沉默FNBP1基因对人HL-7702细胞体外增殖及迁移能力的影响

Effects of Down-regulating FNBPl Gene on Cell Proliferation and Metastasis Ability in HL-7702 Cells

为了研究沉默FNBP1(formin-binding protem1)基因对细胞体外增殖以及对细胞迁移能力的影响,设计并构建了3个靶向FNBP1 siRNA干扰载体(Si-1,Si-2,Si-3),经酶切和DNA测序鉴定后转染人正常的肝细胞HL-7702。RT-PCR和Western blot检测瞬时转染细胞中FNBP1基因的干扰效率,筛选出特异高效的RNAi靶点。并用G418筛选稳定表达了RNAi的细胞株,用XTT法检测细胞体外增殖率,划痕法检测细胞的迁移能力。结果显示:酶切和测序结果证明干扰质粒构建正确;转染特异性的干扰质粒(Si-1、Si-2、Si-3)细胞的FNBP1基因表达量与对照组相比均有所降低,在蛋白质水平表达也明显受到抑制,且重组干扰质粒Si-2的干扰效应较强。XTT检测结果表明,构建的沉默FNBP1重组质粒Si-2能显著抑制HL-7702细胞的体外增殖能力,但并不直接影响HL-7702细胞的迁移能力。

To investigate the effect of silencing FNBP1 （ formin-binding protein 1 ） gene on the proliferation and metas- tasis ability of HL-7702 cells, Three small interference expression vectors （Si-1 ,Si-2 ,Si-3 ） specific to FNBP1 mRNA were designed and constructed, then were transfected into the HL-7702 cells after identified by restriction analysis and DNA sequencing. Analysis of efficiency of FNBP1 siRNA was detected by RT-PCR and Westeru-blot to choose the high- est inhibitory target of FNBP1 among the three vectors. The cell line with stable expression of siRNA of FNBP1 was se- lected by CA18 medium for 15 days. After FNBP1 knockdown, the proliferation of HL-7702 cells was measured by the XTT assay and metastasis ability was measured by wound healing assay. Results are as following, restriction analysis and sequencing proved that siRNA expression vector was constructed correctly; FNBP1 mRNA and protein expression obvi- ously decreased in HL-7702 cells transfected with siRNA （ Si-1, Si-2, Si-3 ） respectively compared with the controlgroups, and Si-2 have the highest inhibitory effect. XTT showed that Si-2 can significantly inhibit the proliferation in HL- 7702 cells, and have no effect on metastasis ability of HL-7702, which strongly indicated that FNBP1 plays an important role in proliferation of HL-7702 cells.