Department of Orthopedics, Renhe Hospital of Three Gorges University, Yichang 443001, P.R. China.
Mol Med Rep. 2013 Feb;7(2):466-70. doi: 10.3892/mmr.2012.1209. Epub 2012 Nov 28.
The present study aimed to determine the effect of small interfering RNA (siRNA)‑induced inhibition of cyclin‑dependent kinase 2 (Cdc2) expression on osteosarcoma MG63 cell proliferation and apoptosis. An siRNA expression plasmid, psilencer 2.1‑U6/Cdc2, targeting the Cdc2 gene, and a control psilencer 2.1‑U6/Scramble plasmid were constructed and transfected into MG63 cells using liposomes. Cdc2 expression in the MG63 cells was investigated by western blot analysis and real‑time polymerase chain reaction. Cell morphology was also examined. The effects of psilencer 2.1‑U6/Cdc2 on MG63 cell proliferation and the cell cycle were detected via MTT and flow cytometry, respectively. Expression levels of apoptosis‑related molecules, B‑cell lymphoma 2 (Bcl‑2) and Bcl‑2‑associated X (Bax) were determined by western blot analysis. MG63 cells stably transfected with the psilencer 2.1‑U6/Cdc2 plasmid (MG63‑siRNA/Cdc2) and negative control cells, MG63‑siRNA/Scramble, were successfully obtained. The silencing efficiencies of the Cdc2‑expressing mRNA and protein in MG63‑siRNA/Cdc2 were 86 and 89% of that of the control MG63‑siRNA/Scramble cells, respectively. Interference of Cdc2 expression inhibited MG63 cell proliferation and was demonstrated to significantly increase and decrease cells in the G2/M and S phases, respectively. Cdc2 expression silencing had negligible effects on Bcl‑2 and Bax expression in MG63 cells. In conclusion, silencing of Cdc2 expression suppresses proliferation of osteosarcoma MG63 cells but has negligible effects on apoptosis.
本研究旨在探讨小干扰 RNA（siRNA）诱导的细胞周期蛋白依赖性激酶 2（Cdc2）表达抑制对骨肉瘤 MG63 细胞增殖和凋亡的影响。构建靶向 Cdc2 基因的 siRNA 表达质粒 psilencer 2.1-U6/Cdc2 和对照 psilencer 2.1-U6/Scramble 质粒，并通过脂质体转染至 MG63 细胞。采用 Western blot 分析和实时聚合酶链反应检测 MG63 细胞中 Cdc2 的表达。观察细胞形态。通过 MTT 和流式细胞术分别检测 psilencer 2.1-U6/Cdc2 对 MG63 细胞增殖和细胞周期的影响。采用 Western blot 分析检测凋亡相关分子 B 细胞淋巴瘤 2（Bcl-2）和 Bcl-2 相关 X（Bax）的表达水平。成功获得稳定转染 psilencer 2.1-U6/Cdc2 质粒（MG63-siRNA/Cdc2）和阴性对照细胞 MG63-siRNA/Scramble 的 MG63 细胞。MG63-siRNA/Cdc2 中 Cdc2 表达 mRNA 和蛋白的沉默效率分别为对照 MG63-siRNA/Scramble 细胞的 86%和 89%。Cdc2 表达抑制干扰抑制 MG63 细胞增殖，并显著增加和减少 G2/M 和 S 期细胞。Cdc2 表达沉默对 MG63 细胞中 Bcl-2 和 Bax 的表达几乎没有影响。总之，沉默 Cdc2 表达可抑制骨肉瘤 MG63 细胞的增殖，但对细胞凋亡几乎没有影响。
