Huang Jian, Li De-huai
Department of Stomatology, The Fourth Affiliated Hospital of Harbin Medical University, Harbin 150001, China.
Zhonghua Kou Qiang Yi Xue Za Zhi. 2011 Oct;46(10):604-7.
To study the effect of diubiquitin (FAT10) down-regulation by small interfering RNA-mediated RNA interference (RNAi) on the biological features of tongue carcinoma cell line Tca8113.
Tca8113 cells were transfected with synthetic small interfering RNA (siRNA) targeting FAT10. Expression of FAT10 mRNA and protein were respectively measured by reverse transcription polymerase chain reaction (RT-PCR) and Western blotting, transfection efficiencies were monitored. The distribution of cell cycle phases was determined using flow cytometry. The proliferative and invasive ability of Tca8113 cells in vitro was evaluated by the colony-forming unit assay and Transwell migration assay respectively.
Both FAT10 mRNA and protein expression were significantly decreased in the experimental group (pU-FAT10-siRNA: mRAN 0.36 ± 0.03, Protein 0.39 ± 0.04) compared with controls (
mRNA 0.95 ± 0.05, Protein 0.69 ± 0.05; pU-siRNA: mRNA 0.92 ± 0.07, Protein 0.64 ± 0.05) (P < 0.05). The cell cycle was arrested in the G(1) phase [pU-FAT10-siRNA: (72.45 ± 5.81)%,
(45.95 ± 3.80)%, pU-siRNA: (45.95 ± 3.80)%]. The proliferation and invasiveness of treated Tca8113 cells were inhibited in vitro (pU-FAT10-siRNA: 41.83 ± 8.19, CONTROL: 317.21 ± 69.48, pU-siRNA: 339.36 ± 73.84).
Delivery of siRNA targeting FAT10 seems efficient in down-regulating FAT10 expression and diminishing the growth, proliferation and invasiveness of Tca8113 cells, suggesting that siRNA-based strategy targeting FAT10 may lay a foundation for the clinical management of tongue carcinoma.
研究小干扰RNA介导的RNA干扰(RNAi)下调双泛素(FAT10)对舌癌细胞系Tca8113生物学特性的影响。
用靶向FAT10的合成小干扰RNA(siRNA)转染Tca8113细胞。分别采用逆转录聚合酶链反应(RT-PCR)和蛋白质免疫印迹法检测FAT10 mRNA和蛋白的表达,监测转染效率。用流式细胞术检测细胞周期各时相分布。分别采用集落形成单位实验和Transwell迁移实验评估Tca8113细胞体外增殖和侵袭能力。
与对照组相比,实验组(pU-FAT10-siRNA:mRNA 0.36±0.03,蛋白0.39±0.04)中FAT10 mRNA和蛋白表达均显著降低(对照组:mRNA 0.95±0.05,蛋白0.69±0.05;pU-siRNA:mRNA 0.92±0.07,蛋白0.64±0.05)(P<0.05)。细胞周期阻滞于G(1)期[pU-FAT10-siRNA:(72.45±5.81)%,对照组:(45.95±3.80)%,pU-siRNA:(45.95±3.80)%]。经处理的Tca8113细胞体外增殖和侵袭能力受到抑制(pU-FAT10-siRNA:41.83±8.19,对照组:317.21±69.48,pU-siRNA:339.36±73.84)。
靶向FAT10的siRNA能有效下调FAT10表达并抑制Tca8113细胞生长、增殖和侵袭,提示基于siRNA靶向FAT10的策略可能为舌癌临床治疗奠定基础。