The effect and mechanism of HK-II gene knock-down on chemosensitivity of colon cancer cells

Objective:After knocking down the expression of hexokinase-Ⅱ(HK-Ⅱ) gene with RNA interference(RNAi) technique,chemotherapeutical effect and mechanism on colon cancer LoVo cell were investigated.Methods:In order to inhibit expression of HK-Ⅱ,a shRNA eukaryotic expression vector was constructed and transfected into LoVo cells.The mRNA and protein expression of HK-Ⅱ gene were assessed using semi-quantitive RT-PCR and Western blot analysis respectively.Then IC50 of LoVo cells by 5-FU and L-OHP were also assessed using MTT assay.The activation of caspase-3 was detected by substrate color reaction.Furthermore,the protein expression of thymidylate synthase(TS) was detected by Western blot analysis.Results:The expression of HK-Ⅱ gene was efficiently blocked by RNAi.The expression inhibition rates were 72.1% and 71.2% at mRNA and protein levels of LoVo cells respectively.The IC50 of 5-FU and L-OHP of the cells transfected with plasmids encoding HK-Ⅱ shRNA were(4.70±0.40)μg/ml and(6.27±0.59)μg/ml respectively,which were significantly lower than that of the normal cultured LoVo cells,(11.93±0.15)μg/ml and(9.77±0.60)μg/ml respectively(P0.01).Compared with those of the normal cultured LoVo cells,the activation of caspase-3 were increased significantly and the protein expression of TS was decreased in the the cells transfected with plasmids encoding HK-Ⅱ shRNA plasmids respectively.Conclusion:Selective knock-down of the HK-Ⅱ gene expression can significantly enhance chemotherapeutical effect of LoVo cell via activation of caspase-3 and decreased expression of TS protein.