Construction and expression of pSUPER-Rac1-siRNA plasmid and its inhibitive effects on HepG2 cells proliferation / 解放军医学杂志

ABSTRACT

Objective pSUPER was used as vector to construct the siRNA targeting the gene of Rac1 to explore its inhibitive effects on Rac1 expression in hepatoma cell HepG2,and observe the proliferation of HepG2.Methods The oligonucleotide possessing 64 bases targeting the hairpin-like RNA of Rac1 gene was chemically synthesized,and inserted into the downstream of H1 promoter of linearized pSUPER vector after annealing.The vector pSUPER-Rac1-siRNA was constructed by sequencing and restrictive digestion of the recombined vector,and then transfected to hepatome cell line HepG2.pSUPER-Control-siRNA was used as a negative contro1.Western blotting was performed to monitor the Rac1 gene in plasmid-transfected cells,and the proliferation of extracorporeal cells after Rac1 expression was detected by MTT assay.Results The siRNA expression vector pSUPER-Rac1-siRNA targeting Rac1 gene was successfully constructed.The expression of Rac1 protein in HepG2 cells was down-regulated effectively by the transfection of pSUPER-Rac1-siRNA with an inhibition rate of 82%.It was revealed by MTT detection that the proliferation rate of cells declined remarkably when the expression of Rac1 protein was inhibited efficiently.Conclusions The hairpin-like RNA expression vector pSUPER-Rac1-siRNA targeting Rac1 has been constructed successfully,which may effectually and specifically down-regulate the gene expression of Rac1 in HepG2 cells.The silencing of Rac1 protein may remarkably inhibit the proliferation of hepatoma cells,implying that the Rac1 may play an important role in the in vitro growth of hepatoma cells.