Lei Lulu, Wang Shan, Liu Miao, et al. Role of PI3K/AKT signaling pathway in cell proliferation and differentiation in neuroblastoma. [J]. Journal of Chongqing Medical University 40(8):1089-1094(2015)
DOI:
Lei Lulu, Wang Shan, Liu Miao, et al. Role of PI3K/AKT signaling pathway in cell proliferation and differentiation in neuroblastoma. [J]. Journal of Chongqing Medical University 40(8):1089-1094(2015)DOI:
Role of PI3K/AKT signaling pathway in cell proliferation and differentiation in neuroblastoma
Objectives:To investigate the effects of phosphatidylinositol 3 kinase(PI3K) /protein kinaseB(AKT) signal pathway on cell proliferation and differentiation in human neuroblastoma SK-N-SH. Methods:Logarithimic phase SK-N-SH cells were subcultured into 96-well plates. The cells in 96-well plates were divided into inhibitor LY294002 group(A),agonist IGF-1 group(B),DMSO group(C),ddH2O group(D),blank group(E) and serum group(F). After 24 h,different concentrations of inhibitor LY294002 were added into group A and the final concentrations were 20,40,50,60,100 ?滋mol/L,respectively. Group B was continued to be cultured with serum-free DMEM for 12 h,then treated with different concentrations(25,50,100,150,200 ng/ml) of agonist IGF-1(which final concentra-tions were 25,50,100,150,200 ng/ml). The negative control group was added with DMSO or ddH2O with the same concentration of LY294002 or IGF-1. The blank group was only added with medium(100 ?滋l/well). All cells were cultured in appropriate media at 37 ℃ in humidified,5%CO2 incubator. The effects of specific inhibitors LY294002 and PI3K/AKT agonist IGF-1 on cell proliferation were detected by MTT assay. Morphological changes were observed under inverted phase contrast microscope. mRNA expression of TrkA in SK-N-SH cells were detected using fluorescence quantitative polymerase chain reaction(FQ-PCR). Results:①MTT assay showed that the growth of SK-N-SH cells was dramatically inhibited by LY294002. Cell proliferation ability in LY294002 group(0.39±0.17) was lower than that in negative control DMSO group(0.78±0.21)(F=3.395,P=0.018). The growth of SK-N-SH cells was promoted by insulin-like growth factors-1(IGF-1). Cell proliferation ability in IGF-1 group(0.72±0.14) was higher than that in blank ddH2O group(0.43±0.08)(F=9.480,P=0.004). ②There was no significant difference in cell differentiation morphology change between LY294002 group and IGF-1 group by microscope observations. ③FQ-PCR results showed that the expression of TrkA mRNA in LY294002 group(0.78±0.05) was lower than that in negative control group(1.56±0.02) and blank group(1.66±0.15)(F=81.89,P=0.000). There were no statistical difference in the TrkA mRNA expression between negative control group and blank group(P=0.224). And there were no statistical difference in the TrkA mRNA expression among IGF-1 group,ddH2O negative control group and blank group(F=0.27,P=0.770). Conclusion:PI3K/AKT signal pathway may be involved in cell proliferation and differentiation of human neuroblastoma SK-N-SH cells. These findings propose the key gene of PI3K/AKT pathway as an expected molecular target for the therapy of human neuroblastoma.
关键词
人神经母细胞瘤SK-N-SH细胞磷脂酰肌醇3 激酶/蛋白激酶 B(PI3K/AKT)增殖分化
Keywords
human neuroblastoma SK-N-SH cellsphosphatidylinositol 3 kinase/protein kinase Bproliferationdifferentiation
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