TLR4通过NF-κB信号途径对宫颈癌细胞增殖的影响

Effect of Toll like- receptor 4 on proliferation of cervical cancer cells by NF-κB signaling pathway

目的:研究Toll样受体4(Toll-like receptor 4,TLR4)小干扰RNA(siRNA)是否通过NF-κB信号途径下调Bcl-2而抑制宫颈癌细胞的增殖。方法:设计并合成针对TLR4基因的3条特异性siRNA)及阴性siRNA,并同时设立空白对照,然后用脂质体LipofectamineTM2000转染细胞。通过半定量RT-PCR和Western印迹法分别检测转染前后He La细胞中TLR4 mRNA和蛋白表达水平,Western印迹法检测p65及Bcl-2蛋白表达水平,MTT法和平板克隆检测细胞增殖率,Annexin V-FITC/PI染色流式细胞术检测细胞凋亡,流式细胞术检测细胞周期。结果:3条针对TLR4基因的siRNA均能抑制TLR4的mRNA和蛋白表达,其中TLR4-siRNA-003的沉默效率最高。转染TLR4-siRNA-003 48 h后细胞中TLR4的mRNA和蛋白表达水平分别下调62%和89%,p65及Bcl-2蛋白水平明显下调,同时细胞增殖明显受到抑制,细胞凋亡率显著升高,细胞周期被阻滞于G1期。结论:TLR4特异性siRNA能够有效沉默TLR4基因表达,并通过NF-κB信号通路下调Bcl-2而显著抑制宫颈癌He La细胞的增殖。TLR4可能成为治疗宫颈癌的一个新靶点。

AIM： To investigate whether specific small interfering RNA（ siRNA） targeting Toll-like receptor 4（ TLR4） gene inhibits the proliferation of cervical cancer cell line He La through NF-κB signaling pathway and down-regulation of Bcl-2 expression. METHODS： Three specific TLR4 siRNAs and 1 negative siRNA were transfected respectively into He La cells. The expression of TLR4 at mRNA and protein levels was measured by reverse transcription-polymerase chain reaction（ RT-PCR） and Western blotting,respectively. The protein expression of p65 and Bcl-2 was detected by Western blotting. The cell proliferation was determined by MTT assay and plate colony formation assay. The apoptotic rate of the cells and the cell cycle were analyzed by flow cytometry. RESULTS： In comparison with the other 2 kinds of TLR4 siRNAs,TLR4-siRNA-003 demonstrated the strongest silencing effect on TLR4 gene expression in the He La cells at 48 h after transfection. The expression of TLR4 at mRNA and protein levels was reduced by 62% and 89%,respectively. The protein levels of p65 and Bcl-2 significantly decreased. The growth rate of He La cells transfected with TLR4-siRNA-003 was significantly inhibited. Moreover,the cell apoptotic rate increased significantly and the cell cycle was arrested at G1 phase as the He La cells were transfected with TLR4-siRNA-003 for 48 h. CONCLUSION： Specific TLR4 siRNA effectively inhibits the expression of TLR4 and inhibits the proliferation of cervical cancer He La cells through NF-κB signaling pathway and down-regulation of Bcl-2 expression,indicating that TLR4 may be a new target for the treatment of cervical cancer.