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目的应用慢病毒介导的RNA干扰技术,通过体外实验探讨CRYAB基因沉默对人骨肉瘤细胞MG-63增殖和迁移能力的影响。方法构建靶向CRYAB基因特异性sh RNA慢病毒载体并转染人骨肉瘤细胞MG-63细胞株,利用Real-time PCR和Western Blotting技术分别从m RNA和蛋白质水平检测其表达的变化。CCK-8法和细胞克隆实验检测沉默CRYAB基因后细胞的增殖能力,Transwell迁移实验检测细胞的迁移能力。结果 Real-time PCR和Western Blotting结果均表明,成功建立稳定转染sh RNA-CRYAB骨肉瘤细胞MG-63细胞株。CCK8实验结果显示sh RNA-CRYAB病毒转染组与阴性对照组相比细胞的增殖能力显著降低(P<0.05),尤其在第四、五、六和第七天表现更明显;细胞克隆实验结果显示,sh RNA-CRYAB病毒转染组和阴性对照组的克隆形成率分别为5.2%和26.0%,具有显著性差异(P<0.05)。Transwell迁移实验结果显示,sh RNA-CRYAB病毒转染组的细胞迁移率明显低于阴性对照组(P<0.01)。结论 sh RNA-CRYAB慢病毒干扰载体能有效抑制CRYAB基因在人骨肉瘤细胞MG-63中表达,进而抑制细胞增殖和迁移。
Objective To investigate the effect of CRYAB gene silencing on the proliferation and migration of human osteosarcoma MG-63 cells by lentivirus-mediated RNAi technique in vitro. Methods The lentiviral vector targeting CRYAB gene was constructed and transfected into human osteosarcoma cell line MG-63. Real-time PCR and Western Blotting were used to detect the expression of m RNA in shrimp MG-63 cells. CCK-8 assay and cell cloning assay were used to detect the proliferation of CRYAB cells. Transwell migration assay was used to detect the cell migration. Results Real-time PCR and Western Blotting results showed that MG-63 cells stably transfected with sh RNA-CRYAB osteosarcoma cells were established successfully. The results of CCK8 showed that the proliferation ability of sh RNA-CRYAB virus transfected group was significantly lower than that of negative control group (P <0.05), especially on the fourth, fifth, sixth and seventh days. The results of cell cloning The clonogenic rates of sh RNA-CRYAB virus transfection group and negative control group were 5.2% and 26.0% respectively, with significant difference (P <0.05). The results of Transwell migration showed that the cell migration rate of sh RNA-CRYAB virus transfected group was significantly lower than that of negative control group (P <0.01). Conclusion sh RNA-CRYAB lentiviral vector can effectively inhibit the expression of CRYAB gene in human osteosarcoma cell line MG-63, and then inhibit cell proliferation and migration.