鞘氨醇激酶1促进SW480细胞迁移机制探讨

Mechanism of sphingosine kinase 1 regulating the migration and invasion of SW480 cells

目的鞘氨醇激酶1(sphingosine kinase 1,SK1)具有调控细胞迁移的重要作用,丝/苏氨酸激酶(serine/threonine kinase,AKT)参与其调控过程。本研究旨在探讨SK1对结肠癌细胞株SW480迁移和侵袭的影响及与AKT信号通路的关系。方法以不同浓度12-豆蔻酸-13-乙酸佛波醇(phorbol 12-myristate-13-acetate,PMA)干预SW480细胞,观察细胞SK1、p-AKT基因和蛋白表达水平变化情况。采用Ttranswell法观察PMA和AKT抑制剂对SW480细胞迁移和侵袭能力的影响。结果 CCK8实验显示,25、50、100nmol/L PMA刺激的SW480细胞培养液吸光度(A)值分别为0.780±0.030、0.844±0.057和0.998±0.045,与空白对照组(A值0.631±0.051)比较均明显增加,F=93.48,P<0.001;PMA可诱导细胞内SK1基因和蛋白表达水平明显增高;Transwell实验结果显示,100nmol/L PMA增强SW480细胞迁移和侵袭(分别为169.8±10.82和89.6±8.44),较对照组(分别为63.6±8.67和39.6±6.12)明显增加,而AKT抑制剂AKTi1/2可减少PMA诱导的迁移和侵袭SW480细胞数量(分别为91.8±9.36和57.2±8.98);PMA增强SW480细胞SK1表达同时促进AKT蛋白磷酸化水平,而AKTi1/2可拮抗PMA的作用。结论 SK1可促进SW480细胞的迁移和侵袭能力,其机制与上调AKT信号通路有关。

OBJECTIVE Sphingosine kinase 1 （SK1） has important effect in cell migration, serine/threonine kinase （AKT） was involved this procedure. The study aimed to investigate the effects of SK1 on migration and invasive ability of SW480 cells, the colon cancer cell line, and explore the relationship with signal AKT. METHODS Phorbol 12-myristate- 13-acetate （PMA） in different doses was administered to SW480 cells. SK1 and p-AKT gene and protein expression was observed by PCR and Western Blot. After cocultured with AKT inhibitor, migration and invasion of SW480 cells were measured by Transwell methods. RESULTS 25,50,100 nmol/L PMA induced SW480 cell proliferation in a dose-depend- ent manner （F=93.48,P〈0. 001） and increased the expression of SK1 gene and protein in a dose-depended manner, 100 nmol/L PMA had the best effect in elevating expression of SK1. In Transwell assay, the cell number of migration and invasion in control group was 63.6±8.67 and 39.6±6.12, 100 nmol/L PMA promoted the processes of SW480 cell migration and invasion（169.8 ±10.82 and 89.6±8.44 respectively）, while AKTil/2, an AKT inhibitor reduced the cell migration and invasion induced by PMA （91.8±9.36 and 57.2±8.98 respectively）. PMA enhances SKI expression in SW480 cell while promoting AKT protein phosphorylation, while AKTil/2 can antagonize PMA. CONCLUSION In this study, it is confirmed that SK1 plays an important role in regulation the migration and invasion of SW480 cells, which may involve the AKT phosphorylation.