南方医科大学学报

南方医科大学学报 ›› 2015, Vol. 35 ›› Issue (09): 1227-.

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FoxM1 shRNA慢病毒载体构建及感染人前列腺癌细胞的稳定细胞株筛选

王一茹，姚斌伟，张艳，张明博，高菡静，唐杰   

  • 出版日期:2015-09-20 发布日期:2015-09-20

Construction of a lentiviral vector of FoxM1 shRNA and its transfection into human
prostate cancer cell lines in vitro

  • Online:2015-09-20 Published:2015-09-20

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摘要： 目的构建携带绿色荧光蛋白（GFP）的人FoxM1 shRNA重组慢病毒载体，并构建筛选FoxM1敲低的人前列腺癌稳定细胞
株，检测FoxM1的表达及对细胞增殖能力的影响。方法设计并合成3对特异性针对人FoxM1基因的shRNA靶序列，构建到
pHBLV-U6-ZsGreen-Puro 慢病毒载体中，测序鉴定载体构建情况，利用慢病毒三质粒系统转染包装293T细胞，荧光显微镜下观
察转染效率。收集到的病毒上清转染人前列腺癌DU-145细胞，Real-time PCR检测各组细胞FoxM1 mRNA水平，经嘌呤霉素
筛选建立FoxM1敲低稳定细胞株，用Western blotting法检测细胞中FoxM1表达，并用MTT法观察稳转细胞的生长增殖活性，
流式细胞术观察稳转细胞凋亡情况，平板克隆实验观察稳转细胞的克隆形成能力。结果经测序鉴定成功构建了3对FoxM1
shRNA慢病毒载体，并进行包装，感染DU-145细胞后Real-time PCR检测结果表明第1对慢病毒载体干扰效率最佳，嘌呤霉素
抗性筛选建立了FoxM1敲低稳定细胞株，荧光显微镜观察感染效率较高，Western blotting结果显示细胞中FoxM1蛋白表达明
显受到抑制，细胞的生长增殖能力明显受限，流式细胞术结果显示FoxM1敲低组细胞凋亡率高于对照组，平板克隆实验结果表
明FoxM1敲低组细胞克隆形成能力下降。结论本研究成功构建了FoxM1 shRNA慢病毒载体，建立了FoxM1敲低稳定前列腺
癌细胞株，抑制细胞内FoxM1的表达能够抑制前列腺癌细胞DU-145的生长增殖、克隆形成能力，并能诱导细胞凋亡。

Abstract: Objective To construct a recombinant lentiviral vector that co-express green fluorescent protein (GFP) and FoxM1
shRNA and establish a prostate cancer cell line with stable FoxM1 down-regulation. Methods Three interfering sequences
targeting FoxM1 were designed and inserted into the lentiviral vector pHBLV-U6-ZsGreen-Puro. After identification by DNA
sequencing, the lentiviral vectors carrying Foxm1 shRNA were packaged in 293 cells. The lentiviral particles were collected to
infect human prostate cancer DU-145 cells, and the transfection efficiency was observed under fluorescence microscope; the
interference efficiency was assessed using real-time PCR. DU-145 cells with stable FoxM1 down-regulation were screened with
puromycin, and the expression level of FoxM1 was detected by Western blotting and the cell growth was observed using MTT
assay. The stably transfected cells were examined for cell apoptosis and cell clone formation capacity with flow cytometry and
colony formation assay. Results DNA sequencing demonstrated successful construction of the 3 FoxM1 shRNA lentivirus
vectors. Real-time PCR showed a high interference efficiency of FoxM1 shRNA1 vector, which resulted in obvious
down-regulation of FoxM1 in DU-145 cells. Western blotting showed that the expression of FoxM1 protein was decreased in
FoxM1 shRNA1 lentivirus-transfected cells, which displayed a suppressed cell proliferation, increased apoptosis rate, and
attenuated clonogenic ability. Conclusion We have successfully established a prostate cancer cell model with stable FoxM1
down-regulation, which shows lowered proliferative and clonogenic activities with increased cell apoptosis.