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

Purpose:To select the effective siRNA eukaryotic expression plasmids (pGPU6/GFP/Neo)of CDK2 gene,then to transfect in the human hepatocellular carcinoma cells Iine(SMMC7721).For studying its effect on the CyclinE gene in the cell cycle and the assistant action of GAPSGF [Polysaccharides component GF extracted by Ganoderma applanatum(Per. ex Gray)].Methods:The siRNAeukaryotic expression plasmids ofCDK2 gene were selected and transfected into SMMC7721 cells with a lipofection method. Real-time PCR method was also utilized to detect the mRNA expression of CyclinE.MTT was used to detect the inhibition of GAPSGF on SMMC7221 and to search the effective dose GAPSGF for anti-tumor in vitro.It was studied that the the effect of GAPSGF on CyclinE mRNA expression of SMMC7721 cell with Real-time PCR method.The assistant effect of GAPSGF was investigated on the inhibition action of CyclinE gene after RNA interference.Results:1.GAPSGF could obviously inhibit the cell proliferation in SMMC7721 at the effective doses of 2.5μg·mL^-1,5μg·mL^-1and 10μg·mL^-1. The inhibition ratio were 20.01%,20.47%and 24.44%.2.GAPSGF could raised the mRNA expression of CyclinE at the effective doses of 2.5μg·mL^-1and 10μg·mL^-1.The mRNA expression of CyclinE are reduced 74%and 68%by 2.5μg·mL^-1and 10μg·mL^-1GAPSGF, respectively.3.The inhibition of RNAi in CDK2 could influence the CyclinE gene expression in cell cycle,The mRNA expression of CyclinE gene are reduced 20%and 36%by siRNA segment 190and siRNA segment 191, respectively.4.Comparing with application of siRNA segment 190 alone,the combining use of GAPSGF and siRNA segment 190 can reduce the mRNA expression of CyclinE.The mRNA expression of CyclinE is reduced 5%by 10μg·mL-1 GAPSGF.Comparing with application of siRNA segment 191 alone,the combining use of GAPSGF and siRNA segment 191 can not reduce the mRNA expression of CyclinE.Conclusion:1.Comparing with the normal hepatocyte,the mRNA expression of CyclinE gene in the hepatoma carcinoma cell have obviously up-regulation.2.GAPSGF can obviously inhibit cell proliferation of SMMC7721 in vitro.The mechanism may be produced by the down-regulation of CyclinE gene expression.3.The mRNA expression of CyclinE gene have obviously relevance on CDK2 gene.Silencing specifically the CDK2 gene by RNAi technique can reduce the mRNA expression of CyclinE gene.4.GAPSGF has certain assistant action for the mRNA expression of CyclinE gene after RNA interference.It is demonstrated that GAPSGF could become a new assistant medicine for treating hepatoma in gene therapy.