Effect Of RNA Interference Targeting Of PI3K On The Proliferation Of Vascular Smooth Muscle Cell And The Intimal Hyperplasia

Objective:To construct shRNA eukaryon plasmid expression vector targeting on rat Pik3cb gene,then determine its transfective efficacy.Construct rat jugular vein-to-artery interposition modals,observes the origin of neointimal cells in autologous vein graft;evaluates its downregulative effects on PI3K/Akt/mTOR signal pathway and the antiproliferative effects on smooth muscle cells,which as a results,reduces intimal hyperplasia.Methods:Six of the sense and antisense RNA oligonucleotide strands targeting Pik3cb mRNA were designed and synthesized individually according to the sequence of the rat Pik3cb, then they were annealed to form double strands and then cloned into pGenesil-1,the sequences were examined all corrected as design.Two effective shRNA named pU6-Pik3cb-shRNA-1 and pU6-Pik3cb-shRNA-2 were selected for this study.Modifed rat vein-to-artery interposition modals using the autologous branch of jugular vein were constructed.Jugular vein grafts were treated with 25%Pluronic F-127 only(group A,n=30),plasmid encoding shRNA targeting Pik3cb p110βsubunit(pU6-Pik3cb-shRNA-1 and pU6-Pik3cb-shRNA-2,group B and C,n= 30,respectively),half pU6-Pik3cb-shRNA-1 and pU6-Pik3cb-shRNA-2(group D,n=30), pGenesil-1 scramble shRNA(group E,n=30),or wortmannin(Group F,n=30).Specimens were harvested at 1,3,7,14 and 28 days post surgery to assessed jugular vein graft neointimal hyperplasia.Immunohistochemical staining was performed with primary antibodies a-smooth actin,CD-34,phosphor-Akt(Thr308)and PCNA,as well as TUNEL method to evaluate the antiproliferative effects of shRNA.A subset(n=18,3 for each group)received the same treatment as above were killed on postoperative day 3 for real time polymerase chain reaction and immunoblotting assays to evaluate expression of Pik3cb mRNA and phospho-Akt(Ser308), phosphor-mTOR(Ser2448)and PCNA.Another subset(n=9)received jugular vein grafts treated with a pGenesil-1 scramble shRNA were killed on postoperative days 1,2 and 3(n=3, respectively)to confirm the transfecive efficency of shRNA eukaryon plasmid expression vector.Results.Two shRNA targeting rat Pik3cb were pricked off and synthesized according to the sequence of Pik3cb.The transfective efficiency is about 60%for vascular smooth muscle cell,and more than 90%for endothelial cell.Modified rat vein-to-artery interposition modal was successfully constructed,and the results suggesting vein graft neintimal cells had an intimate association with endothelial cells or circulating progenitor cells by the immunohistochemical usingα-SM-actin and CD-34. The Pik3cb mRNA and its down stream effective molecules phospho-Akt(Thr308), phospho-Akt(Ser473),phospho-mTOR(Ser2448),as well as PCNA were effectively down regulated by pU6-Pik3cb-shRNA and wortmannin,the mRNA level of transfective groups and the positive control group were reduced 70.3%,51.8%,59.9%and 74.8%,respectively(P＜0.05);the phospho-Akt(Thr308)protein level of transfective groups and the positive control group were reduced 88%,80.4%,85.6%and 90.5%,respectively(P＜0.05);the phospho-Akt(Ser473)protein level of transfective groups and the positive control were reduced 77.8%,70.8%,74.7%and 84.1%,respectively(P＜0.05);the phospho-mTOR(Ser2448)protein level of transfective groups and the positive control were decreased 73.4%,62%,70.5%and 80.6%,respectively(P＜0.05);the PCNA protein level of transfective groups and the positive control were reduced 73.4%,62%,70.5%and 80.6%,respectively(P＜0.05).There has no difference between the untreated and pGenesil-1 scramble shRNA groups(P＞0.05).The cell expression of phospho-Akt(Thr308)were significantly reduced between the transfective groups and the positive control in all stages by means of immunohistochemical, especially at 7-14 days;the masculine cells in untreated and pGenesil-1 scramble shRNA were significantly more than the transfective groups(P＜0.05).The percentage of masculine cells in transfective groups and positive control were 0.6%,0.81%,0.73%and 0.53%at 14 days, respectively,the untreated and pGenesil-1 scramble shRNA were 1.72%and 1.66%, respectively;the percentage of masculine cells in transfective groups and positive control were 0.72%,0.78%,0.77%and 1.08%at 28 days,respectively,the untreated and pGenesil-1 scramble shRNA were 2.19%and 2.01%,respectively;it has no difference between the untreated and pGenesil-1 scramble shRNA groups(P＞0.05).The cell expression of PCNA were significantly reduced between the transfective groups and the positive control in all stages by means of immunohistochemical;the masculine cells in untreated and pGenesil-1 scramble shRNA were significantly more than the transfective groups(P＜0.05).The percentage of masculine cells in transfective groups and positive control at 28 days were 0.64%,0.83%,0.72%and 1.24%,respectively,while the untreated and pGenesil-1 scramble shRNA were 2.31%and 2.53%,respectively;there has no difference between the untreated and pGenesil-1 scramble shRNA groups(P＞0.05).The apoptosis cells were significantly increased in the transfective groups and the positive control by TUNEL in all stages.The percentage of masculine cells in transfective groups and positive control at 14 days were 3.13%,2.56%,2.93%and 3.64%,respectively,while,the untreated and pGenesil-1 scramble shRNA were 1.45%and 1.33%,respectively;the percentage of masculine cells in transfective groups and positive control at 28 days were 2.62%,1.93%, 2.28%and 2.45%,respectively,while the untreated and pGenesil-1 scramble shRNA were 0.66%and 0.84%,respectively;the apoptosis cells has no difference between the untreated and pGenesil-1 scramble shRNA groups(P＞0.05).The proliferation of new vascular endothelium cells were significantly higher at 1～2 weeks after operation,the intimal thickness and neointima area of transfective groups were decreased than the untreated and pGenesil-1 scramble shRNA(P＜0.05).The intimal thickness of the untreated and pGenesil-1 scramble shRNA at 14 days were thicken 6.8 and 6.6 fold than 1 days, while the transfective groups were thicken 3.8,4.5 and 4.1 fold merely,respectively.The neointima area of the untreated and pGenesil-1 scramble shRNA at 14 days were increased 5.2 and 5.1 fold than 1 day,the transfective groups were only increased 2.8,4.6 and 3.1 fold, respectively.The intimal thickness and neointima area of positive control were low before 14 days in positive control,but it increased gradually after 14 days because of the weaken drug effect.Conclusions:The shRNA eukaryon express plasmid vectors targeting rat Pik3cb were successfully constructed.Both pU6-Pik3cb-shRNA-1 and pU6-Pik3cb-shRNA-2 were capable of suppressing intimal hyperplasia through down regulating the PI3K-Akt-mTOR pathway by knockdown Pik3cb,then depressing smooth muscle cells proliferation and promoting their apoptosis.This study presented here provides a new gene therapy strategy to prevent vein graft intimal hyperplasia after bypass grafting.