Hu Zheng, Cai Fang, Cheng Li-Juan, Xia Kun, Xia Jia-Hui, Zhang Zhuo-Hua
National Laboratory of Medical Genetics, Central South University, Changsha 410078, China.
Zhong Nan Da Xue Xue Bao Yi Xue Ban. 2005 Jun;30(3):253-7.
To set up a prostate cancer cell line in which beta-catenin expression is stably suppressed and to investigate the role of Wnt/beta-catenin signaling pathway in prostate tumorgenesis.
We select 3 sites in the complete coden sequence region of beta-catenin gene as the RNAi targets, ligated the annealed double pre-DNA strands into the retroviral vectors pSUPER-retro and transfected them into the packaging cells PA317, and then collected supernatant with retrovirus to infect DU145. After selection by puromycin and culture expansion, the stable cell clones were attained. Expression of the 2 target genes of Wnt/beta-catenin signaling pathway cyclinD1 and c-myc, was detected in the beta-catenin RNAi cells by Western blot. The effect of suppressing beta-catenin by RNAi on cell proliferation was quantified by methylthiazoletetrazolium (MTT) assay.
Western blotting and RT-PCR showed that the expression level of beta-catenin in the 2 stable cell clones apparently decreased. CyclinD1 and c-myc expression decreased in the beta-catenin RNAi cells. MTT showed that the cell number of beta-catenin expression suppression cell clones decreased significantly (P < 0. 05), suggesting the cell proliferation was prevented.
The beta-catenin gene stable suppression cell line was successfully established.
建立β-连环蛋白表达被稳定抑制的前列腺癌细胞系，并研究Wnt/β-连环蛋白信号通路在前列腺肿瘤发生中的作用。
我们在β-连环蛋白基因的完整编码序列区域选择3个位点作为RNA干扰靶点，将退火后的双链pre-DNA连接到逆转录病毒载体pSUPER-retro中，并将其转染到包装细胞PA317中，然后收集含有逆转录病毒的上清液感染DU145。经嘌呤霉素筛选和培养扩增后，获得稳定的细胞克隆。通过蛋白质免疫印迹法检测Wnt/β-连环蛋白信号通路的2个靶基因细胞周期蛋白D1和c-myc在β-连环蛋白RNA干扰细胞中的表达。通过噻唑蓝（MTT）法量化RNA干扰抑制β-连环蛋白对细胞增殖的影响。
蛋白质免疫印迹法和逆转录-聚合酶链反应显示，2个稳定细胞克隆中β-连环蛋白的表达水平明显降低。β-连环蛋白RNA干扰细胞中细胞周期蛋白D1和c-myc的表达下降。MTT法显示，β-连环蛋白表达抑制细胞克隆的细胞数量显著减少（P<0.05），提示细胞增殖受到抑制。
成功建立了β-连环蛋白基因稳定抑制细胞系。
