Title: THE EFFECTS OF SIRNA-CYCLIN E ON CELL PROLIFERATION AND PROTEOME IN HEPATOCELLULAR CARCINOMA HEPG2 CELL LINE
Abstract: Objective:To investigate the effects of small interference RNA(siRNA) of cyclin E gene on the cell growth and cell cycle of HepG2 cells.By using 2-DE and MALDI-TOF MS to identify the proteins with different expressing level before and after transfecting with pU6-cyclinE-shRNA on HepG2 cells.Methods:pU6-cyclinE-shRNA expressive vectors was established by Pgenesil-1 plasmid and cyclin E short hairpin RNA(shRNA) synthesized in vitro.After stable transfection into HepG2 cells by LipofectamineTM 2000,the expression of cyclin E 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 from the HepG2 cells which was transfected with pU6-cyclinE-shRNA and 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 MALDI-TOF MS.Data were then searched with the search engine MASCOT(Matrix Science).Results:After HepG2 cells were transfected with pU6-cyclinE-shRNA,the expression of cyclin E mRNA was suppressed with inhibition rate;cells in G0/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 HepG2 cells in G1 phase and transfected with siRNA-cyclinE on HepG2 cells.The protein spots exhibiting statistically alternations between the two groups through computerized image analysis 20 spots showing 6 or more than 6 fold difference and showing more than 50% frequency were screened.There are 8 proteins including keratin 19,vimentin,neuropolypeptide h3 and so on.Conclusion:pU6-cyclinE-shRNA could suppress the cyclin E expression of HepG2 cells,block more cell arrest in G0 to G1 phase,inhibit proliferation.The protein profile of two groups displayed obviously difference,and selected differential 8 protein spots that is related with DNA modification,cell proliferation,differentiation and metastasis.
Publication Year: 2008
Publication Date: 2008-01-01
Language: en
Type: article
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