PAK4-LIMK1-Cofilin信号通路对非小细胞肺癌迁移及侵袭能力的影响  

Effect of PAK4-LIMK1-cofilin signaling on non-small-cell lung cancer migration and invasion

作　　者：李冬霞[1] 王永玲[1] 蔡松旺[2] 

机构地区：[1]新乡医学院基础医学院,河南新乡453003 [2]中山大学附属第三医院胸外科,广东广州510630

出　　处：《中国病理生理杂志》2015年第12期2130-2135,共6页Chinese Journal of Pathophysiology

基　　金：广东省科技计划(No.2012B031800063);广东省卫生厅基金资助项目(No.B2012111)

摘　　要：目的:探讨p21活化激酶4(PAK4)对非小细胞肺癌(NSCLC)侵袭和迁移能力的影响及相关机制。方法:PAK4-siRNA或阴性对照转染A549和NCI-H520细胞株,实时荧光定量PCR和Western blot分别检测细胞PAK4 mRNA和蛋白表达水平,Transwell小室法检测其对细胞侵袭和迁移能力的影响;Western blot检测其LIMK1、cofilin及其磷酸化水平;免疫共沉淀检测PAK4和LIMK1蛋白是否直接绑定;体外激酶分析实验检测LIMK1是否是PAK4的激酶底物;Western blot检测10例非小细胞肺癌组织中PAK4和磷酸化LIMK1的相关性;PAK4-siRNA和LIMK1质粒共转染A549和NCI-H520细胞后观察细胞迁移和侵袭能力变化。结果:沉默PAK4后A549和NCIH520细胞迁移和侵袭能力明显减弱(P<0.05);LIMK1和cofilin蛋白水平无显著变化,而磷酸化LIMK1和磷酸化cofilin水平显著下调;免疫共沉淀结果示PAK4与LIMK1相互绑定;激酶分析实验结果显示,激酶缺陷型PAK4(K350M)使LIMK1磷酸化的作用显著低于野生型PAK4或活化型PAK4(S445N)(P<0.05)。Western blot检测结果示非小细胞肺癌组织中PAK4表达上调的程度与磷酸化LIMK1含量呈正相关(P<0.05);PAK4-siRNA和LIMK1质粒共转染A549和NCI-H520细胞后,迁移和侵袭细胞数均高于PAK4-siRNA转染组(P<0.05)。结论:PAK4通过直接磷酸化LIMK1而促进非小细胞肺癌的迁移及侵袭能力。AIM： To explore the mechanism of p21-activated kinase 4（ PAK4） on non-small-cell lung cancer（ NSCLC） migration and invasion. METHODS： After A549 and NCI-H520 cell lines were transfected with PAK4-siRNA or negative control,the expression of PAK4 at mRNA and protein levels was detected by real-time PCR and Western blot,respectively. The invasion and migration of A549 cells and NCI-H520 cells were measured by Matrigel invasion assay and Transwell migration assay. LIMK1,cofilin,and their respective phosphorylation were examined by Western blot. The interaction of PAK4 and LIMK1 was investigated by co-immunoprecipitation assay. The relationship between PAK4 and LIMK1 phosphorylation was examined by a protein kinase assay in the A549 cells and NCI-H520 cells. The expression of PAK4 and p-LIMK1 in 10 human NSCLC tissues was examined by Western blot. A549 cells and NCI-H520 cells were individually or commonly transfected with PAK4-siRNA or LIMK1 plasmid in order to observe the cell migration and invasion. RESULTS： After A549 cells and NCI-H520 cells were transfected with PAK4-siRNA for 48 h,the expression of PAK4 at mRNA and protein levels,and the numbers of invasion and migration cells in PAK4-siRNA group were lower than those in control group. Compared with control group,the phosphorylation of LIMK1 and cofilin was lower in PAK4-siRNA group,whereas the total expression levels of LIMK1 and cofilin did not change. The results of co-immunoprecipitation assays showed that PAK4 specifically interacted with LIMK1 in A549 and NCI-H520 cells. LIMK1 phosphorylation in the presence of PAK4（ K350M） was significantly lower than that in the presence of PAK4（ WT） or PAK4（ S445N） in the protein kinase assay. The PAK4 upregulation was positively correlated with the level of p-LIMK1（ P〈0. 05）. After A549 cells and NCI-H520 cells were co-transfected with PAK4-siRNA and LIMK1 plasmid,the migration and invasion cell numbers in cotransfection group were higher than those in PAK4-siRNA transfection group. CON

关 键 词：p21活化激酶4 LIM激酶1 非小细胞肺癌 细胞迁移 细胞侵袭 

分 类 号：R363[医药卫生—病理学]