摘要
目的探讨通过siRNA干扰技术沉默表皮脂肪酸结合蛋白-5(FABP-5)基因表达对人肝癌Bel-7402细胞生物学特性及人参川穹嗪敏感性的影响。方法以人肝癌Bel-7402细胞为研究对象,根据FABP-5mRNA编码序列设计并合成干扰siRNA序列,瞬时转染Bel-7402细胞。将人肝癌Bel-7402细胞分为FABP-5siRNA组、阴性对照组、空白对照组。用RT-PCR和Western blot法分别从mRNA水平和蛋白水平检测FABP-5基因的表达;CCK8法测定细胞体外增殖能力;流式细胞技术检测各组细胞周期和凋亡的变化情况;细胞侵袭小室法检测各组细胞的体外侵袭力;MTT法观察FABP-5基因沉默后Bel-7402细胞对人参川穹嗪的敏感性。结果 RT-PCR和Western blot显示,FABP-5 siRNA组较阴性对照组和空白对照组的FABP-5 mRNA和蛋白表达都明显下降;FABP-5 siRNA组细胞的生长速度明显减慢,细胞周期阻滞在G0/G1期,S期细胞数减少;与阴性对照组和空白对照组相比,FABP-5 siRNA组细胞的凋亡率明显升高(P<0.01),增殖、侵袭能力显著下降(P<0.05);FABP-5 siRNA组细胞对人参川穹嗪的敏感性显著增强(P<0.05)。结论特异性沉默FABP-5基因表达能够降低肝癌细胞的侵袭能力,抑制细胞的增殖,将细胞周期阻滞于G_0/G_1期,使其凋亡显著增加。
Objective To investigate the si RNA silencing epidermal fatty acid binding protein-5（FABP-5） gene expression in human hepatocellular carcinoma Bel-7402 cells and the sensitivity of Ginseng ligustrazine. Methods The interference si RNA sequence was designed and synthesized according to the FABP-5 sequence of m RNA, and then transiently transfected into human hepatoma cell line Bel-7402 cells. The cells were divided into 3 groups： FABP-5 si RNA group, negative control group and blank control group. RT-PCR and Western blot method were used to detect the expression of FABP-5 gene at m RNA and protein level. Cell proliferation in vitro was determined by CCK8 method. Flow cytometry was applied to examine periodic change of the cells of each group and cell apoptosis. Cell invasion was detected with transwell inserts. Each experiment was repeated 3 times. The sensitivity of Ginseng ligustrazine to Bel-7402 cells was observed with MTT assay after FABP-5 gene silencing. Results The results from RT-PCR and Western blot showed that the m RNA of FABP-5 or protein in FABP-5 si RNA group was significantly lower than that in negative control group and blank control group. The growth rate of cells in si RNA group was significantly decreased, cell cycle was arrested in G0/G1 phase, and S phase cells decreased. As compared to the negative control group or blank control group, the apoptosis of cells in FABP-5 si RNA group was significantly increased（P〈0.01） and proliferation or invasion ability decreased significantly（P〈0.05）. The sensitivity of Ginseng ligustrazine in FABP-5 si RNA group cells increased significantly（P〈0.05）. Conclusion The expression of FABP-5 gene reduces the invasion ability of hepatocellular carcinoma cells, inhibits the proliferation of cells, blocks the cell cycle in G0/G1 phase, and increases the apoptosis.
出处
《中国医药生物技术》
2018年第1期41-47,共7页
Chinese Medicinal Biotechnology
