Notch-1下调对双氢青蒿素抗人骨肉瘤细胞株U-2OS存活率的影响

Knockdown of Notch-1 augments inhibitory effect of dihydroartemisinin on viability of human osteosarcoma cell line U-2OS

目的:探讨Notch-1下调对双氢青蒿素抗人骨肉瘤细胞株U-2OS细胞存活率的影响及其作用机制。方法:双氢青蒿素(5、10、15和20μmol/L)孵育U-2OS细胞后,采用免疫印迹和实时荧光定量聚合酶链反应检测骨肉瘤细胞中Notch-1、基质金属蛋白酶(MMP)-2、MMP-9和Notch-1通路下游基因Hes-1的蛋白和mRNA的表达水平。下调Notch-1表达后,采用MTT法、免疫印迹法和细胞迁移实验检测在双氢青蒿素作用下U-2OS细胞的存活率,MMP-2、MMP-9和Hes-1的蛋白表达以及U-2OS细胞的迁移能力。结果:免疫印迹法与逆转录聚合酶链反应结果显示双氢青蒿素呈剂量依赖性降低U-2OS细胞中Notch-1、MMP-2、MMP-9和Hes-1的蛋白和mRNA的表达水平(P<0.05);下调Notch-1表达后,增强了双氢青蒿素抑制U-2OS细胞生长的作用,MMP-2、MMP-9和Hes-1的蛋白表达及细胞迁移能力进一步降低,显著低于加药组(P<0.05)。结论:双氢青蒿素可抑制U-2OS细胞中Notch-1通路的活性;下调Notch-1的表达可增强双氢青蒿素抗骨肉瘤的作用。

AIM： To investigate the effect of Notch-1 knockdown on the growth of dihydroartemisinin-inhibited human osteosarcoma cell line U-2OS. METHODS： U-2OS cells treated with different concentrations of dihydroartemisinin（ 5,10,15 and 20 μmol/L） were collected. The expression of Notch-1,MMP-2,MMP-9 and Hes-1 at mRNA and protein levels was measured by real-time PCR and Western blotting,respectively. U-2OS cells were transfected with Notch-1siRNA for 24 h and incubated with dihydroartemisinin for another 24 h. The cell apoptotic rate,protein expression of MMP-2,MMP-9 and Hes-1,and the migration ability were measured by MTT assay,Western blotting and Transwell experiment,respectively. RESULTS： Dihydroartemisinin（ 5,10,15 and 20 μmol / L） decreased the expression of Notch-1,MMP-2,MMP-9 and Hes-1 at mRNA and protein levels in a dose-dependent manner. Down-regulation of Notch-1 significantly enhanced the effect of dihydroartemisinin on the cell apoptosis,the protein expression of MMP-2,MMP-9 and Hes-1,and migration ability（ P〈0. 05）. CONCLUSION： Notch-1 pathway is involved in the process of dihydroartemisinin-inhibited U-2OS cell growth. Knockdown of Notch-1 augments the inhibitory effect of dihydroartemisinin on U-2OS cell viability.