The construction of rat Pik3cb shRNA and its inhibitory effect on the proliferation of smooth muscle cell

Objective:To construct rat Pik3cb(phosphatidylinositol 3-kinase,catalytic,beta polypeptide) shRNA eukaryon plasmid express vector,and test its downregulating effect on Pik3cb mRNA expression for the application of RNA interference in restraining vein graft restenosis. Methods:Two shRNA of rat Pik3cb were design and synthesize according to the sequence of Pik3cb in the Genbank,annealed to form double strands and then cloned into pGenesil-1,and then the sequences were examed. After shRNAs were transfected into rat thoracic VSMC through METAFECTENETM,the transfection rate was identified by EGFP. pAkt-473 protein expression was detected by Western blot,and cell apoptosis were obtained through flow-cytometry. Results:The two sequences of synthesized shRNA named pU6-Pik3cb-shRNA-1 and pU6-Pik3cb-shRNA-2 were exactly the same with the design. The 48 h and 72 h transfection rate were 15.7% and 10.1%,respectively. The pAkt-473 protein expression significantly reduced,while the apoptosis cells were more significantly increased in the shRNA groups than the control groups(P 0.05). The scramble shRNA did not interfere with the VSMC proliferation. Conclusion:Rat Pik3cb shRNA eukaryon express plasmid vectors were constructed successfully and effectively inhibited the proliferation of rat smooth mouse cells.