RNA干扰COPS3基因抑制骨肉瘤细胞HOS增殖和迁移能力的实验研究

目的 探讨COPS3基因的表达对骨肉瘤细胞系HOS增殖和迁移能力的影响.方法 构建针对COPS3 mRNA的慢病毒表达载体(Lenti-COPS3si)和阴性对照载体(Lenti-NC),并将其感染高表达COPS3基因的人骨肉瘤细胞系HOS.实时定量PCR检测COPS3 mRNA的表达;Western blot测定COPS3的蛋白表达;噻唑蓝(MTT)比色法检测HOS细胞的增殖能力;迁移实验检测HOS细胞迁移能力的改变.结果 与空白对照组和转染Lenti-NC的阴性对照组相比,转染48h后Lenti-COPS3si组COPS3 mRNA和蛋白的表达均明显降低,转染48h后,Lenti-COPS3si组HOS细胞的增殖能力明显下降(P＜0.05).迁移实验结果显示,与空白对照组和阴性对照组相比,细胞的迁移能力下降(P＜0.05).结论 慢病毒载体Lenti-COPS3si可以特异性抑制骨肉瘤细胞系HOS中COPS3的表达,并显著降低细胞的增殖和迁移能力,此技术有望成为抑制骨肉瘤生长和转移的新途径.

RNAi-mediated downregulation of COPS3 in the inhibition of the proliferation and migration In human osteosarcoma cell line (HOS)

Objective To investigate the effects of COPS3 expression on the proliferation and migration m human osteosarcoma cell line （HOS）. Methods A lemiviral vector, which contained small interfering RNA （siRNA） （Lenti-COPS3si） with COPS3mRNA expression and the control siRNA （Lenti-NC） gene, was constructed and transduced into HOS , which expressed COPS3 at high level. Quantitative real-time PCR （qPCR） and Western blot analysis were used to detect the mRNA expression and protein levels of COPS3, respectively. MTT Assay was used to examine changes in cell proliferation. The migration ability of HOS cells was evaluated by Migration Assay. Results 48 hours after the transfection of Lenti-COPS3si into the HOS, in comparison with the COPS3mRNA level of control groups （control group A being unprocessed, and control group B with Lenti negative cell groups）, the group C （with Lenti-COPS3si） demonstrated an effective and specific inhibition in the expression of COPS3mRNA in HOS cells and decrease in its protein level of COPS3. Besides, there was a significant decline in the proliferation rate of the HOS cells （P〈0.05）. The migration assay showed that in comparison with the control groups, the migration ability of HOS cells in group C was significantly reduced （P〈0.05）. Conclusions The Lenti-COPS3si can specifically inhibit COPS3 expression and significantly reduce the proliferation and migration ability of the cells in HOS, which has the potential to become a new pathway down-regulating proliferation and migration of osteosarcoma.