The Effect Of NF-κB Pathway On Proliferation And Apoptosis Of Human Umbilical Vein Endothelial Cells Induced By High Glucose

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 in human umbilical vein endothelial cell (HUVEC). Secondly, we wanted to verify whether the periodic vs. continuous exposure to high glucose may have different effects on HUVEC. Finally, we also wanted to explore the role of NF-κB pathway on the regulation of proliferation and apoptosis of HUVEC induced by high glucose using the RNAi adenovirus vector.Methods: The first step was to design and synthesize a pair of complementary single-strand DNA oligos which targeting 1566 site 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 create 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 knockdown effect induced by the RNAi adenovirus was detected by western blot analysis in HUVEC. 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 HUVEC induced by high glucose. We measured proliferation of HUVEC in the indicated conditions by MTT and BrdU incorporation assay, and tested apoptosis by flow cytometry and TUNEL assay.Results: 1) The RNAi adenovirus exppression vectors targeting to p65 gene were successfully constructed; 2) The results of western blot showed the expression of p65 protein in HUVEC cells could be inhibited efficiently by NF-κB/p65-targeting RNAi adenovirus, and the optimal MOI should be 25 to 50; 3) The decrease of p65 expression in HUVEC cells was first observed 48 hours after infection with RNAi adenovirus, and the decrease of p65 expression was enhanced along with the continuation of infection time, and at least lasted for 6 days; 4) Compared with stable high glucose, intermittent high glucose enhanced apoptosis and proliferation inhibition of HUVEC; 5) P65 protein of nuclear extracts was significantly increased in HG culture in the control group, but only slightly increased in the NF-κB-specific knockdown group, which maintained at basal state; 6) In BrdU assay, knockdown of NF-κB p65 restored (P<0.01)the proliferative damage of HUVEC in high glucose (30.5mmol/L) even to the control level (5.5mmol/L) (P>0.05). It also restored the apoptosis of HUVEC in high glucose to the control level in flow cytometry(P>0.05), but only partially protected HUVEC from apoptosis induced by high glucose in TUNEL assay(P<0.01).Conclusion: The NF-κB p65-targeting RNAi adenovirus is an important tool which can efficiently inhibit the expression of p65 gene in HUVEC. High glucose mediated proliferation inhibition and apoptosis in HUVEC, and it was amplified in the fluctuating glucose condition. Knockdown of NF-κB p65 protected HUVEC from proliferation inhibition and apoptosis by preventing high glucose-induced NF-κB nuclear translocation.