Abstract:

Objective To explore the effects of silencing Ether-go-go 1（Eag1） potassium channel by short hairpin RNA（shRNA） on the cell proliferation， cell cycle and expression of cyclin D1／E in osteosarcoma cells. Methods The successfully constructed Ad5-Eag1-shRNA vector was transfected into MG-63 cells （inhibition group）. The MG-63 cells transfected with nonsense sequence（Ad5-Control-shRNA vector） were assigned as empty transfection group and cells without any treatment was used as control group. The reverse transcription polymerase chain reaction and Western blotting were used to detect the mRNA and protein levels of Eag1 in MG-63 cells transfected with Ad5-Eag1-shRNA. Cell proliferation of each group was detected by CCK-8 assay and clone formation assay，respectively. Changes of cell cycle and protein levels of cyclin D1／E were detected by flow cytometry and Western blotting，respectively. After the establishment of osteosarcoma xenograft model in nude mice，the changes of tumor volume were measured in each group. Results The protein and mRNA levels of Eag1 were lower in inhibition group rather than empty transfection group and control group with statistically significance difference（P＜0. 05）. Compared with other two groups， there were decreased cell proliferation，colony formation and the proportion of cells in S phase and cell cycle protein levels but increased proportion of cells in G0／G1 phase in inhibition groups（P＜0.05）. The tumor volume of the inhibition group was smaller than those of the empty transfection group and the control group（P＜0.05）. Conclusion Eag1 silencing could suppress osteosarcoma progression and cell cycle through cyclin D1 and E pathway and suggest that Eag1 may be a novel molecular target for osteosarcoma therapy. The inhibition of Eag1 gene expression by shRNA can inhibit the proliferation and induce G0／G1 arrest，which may be related to the regulation of cyclin D1 and E pathway， and is expected to become a new target for treatment and diagnosis of osteosarcoma.