Ji Wen, Jiang Zheng
Department of Gastroenterology, First Affiliated Hospital of Chongqing Medical University, YuZhong, Chongqing 400016, P.R. China.
Oncol Lett. 2013 Aug;6(2):367-374. doi: 10.3892/ol.2013.1394. Epub 2013 Jun 13.
The aim of the present study was to employ RNA interference (RNAi) technology to construct and select shRNA-Nanog recombinant plasmids for the inhibition of Nanog gene expression and transfer these plasmids into the human gastric cancer cell line, SGC-7901, as well as to detect the expression of Nanog and the effects on the proliferation, migration, invasion, cell cycle and apoptosis of SGC-7901 cells. The pshRNA-Nanog interference plasmids were constructed and used to transfect SGC-7901 cells using lipofectamine. The expression of the Nanog gene was detected by fluorescence microscopy, RT-PCR and western blotting, and the most markedly inhibited group was identified. The SGC-7901 cells were transfected with recombinant shRNA-Nanog plasmids from the most markedly inhibited group using lipofectamine and the effect on proliferation was determined by CCK-8 assay. The migration and invasion of the SGC-7901 cells was determined by Transwell assays, while the cell cycle and apoptosis were analyzed by flow cytometry. The group with the highest inhibition rate was successfully constructed and identified. It was observed that the proliferation, invasion and migration capacity of the cells was reduced, that the cell cycle was arrested at the S phase and that apoptosis was significantly increased. The Nanog gene in gastric cancer cells is closely associated with cell proliferation, the cell cycle, apoptosis and migration and invasion abilities. The present study establishes the foundations for a novel approach for the genetic treatment of gastric cancer.
本研究的目的是利用RNA干扰（RNAi）技术构建并筛选用于抑制Nanog基因表达的shRNA-Nanog重组质粒，并将这些质粒转染至人胃癌细胞系SGC-7901，同时检测Nanog的表达以及对SGC-7901细胞增殖、迁移、侵袭、细胞周期和凋亡的影响。构建pshRNA-Nanog干扰质粒，并用脂质体转染SGC-7901细胞。通过荧光显微镜、RT-PCR和蛋白质印迹法检测Nanog基因的表达，确定抑制效果最显著的组。使用脂质体将抑制效果最显著组的重组shRNA-Nanog质粒转染至SGC-7901细胞，通过CCK-8法测定对增殖的影响。采用Transwell法检测SGC-7901细胞的迁移和侵袭能力，通过流式细胞术分析细胞周期和凋亡情况。成功构建并鉴定出抑制率最高的组。结果表明，细胞的增殖、侵袭和迁移能力降低，细胞周期停滞于S期，凋亡显著增加。胃癌细胞中的Nanog基因与细胞增殖、细胞周期、凋亡以及迁移和侵袭能力密切相关。本研究为胃癌的基因治疗新方法奠定了基础。
