PI3K/AKT信号通路在SK-N-SH细胞中对增殖和分化的影响

Role of PI3K/AKT signaling pathway in cell proliferation and differentiation in neuroblastoma

摘要:

目的：探讨磷脂酰肌醇3 激酶（phosphatidylinositol 3 kinase，PI3K）/蛋白激酶 B（protein kinase B，AKT）信号通路在人神经母细胞瘤SK-N-SH（human neuroblastoma，SK-N-SH）细胞增殖和分化中的作用。方法：体外培养SK-N-SH细胞，取对数生长期细胞接种于96孔培养板，将培养板中的细胞分为A组抑制剂LY294002组、B组激动剂类胰岛素生长因子-1（insulin-like growth factors-1，IGF-1）组、C组二甲基亚砜（dimethyl sulfoxide，DMSO）组（LY294002阴性对照组）、D组ddH2O组（IGF-1阴性对照组）和空白组E、F组（培养基组），24 h后A组加入不同浓度的抑制剂LY294002，终浓度为20、40、50、60、100 ?滋mol/L，B组换用无血清DMEM继续培养12 h后加入不同浓度激动剂IGF-1，终浓度为25、50、100、150、200 ng/ml，阴性对照组分别加入与LY294002和IGF-1相同浓度的DMSO和ddH2O，空白组为100 ?滋l/孔 培养基，置于37 ℃，5%CO2的孵箱中培养，用四甲基偶氮唑盐比色法（MTT）法检测PI3K/AKT特异性抑制剂LY294002和激动剂IGF-1对SK-N-SH细胞增殖影响，倒置相差显微镜下观察细胞形态学分化改变，荧光定量聚合酶链反应（fluorescence quantitative polymerase chain reaction ，FQ-PCR）检测SK-N-SH细胞TrkA mRNA的表达与变化。结果：①MTT检测结果显示，LY294002明显抑制SK-N-SH细胞生长，LY294002组（0.39±0.17）细胞增值能力较阴性对照DMSO组（0.78±0.21）减弱，差异有统计学意义（P=0.018）。IGF-1促进SK-N-SH生长，IGF-1组（0.72±0.14）细胞增值能力较阴性对照组（ddH2O组，0.43±0.08）增值能力更强，差异有统计学意义（P=0.004）。②显微镜下观察LY294002组和IGF-1组细胞无明显分化形态学改变。③FQ-PCR结果显示，LY294002组（0.78±0.05）酪氨酸蛋白激酶A（tyrosine kinase receptor A，TrkA）信使核糖核酸（messenger ribonucleic acid，mRNA）的表达与阴性对照组（1.56±0.02）、空白组（1.66±0.15）比较，组间差异有统计学意义（F=81.89，P=0.000）。IGF-1组、ddH2O阴性对照组和空白组3组间的TrkA mRNA的表达水平无统计学差异（F=0.27，P=0.770）。结论：PI3K/AKT信号通路可能参与了SK-N-SH增殖和分化过程，该通路关键基因有望成为临床治疗神经母细胞瘤的分子靶点。

Abstract:

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.