Modification Of PI3K Signaling Pathway Under The Driving Of KDR Promoter/Enhancer Influences The Proliferation And Apoptosis Of Rat Vascular Endothelial Cell

Modification of PI3K Signaling Pathway under the Driving of KDR Promoter/Enhancer Influences the Proliferation and Apoptosis of Rat Vascular Endothelial CellObjective:The vascular endothelial cell is one of the important components of the vessel wall. Damage of VEC promotes the restenosis after coronary bypass grafting. The purpose of this study is to construct the KDR specific promoter/enhancer eukaryon expression vector for RNA interference of phosphatidylinositol3-kinase, catalytic, beta polypeptide (Pik3cb) gene in rat vascular endothelial cell (VEC) and evaluate the effects on the proliferation and apoptosis of VEC.Methods:Both the specific promoter/enhancer eukaryon expression vector KDRe/p-pGenesil-10-Pik3cb-shRNA and non-specific eukaryon expression vector CMV-pGenesil-10-Pik3cb-shRNA were designed and constructed according to PI3K p110β subunit gene Pik3cb mRNA sequence. The different shRNA were transfected to vascular endothelial cell by LipofectaminTM2000in different groups, respectively. The samples were divided into5groups:Group A:control group:normal VEC without any treatment; Group B: VEC transfected with KDRe/p-pGenesil-10-Pik3cb-shRNA; Group C:VEC transfected with CMV-pGenesil-10-Pik3cb-shRNA; Group D:VEC transfected with empty plasmid; Group E: VEC treated with wortmannin. At24h、48h and72h after treatments, the expression level of Pik3cb mRNA was measured by real time RT-PCR, and the proliferation and apoptosis levels of VEC were analyzed by CCK-8and flow cytometry, respectively.Results:At24h,48h and72h after treatments:the transfection efficiency were (35.2±4.6)%,(25.7±1.8)%and (16.7±1.6)%, respectively; the real time RT-PCR illustrated the relative expression of Pik3cb mRNA in Group B were (54.82±2.77)%,(50.54±3.98)%and (35.47±4.83), significantly lower than Group A and D (P<0.05), there had no difference between Group B and Group E; the CCK-8analysis revealed the cell inhibition rate of Group B were (21.98±2.25)%,(24.32±3.04) and (26.38±5.06)%, which significantly higher than that of Group A and D (P<0.05), the rate of Group E were (22.21±3.89)%,(26.20±4.86) and (23.07±1.36)%, the difference between Group B and Group E had no statistical significance; the results of flow cytometry showed the apoptosis rates were (9.85±1.34)%,(31.00±7.35)%and (44.50±8.27)%in Group B, and were (0.95±0.50)%,(2.15±0.50)%and (1.80±0.71)%in Group A, Group B were significantly higher than Group A (P<0.05). Conclusion:The transfection of specific promoter/enhancer eukaryon expression vector KDRe/p-pGenesil-10-Pik3cb-shRNA in rat vascular endothelial cell effectively downregulated the expression of Pik3cb mRNA, inhibited the proliferation and promoted the apoptosis of VEC specifically, this can promote early thrombosis and accelerate the proliferation of neointimal. The major mechanism may be inhibition of P13K-Akt-mTOR signal pathway.