siRNA表达载体介导的RNA干扰对肝癌细胞周期蛋白E1表达抑制研究

Study on inhibition of expression of cyclin E1 in hepatocellular carcinoma via RNA interference mediated by siRNA expressing vector

目的探讨经载体介导的RNA干扰对肝癌细胞周期蛋白E1表达抑制效率。方法针对周期蛋白E1基因设计并合成特定的RNA干扰模板片段,连接到pSilencer1.0-U6载体上构建siRNA真核表达载体;脂质体转染法将上述重组质粒转入BEL-7402肝癌细胞株。RT-PCR分析周期蛋白E1表达抑制率;流式细胞术测细胞周期S期和凋亡细胞比率;MTT比色法测定活细胞数并绘制细胞生长曲线。结果重组载体介导肝癌细胞BEL-7402周期蛋白E1抑制率达62%;转染重组质粒32h测得S期细胞为19%。空载体转染对照为27%;转染重组质粒96h流式细胞术测得凋亡细胞为13.1%,高于空载体对照的6.6%;细胞生长曲线作图表明,重组载体转染组细胞生长明显减缓。结论肝癌细胞周期蛋白E1的表达可被载体介导诱发的RNA干扰成功抑制,进而导致细胞生长受抑并诱导凋亡;本研究为探索肝癌的RNAi治疗提供了初步的实验依据。

Objective To explore the inhibition of expression of cyclin E1 in hepatocellular carcinoma via RNA interference mediated by siRNA expressing vector. Methods A RNA interference DNA template targeting cyclin E1 gene was designed and synthesized. By ligation, the fragment was inserted into pSileucer1. 0-U6 to construct the recombinant plasmid pSileucer1. 0-U6-cyclin El. The identified recombinant plasmid was introduced into BEL-7402 cells with lipofactamine. The inhibition of cyclin E1 expression was analyzed by RT-PCR and the ratios of S phase and of apoptotic cell were assessed by flow cytometric detection. The viable cells were counted by MTT colorimetry and a cell growth curve was drew to analyze the inhibition of cell proliferation. Results The inhibition of expression of cyclin E1 in BEL-7402 was up to 62% when mediated by pSileucer1. 0-U6-cyclin El. Thirty-two hours after transfection with recombinant or vector control, the ratio of S phase cells was 19% and 27%, respectively.The apoptotic ratio at 96 hours after recombinant or vector transfection was 13.1% and 6.6%. The cell growth curves indicated that the proliferation of cell transfected with recombinant plasmid was inhibited significantly when compared with control. Conclusion The expression of cyclin E1 could be inhibited successfully by RNA interference induced by siRNA expressing vector.This in turn inhibits the cell growth and induces apoptosis. Our study provided a preliminary resuls in searching of a RNAi therapy of hepatocellular carcinoma.