Effect of RNA interference targeting mammalian target of rapamycin on the proliferation of vascular smooth muscle cell and the intimal hyperplasia

Abstract

To construct RNA interference (RNAi) vector of mammalian target of rapamycin (mTOR) and study the effect on the proliferation of vascular smooth muscle cell (VSMC) and the intimal hyperplasia (IH). The sequence of short hairpin RNA (shRNA) targeting mTOR was designed and synthesized by chemical method and inserted into a retroviral vector pLXIN, then the vector was packaged in PT67 cells. The efficiency of inhibition was verified by Northern blot and Western blot after transfection to VSMC. The changes of p70s 6k and 4E-BP1 were also determined. The proliferation activity of VSMC was determined by flowcytometry and MTT. Autogenous vein graft model were established in 54 rats. Three groups were divided, including gene therapy group, control group and vein graft group. The grafted veins were harvested 7 days, 14 days and 28 days after transplanting respectively. The expression of mTOR was determined by immunohistochemistry stain and Western blot. IH was also observed. The apoptotic VSMC was determined by TUNEL. The shRNA fragments was inserted into pLXIN vector. The shRNA vector was successfully constructed. Additionally, the vector targeting mTOR gene could efficiently decrease the mRNA and protein expression of mTOR and p70s 6k in VSMC, while the expression of 4E-BP1 increased significantly. The cell cycle of VSMC was stunned in phase G(0)/G(1). The protein expression of mTOR in vein graft was inhibited by transfection of pLXIN-shRNA targeting mTOR (P < 0.01) and the IH was also inhibited (P < 0.01), but the apoptosis of VSMC increased significantly (P < 0.01). The mTOR RNAi retroviral vector has been constructed successfully, which can significantly inhibit the proliferation of VSMC and the IH, and promote apoptosis after vein grafting.