摘要目的 探讨SOCS3和Pyk2在非小细胞肺癌(NSCLC)中的表达及其相互作用.方法 分别采用免疫组织化学和免疫荧光染色方法检测SOCS3和Pyk2在100例NSCLC组织、人支气管上皮细胞HBE和6个NSCLC细胞系中的表达.采用甲基化特异性PCR方法检测A549细胞中SOCS3基因甲基化状态.A549细胞经蛋白酶体抑制剂β-lactacystin预处理,再加入去甲基化试剂5-aza-2 ’-脱氧胞苷(5-aza),或转染SOCS3突变质粒,检测Pyk2蛋白、Pyk2 Tyr402和ERK1/2磷酸化水平.采用逆转录(RT)-PCR检测Pyk2 mRNA水平.采用Transwell小室测定细胞迁移情况.结果 100例NSCLC组织中,43例(43.0％) SOCS3阳性表达,65例(65.0％) Pyk2高表达.在NSCLC组织和细胞系中均发现SOCS3和Pyk2表达负相关.5-aza能恢复A549细胞中SOCS3的表达.SOCS3依赖于其SH2和KIR功能区与Pyk2相互作用,从而降低Pyk2蛋白、Pyk2 Tyr402和ERK1/2磷酸化水平,抑制A549细胞迁移.结论 NSCLC中,SOCS3可能通过SOCS-box功能区介导的蛋白酶体途径促使Pyk2蛋白降解,阻断A549细胞迁移.

abstractsObjective To investigate the expression of SOCS3 and Pyk2 and their correlations in non-small cell lung cancer (NSCLC).Methods The expression of SOCS3 and Pyk2 was detected in 100 cases of NSCLC,human bronchial epithelial cells (HBE) and 6 lung cancer cell lines by immunohistochemistry and immunofluorescence staining.The methylation status of SOCS3 was investigated in A549 cells by methylation-specific PCR.A549 cells were either treated with a demethylation agent 5-aza-2'-deoxycytidine (5-aza) or transfected with three SOCS3 mutants with various functional domains deleted.Besides,the cells were pretreated with a proteasome inhibitor β-1actacystin where indicated.The effects of SOCS3 on Pyk2 expression,Pyk2 Tyr 402 and ERK1/2 phnsphorylations were assessed by Western blot.RT-PCR was used to estimate Pyk2 mRNA levels.Transwell experiments were performed to evaluate cell migration.Results SOCS3(43.0％,43/100) and Pyk2 (65.0％,65/100) were expressed in NSCLC.A significant negative correlation was found between SOCS3 and Pyk2 in both NSCLC tissues and cell lines.SOCS3 was aberrantly methylated and 5-aza restored SOCS3 expression.Transfection studies indicated that exogenous SOCS3 interacted with Pyk2,and both Src homology 2 (SH2) and kinase inhibitory region (KIR) domains contributed to Pyk2 binding.Furthermore,SOCS3 was found to inhibit Pyk2-associated ERK1/2 activity in A549 cells.SOCS3 possibly promoted degradation of Pyk2 in a SOCS-box-dependent manner and interfered with cell migration.Conclusions The data indicates that SOCS3 definitely plays roles in regulating Pyk2 signaling,and cell motility.Decreased SOCS3 induced by methylation may confer a migration advantage to A549 cells.