Expression of Ether à go-go 1 and its molecular mechanism of regulating the malignant phenotype of osteosarcoma

Abstract

Objective: To explore the expression of ether à go-go 1 (Eag1) in human osteosarcoma and its molecular mechanisms of regulating the malignant phenotype of osteosarcoma. Methods: The expression levels of Eag1 in osteosarcoma cell lines and human osteosarcoma tissues were detected by reverse transcription polymerase chain reaction (RT-PCR), western blot analysis and immunohistochemistry. The small interfering RNA (siRNA) was used to inhibit the expression of Eag1. The abilities of proliferation and invasion in osteosarcoma cells transfected with Eag1 siRNA were determined by CCK-8, colony formation assay, transwell assay and wound healing assay. The osteosarcoma xenograft model of nude mouse was established and tumor growth curve was drawn. Western blot analysis was performed to detect the expression of vascular endothelial growth factor (VEGF) and signal transducer and activator of transcription 3 (STAT3) in osteosarcoma cells transfected with Eag1 siRNAs. Results: Eag1 was overexpressed in the osteosarcoma cells and tissues. Compared with the scrambled siRNA group, the cell survival rates of Eag1 siRNA1 and Eag1 siRNA2 groups of the two cell lines were significantly lower. [MG-63 cells: scrambled siRNA group (100.0±4.65)%, Eag1 siRNA1 group (63.57±3.89)%, and Eag1 siRNA2 group (54.13±3.70)%; Saos-2 cells: scrambled siRNA group (100.00±5.46)%, Eag1 siRNA1 group (56.70±5.34)%, and Eag1 siRNA2 group (40.27±5.28)% (P<0.001 for all)]. Similar results were obtained from colony formation assay. The colony formation rates of MG-63 cells: the scrambled siRNA group was (92.00±3.46)%, Eag1 siRNA1 group (60.00±3.06)%, and Eag1 siRNA2 group (53.67±2.40)%; the colony formation rates of Saos-2 cells: the scrambled siRNA group was (92.00±5.57)%, Eag1 siRNA1 group (52.33±5.13)%, and Eag1 siRNA2 group (41.67±2.73)%. Compared with the scrambled siRNA group, P<0.001 for all. The tumor volumes of osteosarcoma xenograft in the Eag1 siRNA1 and Eag1 siRNA2 groups were significantly smaller than that in the scrambled siRNA group after 10 days treatment (P<0.01 for all). The invasion assay data showed that MG-63 and Saos-2 cells transfected with Eag1 siRNAs exhibited the ability of cell invasion, when compared with the cells transfected with scrambled siRNA. (Invasive cell number of MG-63 cells: the scrambled siRNA group was 134.00±3.61, Eag1 siRNA1 group 105.20±2.52, and Eag1 siRNA2 group 91.00±3.01; Invasive cell number of Saos-2 cells: the scrambled siRNA group was 132.30±3.23, Eag1 siRNA1 group 114.30±3.48, and Eag1 siRNA2 group 82.67±6.33. Compared with the scrambled siRNA group, P<0.01 for all. The migration rates were (62.48±1.83)%, (35.98±1.23)% and (32.30±1.20)% in the three groups of MG-63 cells, and (70.15±1.42)%, (41.38±1.34)% and (32.40±1.92)% in the three groups of Saos-2 cells, respectively. Compared with the scrambled siRNA group, P<0.001 for all. Notably, the expression levels of VEGF decreased evidently after Eag1 siRNAs transfection, paralleled with reduction in the expression levels of STAT3. Conclusions: Eag1 may promote osteosarcoma cell proliferation and invasion by targeting STAT3-VEGF pathway and may be a potential therapeutic target for osteosarcoma.