Liu Ye, Fan Shun-Wu, Ding Jia-Yi, Zhao Xing, Shen Wei-Liang
Department of Orthopaedics, Sir Run Shaw Hospital, Zhejiang University, Hangzhou 310016, China.
Zhonghua Yi Xue Za Zhi. 2007 Mar 6;87(9):627-33.
To study the impact of small interference RNA (siRNA) targeting cyclin A2 gene on the growth of MG-63 and HSF cells and to explore whether cyclin A2 siRNAs could become a useful tool in the treatment of osteosarcoma.
One pair of siRNA targeting the cyclin A2 mRNA and a pair of nonsense siRNA were designed according the current criteria. SiRNA was chemically synthesized and purified. The siRNA was transfected into osteosarcoma cell line MG-63 and normal human skin fibroblast (HSF) cells via oligofectamine. Cells transfected with nonsense siRNA served as the negative control and those only treated with PBS as the blank control group. Quantitative fluorescence RT-PCR, Western-blot, MTT assay, reverse transcriptase (RT)-PCR, flow cytometry and colony-forming test were employed to evaluate the efficacy of RNA interference. At the same time, the mRNA expression of PCNA and cyclin B1 in siRNA treated MG-63 cells were examined.
1 nmol/L, 10 nmol/L, 50 nmol/L and 100 nmol/L cyclin A2-siRNA can reduced cyclin A2 mRNA and protein expression respectively by 9.43%, 56.35%, 79.17% and 84.30% as compared with that of the blank control group, whereas the negative and blank control groups had similar expression levels. After 48 h treatment with 10 nmol/L siRNA, MG-63 cells were arrested in G0/G1 phase and the proliferation of this tumor cell was suppressed by 39.06% 48 h after transfection. Furthermore, the treated MG-63 cells showed less colony-forming ability. Increasing the siRNA concentration to 50 nmol/L can further inhibit the proliferation of MG-63 cells by 54.94%. In addition, the cyclin A2-depleted MG-63 cells showed decreased levels of PCNA and cyclin B1. In contrast, although cyclin A2 mRNA and protein expression in HSF reduced 58.13% 48 h after treatment by 50 nmol/L siRNA, these cells exhibited only a slight change in cell cycle, and no clear inhibition of proliferation and impaired plate colony-forming ability was observed.
Cyclin A2 gene maybe served as a potential target for tumor therapy. RNA interference induces obvious inhibition of cyclin A2 mRNA and protein expression in MG-63 and HSF cells, which consequently downregulate the proliferation of MG-63 cells. There is few inhibitory effect on the proliferation by siRNAs for HSF cells. These results indicate that siRNAs against cyclin A2 could become a potential antiproliferative tool in future antitumor therapy.
研究靶向细胞周期蛋白A2基因的小干扰RNA（siRNA）对MG-63和HSF细胞生长的影响，探讨细胞周期蛋白A2 siRNAs能否成为治疗骨肉瘤的有效工具。
按照现行标准设计一对靶向细胞周期蛋白A2 mRNA的siRNA和一对无义siRNA。化学合成并纯化siRNA。通过脂质体转染试剂将siRNA转染至骨肉瘤细胞系MG-63和正常人皮肤成纤维细胞（HSF）。转染无义siRNA的细胞作为阴性对照，仅用PBS处理的细胞作为空白对照组。采用定量荧光RT-PCR、Western印迹、MTT法、逆转录（RT）-PCR、流式细胞术和集落形成试验评估RNA干扰的效果。同时，检测siRNA处理的MG-63细胞中PCNA和细胞周期蛋白B1的mRNA表达。
与空白对照组相比，1 nmol/L、10 nmol/L、50 nmol/L和100 nmol/L的细胞周期蛋白A2-siRNA可分别使细胞周期蛋白A2 mRNA和蛋白表达降低9.43%、56.35%、79.17%和84.30%，而阴性对照组和空白对照组表达水平相似。用10 nmol/L siRNA处理48 h后，MG-63细胞停滞于G0/G1期，转染后48 h该肿瘤细胞的增殖受到39.06%的抑制。此外，处理后的MG-63细胞集落形成能力降低。将siRNA浓度增加至50 nmol/L可进一步抑制MG-63细胞增殖54.94%。另外，细胞周期蛋白A2缺失的MG-63细胞中PCNA和细胞周期蛋白B1水平降低。相比之下，尽管50 nmol/L siRNA处理48 h后HSF细胞中细胞周期蛋白A2 mRNA和蛋白表达降低了58.13%，但这些细胞仅在细胞周期上有轻微变化，未观察到明显的增殖抑制和集落形成能力受损。
细胞周期蛋白A2基因可能是肿瘤治疗的潜在靶点。RNA干扰可显著抑制MG-63和HSF细胞中细胞周期蛋白A2 mRNA和蛋白表达，从而下调MG-63细胞的增殖。siRNAs对HSF细胞增殖的抑制作用较小。这些结果表明，针对细胞周期蛋白A2的siRNAs可能成为未来抗肿瘤治疗中一种潜在的抗增殖工具。
