successfullyconstructed the pGenesil-1.2-NF-κB recombinant plasmid expressionvectors.(2)Compared with the control group and the negative control group,the expression of NF-κB p65protein in5-8F cells transfected pGenesil-1.2-NF-κB recombinant plasmid decreased significantly. The control group comparedwith the negative control group, the protein expression of NF-κ B p65showed nodifferent. This suggests that the NF-κB p65siRNA could inhibit the NF-κB p65protein expression.(3) The proliferation inhibition rate in recombinant plasmidexpression vector pGenesil-1.2-NF-κB transfected5-8F cells was (58.43±1.24)%, compared the inhibition rate with blank control group (0) and negativecontrol group (17.89±4.13)%, the difference was statistically significant (P<0.05).The number of cells in S phase were (42.27±0.39)%,compared the with the blankcontrol group (11.3±0.79)%and the negative control group (34.88±0.85)%, thedifference was significant (P<0.05). The cells were arrested in S phase.Conclusion: The siRNA plasmid expression vector against NF-κB p65gene weresuccessfully designed and constructed. After transfected the humannasopharyngeal carcinoma5-8F cells, the NF-κB siRNA plasmid expression vector can effectively inhibit the expression of NF-κB p65in nasopharyngealcarcinoma5-8F cells. The cell proliferation was obviously inhibited and the cellcycle was arrested in S phase. The NF-κB may plays an important role in themalignant proliferation of human nasopharyngeal carcinoma.