小干扰RNA沉默TLR4表达对脂多糖促进人肺癌SPCA1细胞增殖的影响

Effect of Toll-like receptor 4 gene silencing by small interfering RNA on lipopolysaccharide-induced human lung carcinoma SPCA1 cell proliferation

目的:探讨Toll样受体4(TLR4)对脂多糖(LPS)促进人肺癌SPCA1细胞增殖的影响。方法:采用LPS(10μg/ml)刺激细胞,模拟慢性炎症的细胞微环境。将靶向TLR4基因的小干扰RNA(TLR4-siRNA)或阴性对照(NC-siRNA)通过脂质体介导转染人肺癌SPCA1细胞,24 h后加入10μg/ml LPS。按转染siRNA和加入LPS情况,实验分为不做任何处理的Control组、NC+10LPS组以及TLR4-siRNA+10LPS组。采用Real-time PCR和流式细胞术检测SPCA1细胞中TLR4 mRNA及蛋白的表达情况;采用CCK-8法和平板克隆形成实验检测细胞增殖能力,流式细胞术检测分析细胞周期分布情况。结果:与NC+10LPS组相比较,TLR4-siRNA+10LPS组细胞中TLR4 mRNA及蛋白的表达水平显著下降(P<0.01);与Control组和NC+10LPS组相比较,TLR4-siRNA+10LPS组SPCA1细胞的增殖明显减缓(P<0.01),TLR4-siRNA+10LPS组细胞克隆形成能力明显降低[(4.50±1.89)vs(13.33±1.81)、(15.75±1.25)个,P<0.01];TLR4-siRNA+10LPS组细胞周期阻滞于G0/G1期[(61.55±0.55)%vs(53.59±1.59)%、(51.72±0.77)%,P<0.01]。结论:TLR4-siRNA能有效沉默SPCA1细胞中TLR4的表达,能够阻滞细胞的生长,抑制细胞的增殖。

Objective:To determine the effect of Toll-like receptor 4(TLR4) on lipopolysaccharide(LPS)-induced human lung carcinoma SPCA1 cell proliferation. Methods: SPCA1 cells were treated with LPS(10 μg / ml) to mimic a chronic inflammation microenvironment. TLR4 specific small interfering RNA( TLR4-siRNA) and a negative control interfering RNA( NC-siRNA) were transfected into SPCA1 cells by Lipofectamine. At 24 h after transfection,transfectants were treated with 10 μg / ml LPS. At 48 h after LPS treatment,TLR4 mRNA and protein levels were determined by Realtime PCR and FCM respectively,cell proliferation was assessed by CCK-8 assay and colony formation assay,and the cell cycle distribution was examined by FCM. Results: TLR4 mRNA and protein levels were significantly decreased( P <0. 01),proliferation was significantly suppressed( P < 0. 01),and colony formation ability was significantly reduced(P < 0. 01) in SPCA1 cells transfected with TLR4-siRNA as compared with cells transfected with NC-siRNA. The proportion of cells arrested at the G0/ G1 phase was significantly higher( P < 0. 01) in SPCA1 cells transfected with TLR4-siRNA(61. 55 ± 0. 55)% than in NC-siRNA-transfected(53. 59 ± 1. 59)% and control(51. 72 ± 0. 77)%. Conclusion:TLR4-siRNA can effectively silence TLR4 expression in SPCA1 cells,block cell growth and inhibit cell proliferation.