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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 of small interfering RNA (siRNA) on E6 mRNA expression in human papillomavirus type HPV18 in Hep-2 cell line. Methods: The SiRNA eukaryotic expression vector targeting HPV18-E6 gene was constructed by using Ambion pSilencer4.1CMV. The human laryngeal carcinoma Hep-2 cell line carrying HPV18-E6 gene was used as the target cell. The expression of SiRNA was determined by cationic liposome method Vector. The expression of HPV18-E6 gene was detected by RT-PCR, the expression of HPV18-E6 protein was detected by Western blot, and the cell cycle was analyzed by flow cytometry. Results: The RNA interference eukaryotic expression vector psil-svvE6 of human HPV18-E6 gene was successfully constructed and the interference effect on HPV18-E6 gene expression was effectively exerted in Hep-2 cells. Cell cycle arrests in G0 / G1 phase and induces apoptosis. Conclusion: The HPV18-E6 gene may play an important role in the growth of Hep-2 laryngeal carcinoma cells, which is expected to be a target of reversing the immortalization of laryngeal carcinoma cells.