N-Ras基因靶向的shRNA抑制二羟环氧苯并芘诱导的恶变细胞增殖

shRNA Targeting N-Ras Inhibits Proliferation of Transformed Cells Induced by Anti-benzo (a)pyrene-trans-7,8-dihydrodiol-9,10-epoxide(BPDE)

目的用小发卡RNA(small hairpin RNA,shRNA)干扰技术沉默N-Ras基因表达,探讨N-Ras基因表达对二羟环氧苯并芘(BPDE)转化人支气管上皮细胞(16HBE-T)恶性性状的影响。方法将已建立的针对N-Ras基因的shRNA干扰表达质粒载体pGPU6/GFP/Neo-NRas-566转染16HBE-T细胞,经G418筛选,建立稳定沉默N-Ras基因转化细胞系。采用Western blot筛选最佳N-Ras基因沉默细胞系,用流式检测N-Ras沉默细胞周期变化,用软琼脂实验和裸鼠成瘤实验进行细胞表型分析。结果成功培养出稳定沉默N-Ras基因的16HBE-T细胞系,Western blot显示N-Ras蛋白抑制率最高达86%。流式分析发现稳定沉默N-Ras基因表达诱导转化细胞G0/G1期捕获,软琼脂实验和裸鼠成瘤实验发现稳定沉默N-Ras基因降低了体外恶性转化细胞的集落形成率、抑制了体内肿瘤细胞生长。结论N-Ras基因沉默可以降低转化细胞的细胞周期进程和细胞恶性性状表型,提示N-Ras基因在二羟环氧苯并芘诱导细胞恶变过程中具有重要作用。

Objective To explore the effects of N-Ras gene over expression on malignantly transformed human bronchial epithelial cell induced by anti-benzo （a）pyrene-trans-7,8-dihydrodiol -9,10-epoxide （BPDE）（16HBE-T） using small hairpin RNA （shRNA）. Methods The recombinant plasmid pGPU6/GFP/Neo-NRas-566 including N-Ras shRNA was transfected to 16HBE-T cells. Stable N-Ras-silencing clonal 16HBE-T cell lines were selected by G418 and their N-Ras expressions were detected with Western blot to choose the clonal cell line with the highest silencing efficiency. Cell cycle phase and phenotype of stable N-Rassilencing cells were detected by flow cytometry （FCM）, and soft agar growth assay in vitro and tumor growth test in vivo. Results Stable N-Ras-silencing clonal 16HBE-T cell lines were successfully obtained. The results of Western blot assay exhibited that the highest inhibition rate of protein expression was 86%. FCM analysis showed that stable N-Ras-silencing leaded to the G0/G1 arrest in 16HBE-T cells. The result of soft agar growth assay and tumor growth test showed that stable N-Ras-silencing decreased colony formation rate in vitro and inhibited tumor growth in vivo. Conclusion N-Ras silencing slows down cell-cycle progression, and decreased efficiency of transformation and impeded tumor growth. Our findings suggest that over expression of N-Ras gene plays an important role in malignant transformation of cells induced by BPDE.