SOCS3和Pyk2在非小细胞肺癌中的表达及相关性研究

Expression of SOCS3 and Pyk2 and their correlation in non-small cell lung cancer

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-1actacystin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Pvk2蛋白、Pyk2Tyr402和ERK1／2磷酸化水平。采用逆转录（RT）-PCR检测Pyk2mRNA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、Pvk2Tyr402和ERKl／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Pvk2蛋白降解．阻断A549细胞迁移。

Objective 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 ceils （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 phosphorylations 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.