The Effects Of SiRNA-cyclinE On Proteins In Hepatocellular Carcinoma HepG2 Cell Line

Objective To investigate the effects of small interference RNA(siRNA)of cyclin E gene on the cell growth and cell cycle of Hep G2 cells.By using 2 - DE and MALDI - TOF MS to identify the proteins with different expressing level between before and after transfecting with pU6 - cyclinE - shRNA on Hep G2 cells,the object of this part is to screen key molecules tumorigenesis and tumor growth of hepatocellular carcinoma.Methods pU6-cyclinE-shR-NA expressive vectors was established by Pgenesil-1 plasmid and cyclinE short hairpin RNA (shRNA)synthesized in vitro.After stable transfection into Hep G2 cells by LipofectamineTM 2000,the expression of cyclinE was analyzed by RT -PCR,cell growth was measured with MTT assay,cell cycle of the transfected cells was examined by flow cytometry.Proteins extracted form the Hep G2 cells which was ransfected with pU6 - cyclinE - shRNA and the HepG2 cells in G1 phase were separated by two - dimensional gel electrophoresis(2-DE). The stained gels were then scanned using Proxpress2D scanner and analyzed with Proxpress2D software.Selected differential protein spots were excised from preptides were analyzed by MAL-DI-TOF MS.Data were then searched with the search engine MASCOT(Matrix Science).Results After Hep G2 ceils were transfected with pU6 - cyclinE - shRNA,the expression of cyclinE mRNA was suppressed with inhibition rate of 70%;ceils in G1 phase increased after transfecting with pU6 - cyclinE - shRNA,but cells in S and G2/M phases decreased.Average 435±51(n=3)and 376±44(n=3)spots had been detected in protein profile of Hep G2 cells in G1 phase and transfected with siRNA -cyclinE on Hep G2 cells.The protein spots exhibiting statistically alternations between the two groups through computerized image analysis 15 spots showing 6 or more than 6 fold difference and showing more than 50%frequency in patients were screened.There are 8 proteins including keratin 19,vimentin,neuropolypeptide h3 and so on.Conclusion pU6 -cyclinE- shRNA could suppress the cyclinE expression of HepG2 cells,block more cell arrest in GO to G1 phase,inhibit proliferation.The protein profile of two groups displayed obviously difference,and various distinct different proteins may join together in HCC metastasis and tumor growth.