The Effect Of IGF-â…¡ Targeting On Cell Apoptosis In Hepatocellular Carcinoma

Objective: To Inhibit IGF-Ⅱgene over-express by RNAi technique mediated byliposome in hapatocellular carcinoma, and to further verify effective siRNAstargeting human IGF-Ⅱin hapatocellular carcinoma, which is proved inearlier stage empirical study; inhibit tumor cell proliferation and identifywhether over-express of IGF-Ⅱgene impacts on hapatocellular carcinomacell apoptosis。This paper tries to provide scientific evidence for genetherapy on hapatocellular carcinoma.Methods: Three siRNAs targeting human IGF-Ⅱmarked with FAM, and onesiRNA(siRNA4) without any targeted gene (used for a negetive control)were designed and chemically synthesized. Then the synthetic 40nMsiRNAs were respectively transfected into hepatocellular carcinoma cellsHepG-2 by lipofectamin. The comparison would be made between thesiNRA1～3 group and the siRNA4 group, the control group and thelipofectamin group. The efficiency of transfection was examined underfluorescent microscope; the expression level of IGF-ⅡmRNA was detectedby RT-PCR; After the most effective siRNA sequence screened wererespectively transfected into hepatocellular carcinoma cells SMMC-7721,SMMC-7402,HepG-2, The comparison would be made between thescreened siNRA group and the siRNA4 group, the control group, theuntransfected siNRA group and the lipofectamin group. 48 hours aftersiRNA transfection,The cell growth cycle and active were evaluated byFlow cytometric analysis; and the morphological changes was assessed byelectron microscope.Results: It was observed through the FAM that the transfected efficiency was about65～80%. As compared with the siRNA4 group, the control group andlipofectamin group, The result of RT-PCR showed When 40nM siRNA1～3were transfected into HepG-2 cell respectively by lipofectamine, each hadsome degreed inhibition effect and siRNA3 had the highest inhibition effect among the three siRNA sequences.The siRNA3 inhibition effect was thebest when the concentration was 40nM, and the relative inhibition rate was87.02%, The siRNA4 relative inhibition rate was 2.56%.Therefore the mosteffective siRNA sequence screened was: siRNA3, the sense chain: 5'-GUUCUUCCAAUAUGACACC-3', the anti-sense chain: 5'-GGUGUCAUAUUGG AAGA AC-3'. Results demonstrate the expression of IGF-ⅡmRNAwas suppressed,cell proliferation was suppressed; After the over-expressionof the IGF-Ⅱwas silenced,we detected the growth and cell cyles changes.apoptosis was inedntified in the transfected hapatocellular carcinoma cell byFlow cytometric analysis and electron microscope.Conclusions: The transfected efficiency of chemically sythethized siRNAs by lipofectaminwas quite high. Through the experiments we screened a siRNA and decidedits most effective transfection sequences. The synthetic siRNAs targetingIGF-Ⅱinhibited HepG-2 cells proliferation,which is consistent withpreceding experimental result proved effectively.we demonstrated at celllevel the IGF-Ⅱsilencing can reduce the proliferation of SMMC-7721,SMMC-7402,HepG-2 hepatocellular carcinoma cells and arrest cell cycle atG1 phase,then induce the apoptosis of hepatocellular carcinoma cells.These findings explain that overexpressing of IGF-Ⅱplays a very importantrole in apoptosis and compliment the interface mechanism of IGF-Ⅱinapoptosis. The findings of this study shed light on the possible usage ofRNAi in further investigation of hepatocellular carcinoma, which provides avoluble way for the design of new gene therapy.