p21活化激酶6削弱非小细胞肺癌A549细胞侵袭及迁移能力的分子机制  

PAK6 signaling attenuates migratory and invasive abilities of non-small cell lung cancer cells

作　　者：蔡松旺[1] 李冬霞[2] 安军[1] 张军航[1] 

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

出　　处：《中国病理生理杂志》2014年第1期67-71,共5页Chinese Journal of Pathophysiology

基　　金：广东省科技计划(No.2012B031800063);广东省医学科研基金(No.B2012111)

摘　　要：目的:探讨调控p21活化激酶6(PAK6)的微小RNA(miRNA)与非小细胞肺癌侵袭及迁移的关系,并了解PAK6下游分子信号机制。方法:通过生物信息学数据库预测靶向PAK6的miRNA,转染miRNA模拟物及抑制剂,Western blotting及实时定量荧光PCR(real-time PCR)检测A549细胞中PAK6的表达量,萤光素报告酶实验检测预测miRNA对PAK6的调控,并行体外迁移及侵袭实验,检测转染miRNA对PC-3细胞迁移及侵袭能力的影响。转染目标miRNA及siPAK6,Western blotting检测A549细胞基质金属蛋白酶9(MMP-9)表达量。结果:生物信息学预测miR-23a可能是调控PAK6的靶向miRNA。Western blotting检测转染miR-23a组PAK6表达量下降69%,而转染miR-23a抑制剂组PAK6表达量上升52%,差异均有统计学意义。萤光素报告酶实验结果显示,转染miR-23a后野生型PAK6组萤光素酶活性下降52%,而突变组差异无统计学意义(P>0.05)。Real-time PCR检测3组PAK6 mRNA的表达差异无统计学意义(P>0.05)。体外迁移及侵袭实验示,转染miR-23a组迁移细胞数减少73%,侵袭的细胞数减少59%。Western blotting检测发现,转染siPAK6组MMP-9的表达下降85%(P<0.01),转染miR-23a组MMP-9的表达下降76%(P<0.01)。结论:miR-23a通过PAK6-MMP-9信号途径引起非小细胞肺癌细胞迁移及侵袭能力的下降。AIM： To investigate the roles of p21-activated kinase 6 （PAK6） and its target miRNA on the mi- gratory and invasive abilities of non-small cell lung cancer cells. METHODS： miRNA candidates targeting PAK6 were predicted by a target prediction program. The expression of PAK6 was measured by real-time PCR and Western blotting af- ter A549 cells were transfected with miR-23a mimics or inhibitory oligonucleotides. Luciferase reporter assay was used to determine whether PAK6 was the direct target of miR-23a. The abilities of cell migration and invasion were detected by Ma- trigel invasion assay and Transwell migration assay. The expression of PAK6 and matrix metalloproteinase 9 （MMP-9） was analyzed by Western blotting after A549 cells were transfected with siPAK6 or miR-23a mimics. RESULTS： miR-23a was identified by a target prediction program. Exogenetic over-expression of miR-23a resulted in a remarkable decrease in PAK6 expression （69%）, whereas miR-23a inhibitory oligonucleotides induced pronounced increase in PAK6 expression （52%）. The luciferase activity was significantly inhibited by 52% in wild-type PAK6 group, while there was no significant difference in the mutation group. The mRNA level of PAK6 had no change as detected by real-time PCR. Matrigel invasion assay and Transwell migration assay demonstrated there exogenetic over-expression of miR-23a markedly reduced the migra- tion and invasion of PC-3 cells （73% and 59%, respectively）. The MMP-9 expression remarkably decreased by 85% and 76% in the A549 cells transfected with siPAK6 and miR-23a mimics, respectively. CONCLUSION： miR-23a inhibits the migration and invasion of non-small cell lung cancer cells by repressing PAK6-MMP-9 signaling pathway.

关 键 词：微小RNA-23a p21活化激酶6 基质金属蛋白酶9 非小细胞肺癌 

分 类 号：R363[医药卫生—病理学]