构建慢病毒载体介导RNA干扰沉默肝癌细胞Foxq1的表达

Construction of a lentiviral vector to mediate RNA interference of Foxq1 expression in hepatocellular carcinoma cells

目的构建慢病毒载体将Foxq1干扰序列递达到人肝癌7721细胞中,观察该细胞中Foxq1的表达并评估慢病毒载体的有效性。方法体外合成与筛选具有Foxq1干扰功能的核酸片段(siRNA Foxq1),通过重组技术将其插入到慢病毒三质粒系统的转移质粒中,利用包装细胞(293T)将三质粒系统组装为完整的逆转录慢病毒颗粒,然后感染肝癌细胞。采用Western blot和RT-qPCR分别检测Foxq1蛋白和mRNA表达。同时构建与检测不含有siRNA Foxq1序列的直接三质粒系统包装的空病毒载体对照组。结果 Foxq1-834-siRNA具有较强的抑制Foxq1 mRNA功能,其抑制效率达90.17%;基因测序证实体外合成的siRNA Foxq1基因序列成功插入到慢病毒转移质粒中;荧光显微镜观察证明肝癌细胞中持续表达Foxq1-834-siRNA的绿色荧光信号,且信号强度随时间推移逐渐增强;采用RT-qPCR和Western blot检测结果证实慢病毒载体感染的细胞中Foxq1 mRNA和蛋白表达量较对照组明显下降(P<0.001)。结论筛选到有效抑制Foxq1的siRNA Foxq1序列,通过构建慢病毒载体感染细胞能有效的将体外合成的siRNA Foxq1递达到癌细胞中,并能抑制癌细胞中Foxq1的表达,为进一步研究肝癌中Foxq1的功能提供有效的工具。

Purpose To construct a lentiviral vector to deliver Foxql interference sequence to human hepatocellular carcinoma cells （7721 cell）, and to detect the expression of Foxql in the cells to assess the effectiveness of the lentiviral vector. Methods The nucleic acid fragments having Foxql interference function （ siRNA Foxql ） were synthesized and screened in vitro, which were inserted into the transfer plasmid of the lentivirus three-plasmid system by recombinant techniques. After that, packaging cells （293T） were used to assemble the three-plasmid system as a complete retrovirus lentivirus particle, and then the hepatocellular carcinoma cells were infected with it. Western blot and RT-qPCR were used to detect Foxql protein and mRNA expression, respectively. At the same time, the empty viral vector packaged directly by three-plasmid system without the siRNA Foxql sequence was constructed and detected as con- trol group. Results Foxql-834-siRNA could strongly inhibit Foxql mRNA, and the efficiency was 90. 17%. Gene sequencing con- firmed that the gene sequences synthesized in vitro were successfully inserted into the lentiviral transfer plasmid. The continuously ex- pressed green fluorescent signal of Foxql-834-siRNA in hepatoma cells was observed under fluorescence microscope, and the signal strength gradually increased over time. The results of RT-qPCR and Western blot confirmed that Foxql mRNA and protein in cells infected by the lentiviral vector were significantly lower than the control group （ P 〈 0. 001 ）. Conclusion The siRNA Foxql sequence that effectively inhibits Foxql is screened. It is synthesized in vitro, and effectively delivered to human hepatoma cells by the successfully constructed lentiviral vector, which could inhibit the expression of Foxql in hepatoma cells. This provides an effective tool for the further research on the function of Foxql in hepatocellular carcinoma.