Graduate School of the Second Military Medical University, Shanghai, China.
Department of Emergency Trauma, Shanghai East Hospital, Shanghai, China.
Balkan Med J. 2018 Mar 15;35(2):155-162. doi: 10.4274/balkanmedj.2017.0045. Epub 2018 Jan 24.
Even though epidermal growth factor-like domain 7 is known to be overexpressed in osteosarcoma and is associated with poor clinical outcome, few reports are available regarding its mechanism.
The objective of this study was to explore the effect and mechanism of downregulating epidermal growth factor-like domain 7 expression in a human osteosarcoma cell line on the biological function of co-cultured human umbilical vein endothelial cells.
Cell study.
In the present study, human osteosarcoma cell lines U2OS, Saos-2, HOS, and MG63, and normal human osteoblasts were cultured in Dulbecco's Modified Eagle Medium containing 10% fetal bovine serum and 1x antibiotics at 37 °C and 5% CO in an incubator. Of the four osteosarcoma cell lines, U2OS expresses the highest level of epidermal growth factor-like domain 7 mRNA as determined using quantitative reverse transcription polymerase chain reaction. With the knockdown of epidermal growth factor-like domain 7 in U2OS and human umbilical vein endothelial cells by lentivirus, the proliferation and apoptosis of U2OS and human umbilical vein endothelial cells were investigated using MTT and flow cytometry assays. After the co-culture of human umbilical vein endothelial cells and epidermal growth factor-like domain 7-knockdown U2OS, the effects on cell proliferation, apoptosis, adhesion, migration, and the angiogenic ability of human umbilical vein endothelial cells were detected using MTT, flow cytometry, Transwell, and tube formation assays, respectively. The expressions of phosphoinositide 3-kinase, phospho-Akt, total Akt, and vascular endothelial growth factor in human umbilical vein endothelial cells were detected using western blot assay.
Lentivirus with epidermal growth factor-like domain 7 shRNA could not significantly affect the proliferation and apoptosis of both U2OS and human umbilical vein endothelial cells, whereas the knockdown of epidermal growth factor-like domain 7 in U2OS could significantly inhibit the migration, adhesion, and angiogenic ability of co-cultured human umbilical vein endothelial cells. In addition, the expressions of phosphoinositide 3-kinase, phospho-Akt, and vascular endothelial growth factor in human umbilical vein endothelial cells decreased after co-culturing with epidermal growth factor-like domain 7-knockdown U2OS.
Epidermal growth factor-like domain 7-knockdown U2OS cells inhibit the migration, adhesion, and angiogenesis of co-cultured human umbilical vein endothelial cells by diminishing phosphoinositide 3-kinase, Akt signaling pathway activity and vascular endothelial growth factor expression.
表皮生长因子样结构域 7 在骨肉瘤中表达过度，与不良的临床预后相关，但关于其机制的报道较少。
本研究旨在探讨下调人骨肉瘤细胞系中表皮生长因子样结构域 7 的表达对共培养的人脐静脉内皮细胞生物学功能的影响及其机制。
细胞研究。
本研究中，将人骨肉瘤细胞系 U2OS、Saos-2、HOS 和 MG63 以及正常成骨细胞在含 10%胎牛血清和 1x 抗生素的 Dulbecco 改良 Eagle 培养基中于 37°C 和 5%CO2 的孵箱中培养。在这四种骨肉瘤细胞系中，U2OS 通过定量逆转录聚合酶链反应确定表皮生长因子样结构域 7 mRNA 表达水平最高。利用慢病毒下调 U2OS 和人脐静脉内皮细胞中的表皮生长因子样结构域 7，通过 MTT 和流式细胞术检测 U2OS 和人脐静脉内皮细胞的增殖和凋亡。共培养人脐静脉内皮细胞和表皮生长因子样结构域 7 敲低的 U2OS 后，通过 MTT、流式细胞术、Transwell 和管形成试验分别检测对人脐静脉内皮细胞增殖、凋亡、黏附、迁移和血管生成能力的影响。利用 Western blot 试验检测人脐静脉内皮细胞中磷酸肌醇 3-激酶、磷酸化 Akt、总 Akt 和血管内皮生长因子的表达。
表皮生长因子样结构域 7 shRNA 慢病毒对 U2OS 和人脐静脉内皮细胞的增殖和凋亡均无显著影响，而 U2OS 中表皮生长因子样结构域 7 的敲低可显著抑制共培养的人脐静脉内皮细胞的迁移、黏附和血管生成能力。此外，与表皮生长因子样结构域 7 敲低的 U2OS 共培养后，人脐静脉内皮细胞中磷酸肌醇 3-激酶、磷酸化 Akt 和血管内皮生长因子的表达降低。
表皮生长因子样结构域 7 敲低的 U2OS 细胞通过降低磷酸肌醇 3-激酶、Akt 信号通路活性和血管内皮生长因子的表达，抑制共培养的人脐静脉内皮细胞的迁移、黏附和血管生成。
