干扰AURKA基因表达对人骨肉瘤细胞系U2-OS细胞增殖与细胞周期的影响

Therapeutic effects of siRNA targeting AURKA gene on tumor proliferation and changes of cell cycle in human osteosarcoma cell line U2-OS

目的 :探讨AURKA基因表达下调对骨肉瘤细胞增殖和细胞周期分布的影响。方法:脂质体瞬时转染法介导AURKA小干扰RNA(small interfering RNA,siRNA)处理骨肉瘤U2-OS细胞,采用实时荧光定量PCR及Western blot方法检测转染前后AURKA mRNA和蛋白表达,CCK8实验检测细胞活力变化,流式细胞术检测细胞周期分布变化,Western blot检测周期相关蛋白的表达,5-溴脱氧尿嘧啶核苷(Brd U)细胞增殖实验检测细胞增殖情况。结果:AURKA siRNA作用U2-OS细胞后,AURKA mRNA和蛋白的表达水平较正常对照组及阴性对照组显著降低,且差异有统计学意义(P<0.05),两对照组间AURKA表达无明显差异;下调AURKA表达后,细胞活力降低,且作用72 h后抑制率最高,约为(36.63±2.38)%;细胞周期中S期细胞比例下降,G2/M期细胞比例增加,与正常对照组及阴性对照组相比差异有统计学意义(P<0.05),提示存在G2/M期阻滞,S期细胞增殖率下降(P<0.05),周期相关蛋白D1(Cyclin D1)表达水平下降(P<0.05),周期相关蛋白B1(Cyclin B1)表达水平明显增加(P<0.05)。结论:下调AURKA表达可抑制骨肉瘤细胞U2-OS的增殖,同时导致细胞阻滞于G2/M期。

Objective：To investigate the inhibition effect of down-regulation of small interfering RNA （siRNA） targeting AURKA gene on the proliferation and cell cycle distribution of osteosarcoma cells. Methods：AURKA siRNA was synthesized and transfected into osteosarcoma U2-OS cells via Lipofectamine 2000. Then, the AURKA mRNA and protein expression were examined by real-time fluorescence quantificative PCR （qRT-PCR） and Western blot assay, respectively. CCK8 assay and BrdU cell proliferation assay were performed to evaluate cell viability and proliferation, respectively. Cell cycle distribution was observed by flow cytometry. The expressions of cyclin B1 and cyclin D1 were determined by Western blot assay. Results：Both mRNA and protein levels of AURKA in U2-OS cells transfected with AURKA siRNA were significantly decreased compared with those of the other two groups （both P 〈 0.05）. There were no significant differences in the expressions of AURKA between the two control group. CCK8 assay showed that down-regulated AURKA inhibited cell viability and the inhibition rate reached to the highest level at approximately （36.63 ± 2.38）% after 72 h （P 〈 0.05）. The proportion of S phase cells in the cell cycle was decreased and G2/M phase cells was increased compared with those in the normal control group and negative control group, respectively （both P 〈 0.05）, suggesting the presence of G2/M phase arrest and decreased cell proliferation in S phase （P 〈 0.05）. Meanwhile, the expressions of cyclin D1 and cyclin B1 were significantly down-regulated and elevated （both P 〈 0.05）, respectively. Conclusion：Inhibition of AURKA expression suppressed cell growth and led to G2/M arrest of human osteosarcoma cell line U2-OS.