High FFA-induced Proliferation And Apoptosis In Human Umbilical Vein Endothelial Cell Partly Through Wnt/β-catenin Signal Pathway

Objective: The aim of this project was to study the role of Wnt/β-catenin signal pathway in high FFA-induced proliferation and apoptosis in HUVEC.Methods: The first step was to design and synthesize two pairs of complementary single-strand DNA oligos that targeting two various sites of GSK-3βmRNA. Annealling was used to generate double-strand oligos(ds ligos),and then theds oligos were cloned into pENTR?/U6 to generate the Entry clone named pENTR. Recombination reaction in vitro with the pENTR and pAd/BLOCK-iT?-DEST was used to creat the adenovirus plasmid which contains the RNAi cassette. Then, we transfected the adenovirus plasmids digested with PacI into HEK293A cells to product adenovirus,and then infectedthe HEK293A cells with the crude adenovirus to amply the adenoviral stock. Plaque forming assaywas used to titer the adenoviral stock. The GSK-3βgene silencing effect induced by the RNAi adenovirus was detected by westtern blotanalysis and immunohistochemistry assay, also detectded its effect on the protein level ofβ-catenin.In the last part, we transducted the RNAi adenovirus to inhibit the expression of GSK-3βprotein, then to explore the role of Wnt/ GSK-3β/β-catenin signal pathway on the regulation of proliferation and apoptosis in HUVEC cultured with free fatty acids(FFA), by Brdu assay and Flow CytoMeter.Results: 1.One RNAi adenovirus exppression vectors targeting to one sites of GSK-3βgene 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; 3. The results of western blot showed the expression of GSK-3βprotein in HUVEC could be inhibited efficiently by the RNAi adenovirus, and the inhibition effect would last for more than 6 days after infection; 4. The protein level ofβ-catenin could be increased obviously by the RNAi adenovirus, the increase ofβ-catenin was enhanced along with the increae of MOI and the continuation of infection time; 5. The results of Brdu assay and Flow CytoMeter suggested that interfering the GSK-3βwith the RNAi adenovirus may stimulate the proliferation of HUVEC partly; Apoptosis can be seen obviously in HUVEC exposed to FFAs(0.75 mmol/L) for 72 hours; And apoptois may be reversed when interfering with the RNAi adenovirus. It may be conclued that the RNAi adenovirus specific to GSK-3βmay partly protect HUVEC from apoptosis which induced by FFAs.Conclusion: The GSK-3β-targeting RNAi adenovirus is an important tool which can inhibit the expression of GSK-3βgene efficiently, and increase the protein level ofβ-catenin obviously. It can be used in studying the function of Wnt/β-catenin signal pathway. Up-regulation of Wnt/β-catenin signal pathway can partly reverse FFA-induced apoptosis in HUVEC.