鸟氨酸脱羧酶基因沉默抑制宫颈癌细胞生长的研究

Inhibition effects of gene silencing of ornithine decarboxylase on cervical cancer cell growth

目的:探讨鸟氨酸脱羧酶(ODC)基因作为宫颈癌治疗靶基因的可行性。方法:采用慢病毒介导的RNA干扰技术,沉默Hela细胞ODC基因,建立ODC稳定沉默的细胞系Hela/ODC,用定量RT-PCR和免疫印迹检测ODC的表达水平。用MTT实验、克隆形成实验、细胞倍增时间和细胞周期分析评价ODC基因沉默对Hela细胞生长的影响。结果:Hela/ODC的ODC mRNA和蛋白质表达水平均明显下降,mRNA水平下调93.4%。Hela/ODC的增殖受到明显抑制,细胞倍增时间从22.3h延长到32.0h,细胞周期分析S期细胞增加,G0/G1期和G2/M期细胞显著减少。结论:慢病毒载体介导RNA干扰能够稳定沉默ODC基因表达,从而成功抑制肿瘤细胞的生长,表明以ODC基因为靶基因进行宫颈癌的基因治疗是可行的。

OBJECTIVE： To verify the feasibility of using ornithine decarboxylase （ODC） as a target gene in cervical cancer therapy. METHODS： A lentiviral system harboring both enhanced green fluorescent protein reporter gene and the ODC short hairpin RNA expression cassette was constructed. Then the stable ODC gene silencing cell lines Hela/ODC were established. Real-time RT-PCR analysis of mRNA and Western blot analysis of proteins were used to evaluate the expression of ODC gene. The growth-inhibiting characteristics were identified by MTT, clone formation, doubling time and cell cycle analysis, respectively. RESULTS： In the cells studied, ODC mRNA level in Hela/ODC was 93.4% decrease of that in Hela, the reduction of ODC mRNA and protein with lentiviral vectors was significant and specific. Proliferation of Hela/ODC cells was obviously retarded. The cells grew much slower with a delayed population doubling time for about 10 h （from 22.3 h to 32.0 h） in vitro. The ability of single-cell colony growth was also signif- icantly inhibited in ODC-down-regulated cells. Moreover, the proportion of Hela/ODC cells in S phase was increased and the proportions in G0/G1 phase and G2/M phase were both decreased of cell cycle. CONCLUSIONS： Lentiviral vector is capable of down-regulating ODC, resulting in impressive anticancer effects. It offers a powerful new strategy for cancer gene therapy and may be useful in the control of cervical cancer.