The Research On The Expression Of DEPDC7 Gene In Hepatocarcinoma And Of Its Biological Effects

Objective:1.Research the variable expression of DEPDC7 gene in hepatoma carcinoma cells and the localization of DEPDC7 protein.2.Investigate the affection to the proliferation,apoptosis and infestation of hepatoma carcinoma cells by DEPDC7 gene.Method:1.Conventionally culture hepatic cell HL-7702, hepatocarcinoma cells HepG2, SMMC7721 and Huh7.2.Use RT-PCR method to detect the expression difference of DEPDC7 mRNA transcription in hepatocarcinoma cells. Select GAPDH as intemalcontrol, 1.2% aga- rose gel electrophoresis and use Gene Tool software taking semi-quantitative analysis.3.Use Western Blot method to detect the expression difference of DEPDC7 gene in hepatocarcinoma cells at the protein level,and use Gene Tool software taking semi- quantitative analysis.4.Construct recombinant vector pEGFP-C1-DEPDC7 which expresses fused protein DEPDC7 and EGFP. Transfect the hepatocarcinoma HepG2, SMMC-7721, then use laser confocal scanning microscope to localize the DEPDC7 protein.5.Construct recombinant vector pIRES₂-EGFP-DEPDC7, which expresses proteins DEPDC7 and EGFP separately. Transfect the hepatocarcinoma cells HepG2 , SMMC-7721 and Huh7. Use MTT to detect the proliferation before and after transfection, and draw the cell growth curves.6.Transfect hepatocarcinoma cells with pIRES₂-EGFP-DEPDC7 vector ,then use the flow cytometer and DNA ladder to detect the proliferation rate and the apop- tosis rate after transfection.7.Transfect hepatocarcinoma cells with pIRES₂-EGFP-DEPDC7 vector, then use the transwell assay to research the effect of DEPDC7 on invasion. 8.Use the RT-PCR method to detect the DEPDC7 influence on the expression of related invasion gene.Results:1.Through RT-PCR semi-quantitative analysis, the mRNA expression level of DEPDC7 gene in tumor cell significantly reduced,the differences between three groups of hepatocarcinoma cells are statistically significant (P<0.05).2.By Western Blot semi-quantitative analysis, the differences of protein expression levels between hepatic cell HL-7702 and three lines of hepatocarcinoma cells are statistically significant (P<0.05). And the differences among three hepatocarcinoma cells are significantly different. the protein express level of Huh7 is the lowest, has statistical significance (P<0.05).3.Transfect the Hepatocarcinoma cells with the pEGFP-C1-DEPDC7 vector, it is found that the DEPDC7 protein is mainly located on the membrane and a little is found in cytoplasmic.4.As the normal growth curve shows, hepatocarcinoma cells grow faster and be in logarithmic phase within 72hs, and the cells grow slower afer transfection of pIRES₂-EGFP-DEPDC7 vector .5.Overexpression of DEPDC7 gene can suppress the cell proliferation rate but can not cause apoptosis. 6.Overexpression of the DEPDC7 gene can suppress the invasion of tumor cells and influence the expression of gene matrix metalloproteinase.Conclusion:1.The DEPDC7 gene express high in hepatic cell, low in hepatocarcinoma cells. The DEPDC7 protein is mainly located on the cell membrane,some appear in cytoplasm.2.Upgrading the expression of the DEPDC7 gene can not cause cell apoptosis, but can suppress the rates of cell proliferation and invasion, the expression of MMP-9 decreased,and TIMP-1,TIMP-3 raised.