抑癌基因PTEN对人神经母细胞瘤细胞组织因子表达调控的研究

Regulation of tissue factor expression by anti-oncogene PTEN in human neuroblastoma cell line

目的研究10号染色体同源缺失的磷酸酶-张力蛋白同源物(phosphatase and tensinhomolog deleted on chromosome 10,PTEN)对人神经母细胞瘤细胞组织因子(tissue factor,TF)表达的调控。方法以人神经母细胞瘤细胞系 SK-N-SH 和 SK-N-MC 为对象,用 Western blot 法检测 PTEN、TF的表达,用 RT-PCR 方法检测 TF 转录水平,以脂质体 Lipofectamine 2000介导进行 PTEN 基因表达载体pCMV-PTEN 的转染;以特异性抑制剂 Ly294002阻断磷脂酰肌醇-3-激酶(P13K)/AKT 的磷酸化;并以Western blot 法检测磷酸化水平。结果 SK-N-SH 细胞 PTEN 强表达而 TF 弱表达,SK-N-MC 细胞PTEN 基因表达阴性而 TF 强表达。以 PTEN 基因表达载体 pCMV-PTEN 转染 SK-N-MC 细胞,PTEN 表达增强,而 TF 表达水平降低,并伴有 AKT 磷酸化的减弱。用 P13K/AKT 信号途径特异性抑制剂Ly294002处理后,TF 的表达可呈剂量依赖性降低。结论 PTEN 基因的表达可能通过抑制 P13K/AKT信号途径而下调人神经母细胞瘤细胞中 TF 的表达,PTEN 基因的缺失可能是 TF 异常高表达的原因。

Objective To explore the role of PTEN gene in the regulation of tissue factor （TF） expression in human neuroblastoma cells. Methods Expression of PTEN or TF was determined by Western blotting. Transcription of TF was examined by RT-PCR. PTEN gene expressing vector pCMV-PTEN was transfected with Lipofectamine2000. Phosphorylation of AKT was inhibited by LY294002 and then examined by Western blotting. Results Human neuroblastoma cell line SK-N-SH was PTEN-positive and expressed low level TF, whereas an other neuroblastoma cell line SK-N-MC was PTEN-negative but expressed high level TF. TF level was downregulated in SK-N-MC cells by enforced expression of PTEN in a dose dependent manner. Inhibition of TF was achieved along with inactivation of AKT. Furthermore treatment with PI3K/AKT inhibitor LY294002 also resulted in decrease of TF expression in a dose-dependent manner. Conclusion Expression of TF is inhibited by PTEN gene via inactivating PI3K/AKT pathway, loss of PTEN might be the explanation of abberant high-level TF in human neuroblastoma. It may be at least one of the mechanisms by which loss of PTEN expression confers to cancer progression.