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目的:构建针对HPV16 E6基因的RNA干扰表达载体,并研究其对宫颈癌HPV16 E6基因的抑制作用。方法:针对HPV16 E6基因序列设计shRNA(short hairpin RNAs,shRNAs)片断,构建针对HPV16 E6基因的RNA干扰(RNA interference,RN Ai)质粒表达载体,脂质体法转染宫颈癌Caski细胞株,应用荧光定量PCR及流式细胞术检测其对HPV16 E6 mRNA及蛋白表达的影响。结果:经PCR及测序证实3种表达载体构建成功,细胞试验表明3种表达载体均抑制了HPV16 E6的mRNA及蛋白的表达,其中CaskiB细胞HPV16E6mRNA的抑制率为89.5%,蛋白抑制率达98.1%。结论:RNAi表达载体可以有效的抑制HPV16 E6基因的表达。
OBJECTIVE: To construct RNA interference expression vector targeting HPV16 E6 gene and study its inhibitory effect on cervical cancer HPV16 E6 gene. Methods: The short hairpin RNAs (shRNAs) fragments were designed according to the sequence of HPV16 E6 gene. RNA interference (RNAi) gene expression vector was constructed for HPV16 E6 gene. Caski cell line was transfected into cervical cancer cells by liposome. Fluorescent quantitative PCR and flow cytometry were used to detect the expression of HPV16 E6 mRNA and protein. Results: The three kinds of expression vectors were successfully constructed by PCR and sequencing. The results of cell assay showed that all the three vectors inhibited the expression of HPV16 E6 mRNA and protein. The inhibitory rate of HPV16 E6 mRNA and protein in Caski B cells was 89.5% and 98.1% . Conclusion: RNAi expression vector can effectively inhibit the expression of HPV16 E6 gene.