The Study Of RNA Interference On CDK2 Gene Expression In Hepatocellular Carcinoma Cells And Assistant Action Of GAPSGF

Purpose:To study the effect of GAPSGF on CDK2 gene in cell cycle regulatory;and its assistant effect on the inhibition action of RNAi in CDK2 gene in SMMC7221 cells.To further study GASPGF anti-tumor function mechanism,and to provide a new technology platform and an advanced method for hepatoma gene therapy.Methods:MTT was used to detect the inhibition of GAPSGF in cell proliferation of SMMC7221 and to search the effective dose for anti-tumor in vitro.The siRNA eukaryotic expression plasmids of CDK2 gene were constructed with gene recombination technology and transfected into SMMC7721 cells with a lipofection method.It was identified that the effect of GAPSGF on CDK2 mRNA and protein expression of SMMC7721 cells with RT-PCR and western blotting methods;and the assistant effect of GAPSGF was also investigated on the inhibition action of RNAi in CDK2 gene.Results:1.GAPAGF could inhibit the cell proliferation in SMMC7721 at the effective dose of 2.5μg·mL^-1,5μg·mL^-1and 10μg·mL^-1.The inhibition ratio were 20.01%,20.47%and 24.44%,respectively.2.Comparing with SMMC7721 group,the CDK2 mRNA expression of 2.5μg·mL^-1,5μg·mL^-1and 10μg·mL^-1GAPSGF groups decrease 45%,40% and 42%,respectively,the CDK2 protein expression of 2.5μg·mL^-1and 10μg·mL^-1GAPSGF groups decreased 53.9%and 18.0%,respectively.3.The siRNA190 and siRNA191 eukaryotic expression plasmids of CDK2 gene have been constructed and transfected successfully into SMMC7721 cells,transfection rate was over 90%.4.It was raised 47.1%by 10μg·mL^-1GAPSGF that siRNA190 inhibit the protein expression of CDK2;it was increased 3%by 2.5μg·mL^-1 GAPSGF that siRNA191 inhibit mRNA expression of CDK2,and the protein expression was raised 13.3%.Conclusion:1.GAPAGF can inhibit cell proliferation of SMMC7721 in vitro.The mechanism may be produced by the down-regulation of CDK2 gene expression.2.siRNA eukaryotic expression plasmids of CDK2 gene can be constructed and transfected into SMMC7721 cells successfully,and it can specifically silence CDK2 gene.It is indicated that CDK2 gene may be a new target for treating hepatoma in the gene therapy.3.GAPSGF has certain assistant action for the RNA interfere expression of CDK2.It is demonstrated that GAPSGF could become a new assistant medicine for treating hepatoma in gene therapy.