Huang Hong-jie, Ping Fei-yun, Hu Ji-an, Zhao Shi-fang
Department of Oral and Maxillofacial Surgery, School of Stomatology, Zhejiang University, Hangzhou 310006, China.
Zhonghua Kou Qiang Yi Xue Za Zhi. 2009 Jun;44(6):365-9.
To investigate the effects of epidermal growth factor receptor (EGFR) gene silencing mediated by short hairpin RNA (shRNA) on proliferation and apoptosis of human tongue carcinoma cells.
shRNA eukaryotic expression vector targeting the specific sequence of human EGFR gene was constructed and termed shEGFR. The control vector targeting the unrelated sequence was also constructed and termed shNC. The vectors were transiently transfected into Tca8113 cells of human tongue squamous cell carcinoma by Lipofectamine 2000, respectively. The mRNA and protein levels of EGFR in Tca8113 cells were detected by reverse transcriptase polymerase chain reaction (RT-PCR) and Western blotting. The cell proliferation of Tca8113 cells was evaluated by methyl thiazolyl tetrazolium (MTT) assay. The apoptosis of Tca8113 cells was assessed by flow cytometry.
EGFR expression in Tca8113 cells transfected with shEGFR were obviously decreased at mRNA level (81.6%) and protein level (72.0%) (P < 0.05) 48 h after transfection of shEGFR compared with untransfected Tca8113 cells. The proliferation activity of Tca8113 cells transfected with shEGFR was significantly lower than that of Tca8113 cells transfected with shNC and untransfected Tca8113 cells (P < 0.05). The early apoptotic rate of Tca8113 cells transfected with shEGFR was significantly higher than that of Tca8113 cells transfected with shNC and untransfected Tca8113 cells [(39.4 +/- 7.7)%, (4.3 +/- 1.2)%, (2.5 +/- 0.9)%, P < 0.05] 48 h after transfection of shEGFR.
EGFR gene silencing mediated by shRNA may inhibit cell proliferation and induce apoptosis in human tongue carcinoma cells.
探讨短发夹RNA(shRNA)介导的表皮生长因子受体(EGFR)基因沉默对人舌癌细胞增殖和凋亡的影响。
构建靶向人EGFR基因特定序列的shRNA真核表达载体,命名为shEGFR。同时构建靶向无关序列的对照载体,命名为shNC。分别用Lipofectamine 2000将载体瞬时转染入人舌鳞状细胞癌Tca8113细胞。采用逆转录聚合酶链反应(RT-PCR)和蛋白质印迹法检测Tca8113细胞中EGFR的mRNA和蛋白水平。采用甲基噻唑基四氮唑(MTT)法评估Tca8113细胞的增殖情况。采用流式细胞术检测Tca8113细胞的凋亡情况。
与未转染的Tca8113细胞相比,转染shEGFR后48 h,Tca8113细胞中EGFR的mRNA水平(81.6%)和蛋白水平(72.0%)明显降低(P<0.05)。转染shEGFR的Tca8113细胞的增殖活性明显低于转染shNC的Tca8113细胞和未转染的Tca8113细胞(P<0.05)。转染shEGFR后48 h,转染shEGFR的Tca8113细胞的早期凋亡率明显高于转染shNC的Tca8113细胞和未转染的Tca8113细胞[(39.4±7.7)%,(4.3±1.2)%,(2.5±0.9)%;P<0.05]。
shRNA介导的EGFR基因沉默可能抑制人舌癌细胞的增殖并诱导其凋亡。