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目的:检测沉默Ether à go-go 1(Eag1)基因对骨肉瘤增殖和血管生成的影响及其可能的调控机制。方法:构建抑制Eag1表达的短发卡RNA(short hair RNA,shRNA)重组腺病毒载体Ad5-Eag1-shRNA并感染骨肉瘤细胞株。采用实时定量聚合酶链反应(real-time polymerase chain reaction,Real-time PCR)和蛋白印迹技术(Western blot analysis,WB)检测Ad5-Eag1-shRNA的感染效能。采用CCK-8法和裸鼠成瘤实验检测感染前后骨肉瘤细胞增殖和裸鼠骨肉瘤体积的变化。采用免疫组化技术和WB技术分别检测感染前后裸鼠肿瘤组织中微血管密度(microvessel density,MVD)和血管内皮生长因子(vascular endothelial growth factor,VEGF)表达的变化。采用WB技术检测感染前后骨肉瘤细胞中VEGF、磷脂酰肌醇3-激酶(phosphoinositide 3-kinase,PI3K)和蛋白激酶B(protein kinase B,AKT)的表达变化。结果:构建的重组腺病毒载体Ad5-Eag1-shRNA具有良好的感染效能。感染Ad5-Eag1-shRNA可沉默骨肉瘤中Eag1的表达并能有效地抑制骨肉瘤细胞增殖和裸鼠骨肉瘤生长(均P<0.001),同时下调裸鼠骨肉瘤组织中MVD密度和VEGF的表达(均P<0.001)。感染Ad5-Eag1-shRNA可下调骨肉瘤细胞中VEGF的表达(P<0.05)并抑制PI3K和AKT的磷酸化(均P<0.001)。结论:沉默Eag1可能通过调控VEGF/PI3K/AKT信号通路抑制骨肉瘤增殖和血管生成。
OBJECTIVE: To investigate the effect of silencing the gene of Ether-go-go 1 (Eag1) on the proliferation and angiogenesis of osteosarcoma and its possible regulatory mechanism. Methods: Recombinant adenovirus vector Ad5-Eag1-shRNA was constructed and transfected into osteosarcoma cell lines with short hair RNA (shRNA), which inhibits the expression of Eag1. The infectivity of Ad5-Eag1-shRNA was detected by real-time polymerase chain reaction (Real-time PCR) and Western blot analysis (WB). The changes of osteosarcoma cell proliferation and the volume of osteosarcoma in nude mice before and after infection were detected by CCK-8 method and nude mice tumorigenesis test. The changes of the microvessel density (MVD) and the expression of vascular endothelial growth factor (VEGF) in the tumor tissue of nude mice before and after infection were detected by immunohistochemistry and WB respectively. The expression of VEGF, phosphoinositide 3-kinase (PI3K) and protein kinase B (AKT) in osteosarcoma cells before and after infection were detected by WB. Results: The constructed recombinant adenovirus vector Ad5-Eag1-shRNA had good infectivity. Infection of Ad5-Eag1-shRNA silences the expression of Eag1 in osteosarcoma and effectively inhibits the proliferation of osteosarcoma cells and the growth of osteosarcoma in nude mice (all P <0.001), and down-regulates the MVD density and the expression of VEGF in osteosarcoma of nude mice (All P <0.001). Infection of Ad5-Eag1-shRNA downregulated the expression of VEGF in osteosarcoma cells (P <0.05) and inhibited the phosphorylation of PI3K and AKT (all P <0.001). Conclusion: Silencing Eag1 may inhibit osteosarcoma proliferation and angiogenesis by regulating VEGF / PI3K / AKT signaling pathway.