慢病毒介导靶向PRL-3基因的siRNA抑制肝癌细胞的侵袭性  

Lentiviral-mediated siRNA against PRL-3 Suppresses Invasion Potency of Human Hepatocellular Carcinoma Cells

作　　者：黄德好[1] 张琪[1] 李华[1] 陈伟[1] 陈规划[1] 

机构地区：[1]中山大学附属第三医院肝移植科,广东广州510630

出　　处：《中山大学学报（医学科学版）》2010年第3期309-314,共6页Journal of Sun Yat-Sen University:Medical Sciences

基　　金：国家重点基础研究发展规划(国家973规划;2009CB522404)

摘　　要：【目的】探讨肝再生磷酸酶-3(PRL-3)的表达对肝癌细胞侵袭能力的影响。【方法】设计、合成4对针对PRL-3基因siRNA的寡核苷酸序列,分别构建质粒,转染293T细胞,据其对PRL-3的抑制率,筛选最有效的PRL-3基因RNAi靶序列,设计并合成其siRNA的双链DNAOligo,与含绿色荧光蛋白(GFP)编码基因的pGCL-GFP线性化载体连接产生重组慢病毒质粒LV-PRL3-SiRNA,并行PCR和测序鉴定。将LV-PRL3-SiRNA,pHelper1.0和pHelper2.0三种质粒DNA共转染293T细胞,包装产生慢病毒,以293T细胞GFP蛋白的表达水平经孔稀释法测定病毒滴度。重组慢病毒感染肝癌SMCC7721细胞,行realtime-PCR和western-blot验证PRL-3基因的沉默效果。用Transwell侵袭试验检测SMCC7721细胞体外侵袭力的改变。【结果】成功构建PRL-3基因RNA干扰慢病毒载体并获得相应的慢病毒,浓缩病毒悬液的滴度为6×108Tu/mL。和对照组相比,RNA干扰组SMMC7721细胞PRL-3基因的mRNA水平和蛋白水平的抑制率分别为75%和63%;其穿过人工基底膜的平均数(14.2±0.8)明显少于空白对照组(33.0±1.6)及非特异性siRNA感染组(31.2±0.8),侵袭抑制率为58%(P<0.01)。【结论】降低PRL-3的表达可抑制肝癌细胞的侵袭能力。【Objective】To construct recombinant lentiviral vector expressing siRNA targeting PRL-3 gene and explore its influence on invasive potency of infected hepatocellular carcinoma cell line SMCC7721.【Methods】Four sequences of siRNA targeting PRL-3 gene were designed,of which both sense and anti-sense DNA Oligo were designed,synthesized and corresponding plasmids were constructed.The most effective sequence of siRNA was screened by efficiency of PRL-3 gene knock-down in transfected 293T cells and its DNA Oligo was cloned into the pGCL-GFP vector,which contained coding gene of green fluorescent protein（GFP）.The resulting recombinant lentiviral vector expressing siRNA targeting PRL-3 gene was called LV-PRL3-SiRNA,and it was confirmed by PCR and DNA sequencing.293T cells were co-transfected with LV-PRL3-SiRNA,pHelper 1.0 and pHelper 2.0 and the lentivirus was produced.The titer of virus was tested according to the expression level of GFP in 293T cells.SMCC7721 cells were infected with recombinant lentivirus and the silencing effect of PRL-3 gene was assessed by real-time PCR and western-blot.The invasive activity of infected SMCC7721 cells was analyzed by Transwell invasion assay.【Results】Recombinant lentiviral vector expressing siRNA targeting PRL-3 gene was successfully established and confirmed by DNA sequencing.The recombinant lentivirus with the most effective PRL-3 gene knock-down were harvested from 293T cells with a viral titer of 6×108 Tu/mL.Results of real-time PCR and Western blot showed that compared with control group,PRL-3 expression in SMCC7721cells infected with LV-PRL3-SiRNA decreased by 75%and 63%at mRNA and protein level respectively.After PRL-3 gene was knocked down,invasion potency of SMCC7721 cells was significantly suppressed.【Conclusions】Down-regulation of PRL-3 expression can weaken invasion potency of hepatocellular carcinoma（HCC）cells.

关 键 词：RNA干扰 慢病毒 PRL-3 肝细胞癌 

分 类 号：R735.7[医药卫生—肿瘤]