Construction Of NF-κB-targeting RNAi Adenovirus Vector And The Effect Of NF-κB Pathway On Proliferation And Apoptosis Of Vascular Endothelial Cells

Objective: The aim of this project was to construct RNAi combinant adenoviral expressive vector specific to p65 gene and to observe its gene knockdown effect on the expression of p65.Secondly, we also wanted to explore the role of NF-κB pathway on the regulation of proliferation and apoptosis of vascular endothelial cells using the RNAi adenovirus vector.Methods: The first step was to design and synthesize three pairs of complementary single-strand DNA oligos which targeting three various sites of p65 mRNA. Annealling was used to generate double-strand oligos (ds oligos), and then the ds oligos were cloned into pENTR?/U6, the entry vector, to generate the Entry clone named pENTR. Recombination reaction in vitro with the pENTR and pAd/BLOCK-iT?-DEST, the adenovirus backbone vector, was used to creat the adenovirus plasmid which contains the RNAi cassette. Then, we transfected the adenovirus plasmid digested with PacI into HEK293A cells to product adenovirus, and infected the HEK293A cells with the crude adenovirus to amply the adenoviral stock. Plaque forming assay was used to titer the adenoviral stock .The p65 gene kockdown effect induced by the RNAi adenovirus was detected by western blot analysis.In the last part, we transducted the RNAi adenovirus to inhibit the expression of p65 protein and to explore the role of NF-κB pathway on the regulation of proliferation and apoptosis of ECV304 cells, the vascular endothelial cell strain, with MTT assay and Flow CytoMeter.Results: 1) Three RNAi adenovirus exppression vectors targeting to three various sites of p65 gene were produced, and the sequence and correct site of ds oligos inserted were confirmed by PCR and sequencing assay; 2) The adenovirus were packaged and amplified in HEK293A cells with high titer. The results of plaque assay showed that the titer of second filial generation adenovirus range from 3.0×109pfu to 2.5×1010pfu; 3) The results of western blot showed the expression of p65 protein in ECV304 cells could be inhibited efficiently by there various RNAi adenovirus, and the optimal MOI should be 15 to 25; 4) The decrease of p65 exppression in ECV304 cells was first observed 48 hours after infection with RNAi adenovirus, and the decrease of p65 exppression was enhanced along with the continuation of infection time; 5) The results of MTT assay suggested that blocking the NF-κB pathway with the RNAi adenovirus could decrease the proliferation of ECV304 cells efficiently, but with slight affect on the apoptosis of the cells.Conclusion: The NF-κB/p65-targeting RNAi adenovirus is an important tool that can inhibit the expression of p65 gene efficiently, used in studying the function of NF-κB pathway. The NF-κB pathway plays an important role in the regulation of proliferation of ECV304 cells.