摘要
目的:通过体外RNAi干扰技术靶向抑制原癌基因Bmi-1表达,观察其对胆囊癌细胞增殖的效应及对细胞周期的影响。方法:针对Bmi-1不同位点构建4个miRNABmi-1重组质粒,采用RT-PCR和Western blot检测各组Bmi-1 mRNA和蛋白表达,选择干扰效果最明显组质粒转染胆囊癌GBC-SD细胞,48 h后采用BrdU及流式细胞仪检测细胞增殖及细胞周期变化。结果 :RT-PCR显示miRNABmi-1重组质粒1、3、4组mRNA表达明显低于对照组(P<0.05);Western blot显示miRNABmi-1重组质粒2、3、4组蛋白表达明显低于对照组(P<0.05);选取第4组(mRNA及蛋白表达抑制效果最强)质粒转染GBC-SD细胞,BrdU显示mRNABmi-1-4组细胞增殖抑制明显,增殖率为46.63±5.31,明显低于对照组(P<0.05);细胞周期显示miRNABmi-1-4组G0/G1期细胞增加(72.20±1.71),G2/M和S期细胞减少(18.30±7.21,9.50±6.01),与对照组比较,差异具有显著性(P<0.05)。结论:靶向沉默Bmi-1表达,能有效抑制胆囊癌细胞增殖,使细胞周期阻滞于G0/G1期,Bmi-1可能成为胆囊癌基因治疗的有效靶点。
Objective Via targeted inhibition of oncogene Bmi-1 expression by RNAi interfering technology in vitro, to observe its effect on the proliferation and cell cycle of gallbladder cancer cells. Methods Four miRNABmi-1 recombinant plasmids were constructed according to different Bmi-1 sites. RT-PCR and Western blot were used to mRNA and protein expression of Bmi-1 in gallbladder cancer cells were measured by RT-PCR and Western blot. mRNA and protein expression of Bmi-1 in gallbladder cancer cells. The most effective interfering plasmids in the miRNABmi-1 groups were transfected into GBC-SD cells. Cell proliferation and cell cycle were analyzed 48 h after transfection by BrdU and flow cytometry. Results Bmi-lmRNA expression in miRNAhmil-1,-3 and -4 was significantly lower than the control group （P 〈 0.05）;and Bmi-1 protein expression in miRNAbmil-2,-3 and -4 was significantly lower than the control group （P 〈 0.05）. The recombinant plasmid in miRNAhmil-4, with the strongest inhibitive effect of Bmi-lmRNA and protein expression, was transfected into GBC-SD cells, then the cell proliferation rate （46.63 ± 5.31 ） was significantly lower in mRNABmil-4 group than the control groups （P 〈 0.05） ; G0/G1 phase cells increased （72.20 ± 1.71） and G2/M and S phase cells decreased （18.30 ± 7.21, 9.50± 6.01） in miRNABmil-4 group. Both were significantly different from the control groups （P 〈 0.05）. Conclusions Targeting and silencing Bmi-1 expression can effectively inhibit the proliferation of GBC-SD cells and restrain the cell cycle atin GO/G1 phase. Bmi-1 gene may be a novel target for geneic therapy of gallbladder carcinoma.
出处
《实用医学杂志》
CAS
北大核心
2014年第5期697-702,共6页
The Journal of Practical Medicine
基金
云南省科技计划项目(编号:2011FZ124)
云南省科技厅-昆明医科大学应用基础研究联合专项(编号:2012FB050)
昆明医科大学博士研究生创新基金项目(编号:2012D05)
