HPV18-E6基因SiRNA对喉癌细胞的生长抑制作用

Inhibitory Effects of HPV18-E6 Gene SiRNA on Growth of Hep-2 Cells

目的:探讨小分子干扰RNA(SiRNA)对喉癌细胞系Hep-2细胞中人乳头状瘤病毒HPV18型E6基因mRNA表达的干扰作用。方法:用Ambion公司pSilencer4.1CMV构建针对HPV18-E6基因的SiRNA真核表达载体,以携带HPV18-E6基因的人喉癌Hep-2细胞系为靶细胞,通过阳离子脂质体法转染SiRNA表达载体。RT-PCR分析转染后细胞HPV18-E6基因表达;Westernblot试验观察干涉后HPV18-E6蛋白的表达;流式细胞仪分析细胞增殖周期的改变。结果:成功构建了人HPV18-E6基因的RNA干涉真核表达载体psil-svvE6,并在Hep-2细胞中有效地发挥了对HPV18-E6基因表达的干涉作用。细胞周期阻滞于G0/G1期,并诱导细胞凋亡。结论:HPV18-E6基因在喉癌细胞Hep-2生长中可能起到非常重要的作用,有望成为逆转喉癌细胞永生化的靶点。

Objective： To investigate the interference efficiency of HPV 18-E6 small interfering RNA （SiRNAs） in human laryngeal carcinoma cancer cells line Hep-2. Methods： The HPV18-E6 gene SiRNA eukaryotic expression vector was constructed by inserting SiRNA cDNA into SiRNA insertion position ofpSilencer 4. 1-svv vector. The HPV18-E6 SiRNA vector was transfected into HEP-2 cells line by lipofectamine 2000. The expression level of HPV18E6 mRNA was detected by using real-time quantitative reverse transcription polymerase chain reaction （real-time RT-PCR）, The effect of HPV 18-E6 SiRNA on cell cycle and apoptosis was determined by flow cy- tometry. The expression of HPV18-E6 SiRNA on Hep-2 cells was detecter by Western blot, Results： We successfully designed expression vector of SiRNA specifically targeting to HPV18-E6 mRNA. And the growth of Hep-2 cells was significantly suppressed by HPV18-E6 SiRNA. Before transfection, the expression of HPV18-E6 mRNA level was 1.04 ± 0,28, However, the expression of HPV18-E6 mRNA level was 0.15±0.03 36 hours after transfection. The FCM shows the number of cells in G1 phase was increased after HPV18-E6 SiRNA transfection, which indicates the cell division was blocked in G1 pre-DNA-synthetic gap. Conclusion： The HPV18-E6 gene expression may play a important role in the growth of laryngeal carcinoma cells,and it might be a new target of the treatment for laryngeal carcinoma,