慢病毒介导的siRNA沉默Foxp1对肝癌细胞增殖、凋亡和迁移的影响

Effects of Lentiviral-Mediated Foxp1 siRNA to Proliferation, Apoptosis and Migration of Hepatoma Carcinoma Cell

目的探讨Foxp1对肝癌细胞增殖、凋亡和迁移的影响。方法体外合成干扰Foxp1功能的小核酸片段(siRNA Foxp1),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反转录慢病毒载体(lenti-pLL3.7-Foxp1-siRNA),感染高表达Foxp1的肝癌细胞株。同时构建不含有Foxp1-siRNA序列的直接三质粒系统包装的空病毒载体对照组。荧光显微镜观察慢病毒载体介导siRNA感染细胞的效果。Western blot和Real-time QPCR(RTQPCR)分别检测肝癌细胞中Foxp1蛋白表达和mRNA水平。CCK-8实验、流式细胞仪、细胞划痕愈合实验和侵袭小室实验分别检测细胞增殖、凋亡和迁移的改变。结果与对照组细胞比较,肝癌细胞感染lenti-pLL3.7-Foxp1-siRNA后,细胞中Foxp1蛋白和mRNA表达显著下降,增殖活性受到显著抑制,细胞凋亡明显增加,在二维空间和三维空间的迁移细胞显著减少(均P<0.01)。结论 Foxp1作为一种多功能转录因子,促进肝癌细胞的增殖和迁移,抑制其凋亡。

Objective To explore the effects of Foxp1 on proliferation, apoptosis and migration of hepatoma carcinoma cells. Methods Small nucleic acid fragments which could interfere Foxp1(siRNA Foxp1) were synthesized in vitro, and it was inserted into the transfer plasmid of the lentiviral three-plasmid system by recombinant techniques, using the packaging cells(293T) to assemble the three-plasmid system as a complete retrovirus lentivirus vector(lenti-pLL3.7-Foxp1-siRNA), which was used to infect hepatoma carcinoma cell lines with high expression of Foxp1; at the same time, the empty viral vector without the the Foxp1-siRNA sequence directly packaged by three-plasmid system was constructed as control group. The infected effect of lentiviral vector-mediated siRNA was detected by fl uorescence microcopy. Expression of Foxp1 in hepatoma carcinoma cells were detected by western blot and real-time QPCR(RT-QPCR) at protein and mRNA levels. CCK-8 experiments, fl ow cytometry, the wound healing assay and transwell assay were used to investigate the changes in cell proliferation, apoptosis and migration. Results Compared with control group, expression of Foxp1 protein and mRNA significantly decreased in hepatoma carcinoma cells infected with lentipLL3.7-Foxp1-siRNA; meanwhile, the vitality of cells was notably inhibited, and the apoptosis strikingly increased, the migration in the two-dimensional space and three-dimensional space signifi cantly decreased(respectively, P<0.01). Conclusion As a kind of multi functional transcription factor, Foxp1 could promote the proliferation and migration of hepatoma carcinoma cells, and inhibit its apoptosis.