靶向β-catenin的shRNA抑制结肠癌细胞增殖的体外实验研究

ShRNA-mediated gene silencing of lS-catenin inhibits growth of human colon cancer cell Colo205 in vitro

目的探讨靶向β-catenin的shRNA对结肠癌细胞Colo205的WNT通路的阻断和该基因沉默效应对结肠癌细胞在体外的生长抑制作用。方法构建靶向β-catenin的shRNA载体质粒和阴性对照载体.然后分别转染人结肠癌细胞Colo205。用RT-PCR和Western印迹法检测β-catenin的mRNA和蛋白的表达情况，细胞免疫荧光染色法检测β-catenin蛋白的表达。噻唑蓝实验评价转染后各组细胞的体外增殖情况。软琼脂集落形成实验检测各组细胞的非锚着依赖性生长的能力。结果成功构建了靶向β-catenin的shRNA和阴性对照质粒载体。特异性靶向β-catenin的shRNA能够明显下调β-catenin mRNA，并抑制β-catenin蛋白的表达，其抑制率分别为47.89％和45.26％（P〈0.05）。转染了特异性shRNA的实验组（CAT组）细胞增殖在转染后呈现随时间延长而进行性抑制的现象。在转染后72h，CAT组的细胞存活率为48.5％。与空白对照组的91.3％相比明显降低（P〈0.05）。在软琼脂里CAT组的细胞克隆数目（9个）明显少于空白对照组（46个）和阴性对照组（43个）（P〈0.05）。结论靶向β-catenin的特异性shRNA对结肠癌Colo205细胞具有沉默β-catenin基因表达从而阻断WNT信号通路的作用，并且能够有效地在体外抑制结肠癌细胞的增殖。

Objective To observe the gene silencing and disruption of WNT pathway mediated by the specific shRNA targeted against β-catenin and its effect on cell proliferation of the human colon cancer cell line Colo205. Methods The shRNA plasmid vectors against β-catenin were constructed and transfected into Colo205 cells with Lipofectamine^TM 2000. The expression of β-catenin was detected by RT-PCR and Western blot. Immunofluorescence staining was also performed to detect the β-catenin protein expression in cells. The cell proliferation inhibition was determined by MrlT assay and soft agar colony formation assay. Results The shRNA vectors targeted against β-catenin were successfully constructed and efficiently suppressed the expression of β-catenin mRNA and protein （P〈0.05）. The expression inhibition rates were 47.89% and 45.26% at the mRNA and protein level respectively. Immunofluorescence microscopy also demonstrated the inhibition of β-catenin protein induced by these specific shRNAs. The MTT assay indicated that the specific shRNA resulted in significant inhibition of cell growth on the culture plates in time-dependent manner. At 72 h post- transfection, the cell viability of CAT group was 48.5%, which was significantly different as compared with that of blank control group＇s 91.3%（P〈0.05）. In the soft agar, there were 9, 46, 43 cell colonies in the CAT, blank, and negative control groups respectively, which were significantly different （P〈 0.05）. Conclusions The specific shRNAs targeted against β-catenin has a gene silencing effect and blocks the WNT signaling pathway, which can inhibit the growth of Colo205 cells.