Wortmannin抑制PI3K信号通路对人甲状腺滤泡癌细胞迁移侵袭能力的影响

Effect of PI3K inhibitor wortmannin on invasion and migration of human thyroid follicular carcinoma cell line

目的利用特异性PI3K抑制剂wortmannin抑制PI3K信号通路,研究其对人甲状腺滤泡癌细胞CGTHW-1迁移和侵袭能力的影响,并初步探索其作用机制。方法培养CGTHW-1细胞,用MTT法筛选wortmannin最佳作用浓度及时间。将细胞分为实验组和对照组(分别在血清饥饿和含血清两种条件下培养)。对照组细胞未给予任何处理,实验组细胞用wortmannin进行处理。通过划痕实验、Transwell小室侵袭实验分别观察wortmannin对细胞迁移及侵袭能力的影响。利用FITC标记的鬼笔环肽进行微丝免疫荧光染色,观察在wortmannin作用下细胞的形态学变化。免疫细胞化学SP法及半定量RT-PCR法检测wortmannin对细胞Rac1蛋白、mRNA表达的影响。结果在含血清培养液培养条件下,经wortmannin处理后,划痕伤口愈合速度减慢,CGTHW-1细胞侵袭至小室下层细胞数(13.8±3.03个)明显低于对照组(41.6±6.95个,P<0.01);wortmannin显著降低Rac1蛋白及mRNA的表达(P<0.05)。而在血清饥饿条件下,两组细胞迁移均受抑制,侵袭细胞数组间差异无统计学意义(P>0.05)。结论wortmannin能有效抑制人甲状腺癌细胞迁移侵袭,其作用机制可能是通过抑制PI3K下游分子Rac1,从而引起细胞骨架重构。PI3K及Rac1可能成为抑制甲状腺癌转移的关键性分子。

Objective To investigate the effect of phosphatidylinositol 3-kinase（PI3K） inhibitor wortmannin on migration and invasion of human thyroid follicular carcinoma cell line CGTHW-1 and its mechanism.Methods MTT colorimetric assay was used to select the optimal concentration and time of giving wortmannin.CGTHW-1 cells used in present study were divided into control group and experimental group（cultured in the medium with or without blood serum）.Cells of experimental group were treated with wortmannin,and that of control group were given no treatment.The migration and invasion ability of CGTHW-1 cells was detected by scratch test and Transwell chamber assay respectively.Microfilament fluorescence staining with fluorescein isothiocyanate（FITC）-labeled phalloidin was performed to observe the morphologic changes in CGTHW-1 cells.The expressions of Rac1 protein and mRNA in CGTHW-1 cells were examined by immunocytochemistry staining and semiquantitative RT-PCR.Results When CGTHW-1 cells were cultured in serum medium,the migrating speed of the cells slowed down,and the number of cells passing through the Transwell chamber reduced significantly after being treated with wortmannin in experimental group（13.8±3.03） than in control group（41.6±6.95,P〈0.01）.The expression of protein and mRNA of Rac1 was significantly down-regulated in CGTHW-1 cells after wortmannin treatment（P〈0.05）.However,when cells were cultured in serum-free medium,no significant difference was found in the migration and invasion ability between the two groups（P〉0.05）.Conclusions Wortmannin can effectively depress the migration and invasion of human thyroid follicular carcinoma cells,and the depression effect may be due to cytoskeleton rearrangement induced by inhibiting the expression of Rac1（the downstream molecule of PI3K）.PI3K and Rac1 are probably potential targets for human thyroid cancer treatment,which may provide a theoretical evidence for the drug treatment of thyroid cancer.