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目的研究 siRNA 对骨肉瘤 MG-63细胞株及正常成纤维细胞细胞周期蛋白 cyclin A2基因表达的抑制及对细胞增殖的影响。方法设计并化学合成靶向 cyclinA2基因的 siRNA,用oligofectamine 将其转染到骨肉瘤细胞系 MG-63及正常人成纤维细胞(HSF)中,用无义 siRNA 转染作为阴性对照,以单加缓冲液磷酸盐缓冲液(PBS)作为空白对照,实时-聚合酶链反应(PCR)、Western印迹方法检测 cyclinA2基因沉默效果,采用 MTT、RT-PCR、流式细胞、平板克隆培养等方法评价cyclinA2基因表达后被抑制后细胞的生长状态,同时检测细胞 PCNA 及 cyclinB1基因表达的改变。结果 1、10、50、100 nmol/L 浓度的 cyclinA2-siRNA 分别使 MG-63细胞 cyclinA2基因表达降低了9.4%,56.4%,79.2%和84.3%,同时 cyclinA2蛋白表达也相应降低。转染10 nmol/L及50 nmol/LsiRNA 后48 h,MG-63细胞增殖抑制分别达到39.1%及54.9%,细胞周期阻滞于 G0/G1期,平板克隆形成率降低,细胞中增殖细胞核抗原(PCNA)及 cyclinB1的 mRNA 表达水平明显下降。对正常成纤维细胞 HSF,50 nmol/L cyclinA2-siRNA 可抑制58.1%的 cyclinA2基因及蛋白表达,其导致细胞周期出现轻度改变,但细胞的增殖及平板克隆形成能力未受影响。结论 cyclinA2是肿瘤细胞增殖所依赖的关键基因,针对 cyclinA2基因的 siRNA 能有效降低细胞中 cyclinA2 mRNA 及蛋白的表达,其能抑制骨肉瘤细胞的生长而对正常细胞的增殖影响很小,靶向 cyclinA2的 siRNA 有望成为治疗骨肉瘤的新途径。
Objective To investigate the effect of siRNA on cyclin A2 gene expression in osteosarcoma MG-63 cells and normal fibroblasts and its effect on cell proliferation. Methods siRNA targeting cyclinA2 gene was designed and synthesized. The recombinant plasmid was transfected into osteosarcoma cell line MG-63 and normal human fibroblast (HSF) with oligofectamine, transfected with nonsense siRNA as a negative control, CyclinA2 gene silencing was detected by real-time polymerase chain reaction (PCR) and Western blotting in PBS buffer as control. The expression of cyclinA2 was evaluated by MTT, RT-PCR, flow cytometry and plate clone culture The gene expression was inhibited after the growth of cells, while detecting cell proliferation and cyclinB1 gene expression changes. Results cyclinA2-siRNA at 1, 10, 50 and 100 nmol / L reduced cyclin A2 expression in MG-63 cells by 9.4%, 56.4%, 79.2% and 84.3%, respectively. Proliferation of MG-63 cells reached to 39.1% and 54.9% at 48 h after transfection with 10 nmol / L and 50 nmol / L siRNA, respectively. The cell cycle arrest was at G0 / G1 phase and the rate of plate clone formation was decreased. The proliferating cell nuclear antigen (PCNA) and cyclinB1 mRNA expression levels decreased significantly. For normal fibroblast HSF, cyclinA2-siRNA inhibited 58.1% of cyclinA2 gene and protein expression, which led to mild changes in cell cycle, but cell proliferation and plate clone formation ability was not affected. Conclusions cyclinA2 is the key gene of tumor cell proliferation. The siRNA targeting cyclinA2 gene can effectively reduce the expression of cyclinA2 mRNA and protein, which can inhibit the growth of osteosarcoma cells and has little effect on the proliferation of normal cells. The expression of cyclinA2 SiRNA is expected to become a new way to treat osteosarcoma.