CyclinE干扰RNA对肝癌HepG2细胞增殖和蛋白质组的影响

THE EFFECTS OF SIRNA-CYCLIN E ON CELL PROLIFERATION AND PROTEOME IN HEPATOCELLULAR CARCINOMA HEPG2 CELL LINE

目的:研究siRNA对HepG2细胞株cyclinE表达的抑制及细胞生长的影响,并用双向凝胶电泳和质谱技术分析鉴定转染前后细胞内差异表达的蛋白质。方法:构建质粒表达载体pU6-cyclin E-shRNA,稳定转染肝癌HepG2细胞株,获得HepG2-cyclin EshRNA细胞系,RT-PCR检测cyclinE mRNA表达,MTT测定细胞的增殖活性,流式细胞仪检测细胞周期。裂解、抽提HepG2-cyclin EshRNA细胞和G1期肝癌HepG2细胞全蛋白,用双向电泳技术进行分离后,用Proxpress2D分析软件对两组细胞全蛋白质表达谱进行差异分析,并应用MALD I-TOF-MS和数据库搜索鉴定差异显著的蛋白点。结果:HepG2-cyclin E-shRNA细胞与转染空质粒HepG2细胞和未转染的HepG2细胞相比,生长速度减慢,出现G0/G1期细胞增多,G2/M和S期细胞减少。双向电泳后软件分析G1期肝癌HepG2细胞株平均检测到435±51(n=3)个蛋白点,HepG2-cyclinEshRNA细胞376±44(n=3)个,匹配分析,筛选出两组间差异6倍及以上且出现频率大于等于50%的蛋白点20个。应用质谱鉴定出角蛋白19、波形蛋白、神经多肽h3等8个G1期HepG2细胞高表达蛋白点。结论:在HepG2细胞株中,导入cyclinE干扰RNA,可有效抑制cyclinE的表达,进而使细胞增殖减缓,逆转其恶性表型。两组细胞蛋白质谱有明显差异,鉴定出G1期HepG2细胞高表达蛋白点8个,这些蛋白的高表达引起肿瘤的基因修饰异常、分化异常、增殖紊乱,肿瘤侵袭转移。

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 - shR- NA on HepG2 cells. Methods：pU6- eyelinE -shRNA expressive vectors was established by Pgenesil - 1 plasmid and cyclin E short hairpin RNA （shRNA） synthesized in vitro. After stable transfeetion into HepG2 cells by TM Lipofectamlne^TM 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 cytome- try. 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; ceils 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 ceils 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 ceils, block more cell arrest in Go 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 modifica- tion, cell proliferation, differentiation and metastasis.