ABSTRACT
Objective To investigate the effects of silenced Racl on invasion and migration in LOVo cells.Methods The expression of Racl mRNA and protein in colorectal cancer cells(including LoVo SW480.SW620.SW1116,HT29)were detected by RT-PCR and Western blot,respectively.The changes of cytoskeleton were observed in LoVO cells after transfected with Racl-shRNA.then invasion and migration were recorded respectively in LoVo cells after transfected with Racl-N17 and Racl-L61.Results Racl mRNA and protein were overexpressed in all selected colorectal cancer cells.Deletion of Racl decreased the cross-linked actin network and pseudopodia,and inhibited the invasion and migration in LoVo cells.The migration experiment showed that the migrated cells were higher in Racl-shRNA[(75±5)cells].Racl-N17 [(93±5)cells]and Racl-L61[(267±7)cells]groups compared with control group[(214±8)cells,P＜0.01,＜O.01 and＜0.05,resprectively].The invasion experimental study revealed that the migrated cells were higher in Racl-shRNA[(35±5)cells],Racl-N17[(42±5)cells]and Racl-L61[(86±7)cells] groups compared with control group[(73±6)cells,P＜0.01,＜0.01 and＜0.05,resprectively].Condusion Deletoin of Racl can inhibit the invasion and migration in LDVo cells.
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.