RNA干扰双泛素对舌鳞状细胞癌细胞生物学行为的影响

Effect of diubiquitin gene silencing by small interfering RNA on proliferation and invasion of tongue carcinoma Tca8113 cells

目的应用RNA干扰技术抑制人舌鳞状细胞癌细胞株Tca8113中双泛素（diubiquitin）的表达，探讨双泛素表达下调对舌鳞状细胞癌细胞生物学行为的影响。方法构建针对双泛素特异性小干扰RNA（small interfering RNA，siRNA）真核表达载体pu-双泛素-siRNA，将其转染至Tca8113细胞，采用反转录聚合酶链反应（RT-PCR）、蛋白质印迹法检测转染后的Tca8113细胞中双泛素的表达。通过流式细胞仪分析siRNA对舌鳞状细胞癌细胞周期的影响，并通过细胞侵袭能力实验观察siRNA对Tca8113细胞恶性生物学行为的变化。结果siRNA干扰Tca8113细胞后，双泛素的表达无论是在mRNA水平还是在蛋白质水平均显著下降[mRNA：实验组（0．36±0．03）、空白对照组（0．92±0．07）、空载体组（0．95±0．05）；蛋白：实验组（0．39±0．04）、空白对照组（0．64±0．05）、空载体组（0．69±0．05）]（P〈0．05），细胞周期被阻滞在G．期，细胞生长缓慢，体外侵袭能力下降（P〈0．05）。结论通过RNAi技术阻断双泛素的表达，可抑制Tca8113细胞的生长、增殖、侵袭，提示双泛素在舌鳞状细胞癌的发生、发展过程中起重要作用。

Objective To study the effect of diubiquitin （FATIO） down-regulation by small interfering RNA-mediated RNA interference （RNAi） on the biological features of tongue carcinoma cell line Tca8113 . Methods Tca8113 cells were transfected with synthetic small interfering RNA（siRNA） targeting FATI0. 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 Tea8113 cells in vitro was evaluated by the colony-forming unit assay and Transwell migration assay respectively. Results Both FAT10 mRNA and protein expression were significantly decreased in the experimental group （ pU-FATI0-siRNA ： reRAN 0. 36 ± 0. 03, Protein 0. 39 ±0. 04） compared with controls （ Control ： 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 G1 phase[ pU-FAT10-siRNA： （72. 45±5.81 ）% ,Control：（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）. Conclusions Delivery of siRNA targeting FAT10 seems efficient in down-regulating FATIO expression and diminishing the growth, proliferation and invasiveness of Tea8113 cells, suggesting that siRNA-based strategy targeting FATIO may lay a foundation for the clinical management of tongue carcinoma.