慢病毒介导COX-2基因的shRNA对子宫内膜癌侵袭力的影响

The influence of lentiviral-mediated COX-2 shRNA on the invasiveness in endometrium adenocarcinoma

目的:探讨慢病毒介导COX-2基因的shRNA对子宫内膜癌HEC-1B细胞侵袭力的影响。方法:设计、合成4对针对COX-2基因shRNA的寡核苷酸序列,分别构建质粒,转染293T细胞,据其对COX-2的抑制率,筛选最有效的COX-2基因RNAi靶序列,设计并合成其shRNA的双链DNA Oligo,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-COX-2-ShRNA,并行PCR和测序鉴定。将LV-COX-2-ShRNA,pHelper 1.0和pHelper 2.0三种质粒DNA共转染293T细胞,包装产生慢病毒,以293T细胞GFP蛋白的表达水平采用逐孔稀释滴度法测定病毒滴度。重组慢病毒感染人子宫内膜癌HEC-1B细胞,行RT-PCR和western-blot验证COX-2基因的沉默效果,用Transwell侵袭试验检测HEC-1B细胞体外侵袭力的改变。结果:成功构建COX-2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5×107Tu/ml。和对照组相比,RNA干扰组HEC-1B细胞COX-2基因的mRNA水平和蛋白水平的抑制率分别为61.87%和67.48%;其穿过人工基底膜的平均数为16.6,明显少于空白对照组50.2及非特异性siRNA感染组47.2,侵袭抑制率为64.8%(P<0.01)。结论:RNA干扰可有效抑制HEC-1B细胞的侵袭能力。

Objective：To explore the influence of lentiviral-mediated COX-2 shRNA on the invasiveness in endometrial adenocarcinoma cell line HEC-1B.Methods：Four sequences of siRNA targeting COX-2 gene were designed,of which both sense and anti-sense DNA Oligo were designed,synthesized and corresponding plasmids were constructed.The most efective sequence of siRNA was screened by eficiency of COX-2 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 COX-2 gene was called LV-COX-2 SiRNA.and it was confirmed by PCR and DNA sequencing.293T cells were co-transfected with LV-COX-2-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.HEC-1B cells were infected with recombinant lentivirus and the silencing effect of COX-2 gene was assessed by rea1-time PCR and western-blot.The invasive activity of infected HEC-1B cells was analyzed by Transwell invasion assay.Results：Recombinant lentiviral vector expressing siRNA targeting COX-2 gene was successfully established and confirmed by DNA sequencing.The recombinant lentivirus with the most effective COX-2 gene knock-down were harvested from 293T cells with a viral titer of 5×l07Tu/ml.Results of real-time PCR and Western blot showed that compared with contro1 group.COX-2 expression in HEC-1B cells infected with LV-COX-2-SiRNA decreased by 61.87% and 67.48% at mRNA and protein level respectively.After COX-2 gene was knocked down,invasion potency of HEC-1B cells was significantly suppressed.Conclusion：Down-regulation of COX-2 expression can weaken invasion potency of endometrial carcinoma cells.