Department of Orthopedics, The People's Hospital of Guangxi Zhuang Autonomous Region, Nanning, Guangxi 530021, P.R. China.
Department of Orthopedics, Zhejiang Provincial Hospital of TCM, Hangzhou, Zhejiang 310000, P.R. China.
Mol Med Rep. 2018 Jan;17(1):422-427. doi: 10.3892/mmr.2017.7866. Epub 2017 Oct 25.
The present study aimed to investigate the effect of Dermatopontin (DPT) gene silencing on the apoptosis and proliferation of osteosarcoma MG‑63 cells. Three eukaryotic expression vectors of short hairpin (sh)RNA fragments targeting different loci of DPT were designed and transfected into an osteosarcoma cell line MG‑63. The cells were assigned to a blank, shRNA‑control, DPT‑shRNA‑a, DPT‑shRNA‑b or DPT‑shRNA‑c group. The shRNA with the highest silencing efficiency was screened using reverse transcription‑quantitative polymerase chain reaction and western blotting. The screened shRNA was transfected into MG‑63 cells. The proliferation, cell cycle and apoptosis of MG‑63 cells were measured using a Cell Counting Kit‑8 assay, flow cytometry and Annexin V‑fluorescein isothiocyanate assay. The recombinant plasmids containing DPT shRNA were successfully constructed. DPT gene silencing was able to significantly reduce the proliferation rate of MG‑63 cells (P<0.05). The proportion of cells in the G0/G1 phase and in the G2/M phase increased significantly (both P<0.05), while the proportion of cells in the S phase decreased (P<0.05). Furthermore, the cell apoptosis rate increased significantly (P<0.05). These results demonstrate that DPT gene silencing is able to reduce the proliferation of MG‑63 cells, slow down cell cycle progression and promote apoptosis, hence may become a novel target for the treatment of osteosarcoma.
本研究旨在探讨沉默 Dermatopontin(DPT)基因对骨肉瘤 MG-63 细胞凋亡和增殖的影响。设计了针对 DPT 不同基因位点的三个短发夹(sh)RNA 片段的真核表达载体,并转染入骨肉瘤细胞系 MG-63。将细胞分为空白组、shRNA-对照 组、DPT-shRNA-a 组、DPT-shRNA-b 组和 DPT-shRNA-c 组。采用逆转录-定量聚合酶链反应和 Western blot 筛选沉默效率最高的 shRNA。将筛选出的 shRNA 转染入 MG-63 细胞。采用细胞计数试剂盒-8 法、流式细胞术和 Annexin V-荧光素异硫氰酸酯法检测 MG-63 细胞的增殖、细胞周期和凋亡。成功构建了含有 DPT shRNA 的重组质粒。DPT 基因沉默可显著降低 MG-63 细胞的增殖率(P<0.05)。G0/G1 期和 G2/M 期细胞比例显著增加(均 P<0.05),而 S 期细胞比例减少(P<0.05)。此外,细胞凋亡率显著增加(P<0.05)。这些结果表明,DPT 基因沉默可降低 MG-63 细胞的增殖,减缓细胞周期进程并促进凋亡,因此可能成为骨肉瘤治疗的新靶点。