Key Laboratory for Biotechnology on Medicinal Plants of Jiangsu Province, School of Life Science, Jiangsu Normal University, Xuzhou 221116, PR China.
Department of Oncology, Beijing Hospital, Beijing 100730, PR China.
Biomed Pharmacother. 2017 Dec;96:1324-1334. doi: 10.1016/j.biopha.2017.11.079. Epub 2017 Dec 6.
The purpose of this study was to explore the role by which the DNA-dependent protein kinase complex catalytic subunit (DNA-PKcs) influences osteosarcoma MG-63 cell apoptosis, proliferation, migration and invasion. Osteosarcoma tissues and adjacent normal tissues were obtained from 57 osteosarcoma patients. Human osteosarcoma MG-63 cells were assigned into designated groups including the blank, siRNA-negative control (NC) and siRNA-DNA-PKcs groups. RT-qPCR and Western blotting methods were employed to evaluate the mRNA and protein expressions of DNA-PKcs. A cell counting kit-8 (CCK-8) assay was performed to assess cell viability. The evaluation of cell migration and invasion were conducted by means of Scratch test and Transwell assay. Flow cytometry with PI and annexin V/PI double staining was applied for the analysis of the cell cycle and apoptosis. Twenty-Four Balb/c nude mice were recruited and randomly divided into the blank, siRNA-NC and siRNA-DNA-PKcs groups. Tumorigenicity of the Balb/c nude mice was conducted to evaluate the rate of tumor formation, as well as for the assessment of tumor size and weight, and confirm the number of lung metastatic nodules in the mice post transfection. Osteosarcoma tissues were found to possess greater expression of DNA-PKcs than that of the adjacent normal tissues. DNA-PKcs expression in osteosarcoma tissues were correlated with the clinical stage and metastasis. Compared with the blank and siRNA-NC groups, proliferation, miration, as well as the invasion abilities of the MG-63 cells increased. Furthermore, an increase in apoptosis and cells at the G1 stage in the MG-63 cells was observed, while there were reductions in the cells detected at the S stage. The mRNA and protein expressions of CyclinD1, PCNA, Bcl-2 decreased while those of Bax increased in the siRNA-DNA-PKcs group. The tumor formation rate, tumor diameter, weight and lung metastatic nodules among the nude mice in the siRNA-DNA-PKcs group were all lower than those in the blank and siRNA-NC groups. The observations and findings of the study suggested that the silencing of DNA-PKcs inhibits the proliferation, migration and invasion, while acting to promote cell apoptosis in MG-63 cells and osteosarcoma growth in nude mice.
本研究旨在探讨 DNA 依赖性蛋白激酶复合物催化亚基 (DNA-PKcs) 在骨肉瘤 MG-63 细胞凋亡、增殖、迁移和侵袭中的作用。收集 57 例骨肉瘤患者的骨肉瘤组织和相邻正常组织。将人骨肉瘤 MG-63 细胞分为空白组、siRNA 阴性对照组（siRNA-NC）和 siRNA-DNA-PKcs 组。采用 RT-qPCR 和 Western blot 法检测 DNA-PKcs 的 mRNA 和蛋白表达。细胞计数试剂盒-8（CCK-8）法检测细胞活力。划痕实验和 Transwell 实验评估细胞迁移和侵袭能力。流式细胞术碘化丙啶（PI）和 Annexin V/PI 双染法分析细胞周期和凋亡。招募 24 只 Balb/c 裸鼠，随机分为空白组、siRNA-NC 组和 siRNA-DNA-PKcs 组。裸鼠成瘤性实验评估成瘤率、肿瘤大小和重量，以及转染后裸鼠肺转移结节数。骨肉瘤组织中 DNA-PKcs 的表达高于相邻正常组织。骨肉瘤组织中 DNA-PKcs 的表达与临床分期和转移有关。与空白组和 siRNA-NC 组相比，MG-63 细胞的增殖、迁移和侵袭能力增强。此外，MG-63 细胞中凋亡和 G1 期细胞增加，S 期细胞减少。siRNA-DNA-PKcs 组细胞中细胞周期蛋白 D1（CyclinD1）、增殖细胞核抗原（PCNA）、B 细胞淋巴瘤-2（Bcl-2）mRNA 和蛋白表达降低，Bax 表达增加。siRNA-DNA-PKcs 组裸鼠的肿瘤形成率、肿瘤直径、重量和肺转移结节均低于空白组和 siRNA-NC 组。研究结果表明，沉默 DNA-PKcs 可抑制 MG-63 细胞增殖、迁移和侵袭，促进细胞凋亡，抑制裸鼠骨肉瘤生长。
