沉默FNBP1基因对人HL-7702细胞体外增殖及迁移能力的影响

发布时间：2018-10-26 12:32

【摘要】：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。
[Abstract]:In order to study the effect of silencing FNBP1 (formin-binding protem1) gene on cell proliferation and cell migration in vitro, three FNBP1 siRNA interference vectors (Si-1,Si-2,Si-3) were designed and constructed. After restriction endonuclease digestion and DNA sequencing, HL-7702.RT-PCR and Western blot were used to detect the interference efficiency of FNBP1 gene in the transient transfection cells, and specific and efficient RNAi targets were screened out. The cell lines expressing RNAi stably were screened by G418. The proliferation rate of cells in vitro was detected by XTT method and the migration ability of cells was detected by scratch method. The results showed that the construction of interfering plasmid was correct by restriction endonuclease digestion and sequencing. Compared with the control group, the expression of FNBP1 gene in the transfected interfering plasmid (Si-1,Si-2,Si-3) cells was decreased, and the expression of FNBP1 gene was also inhibited at the protein level. The results of XTT analysis showed that the silencing FNBP1 recombinant plasmid Si-2 could significantly inhibit the proliferation of HL-7702 cells in vitro, but had no direct effect on the migration ability of HL-7702 cells.
【作者单位】：重庆医科大学分子医学及肿瘤学研究中心细胞生物学及遗传学教研室;
【基金】：国家自然科学基金项目(20803098) 重庆市教育委员会科学技术研究项目(KJ080301)
【分类号】：R346

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