p21活化激酶6对人非小细胞肺癌A549细胞侵袭及迁移能力的影响

Effects of p21-activated kinase 6 on invasion and migration of human non-small-cell lung cancer A549 cells

目的:探讨p21活化激酶6(PAK6)对非小细胞肺癌侵袭及迁移能力的影响及机制研究。方法:实时荧光定量PCR检测人非小细胞肺癌A548细胞、人支气管上皮细胞、非小细胞肺癌组织及癌旁组织中PAK6mRNA的表达。A549细胞分别转染siRNA-PAK6(siPAK6组)和阴性对照(对照组),实时荧光定量PCR技术检测PAK6 mRNA,Western blotting检测PAK6表达量;并行体外迁移及侵袭实验,检测转染siRNA-PAK6对A549细胞侵袭及迁移能力的影响;共聚焦显微镜观察细胞骨架的改变。结果:A549细胞中PAK6 mRNA的表达量明显高于人支气管上皮细胞中PAK6 mRNA的表达量(3.50±1.16 vs 1.12±0.42,P<0.05),非小细胞肺癌组织中PAK6 mRNA的表达量明显高于癌旁组织中PAK6 mRNA的表达量(5.13±1.33 vs 1.08±0.37,P<0.05)。A549细胞转染siPAK6后,PAK6蛋白表达量下降72%(P<0.05),PAK6 mRNA水平明显下降(3.72±0.75 vs 0.69±0.21,P<0.05)。A549细胞转染siPAK6组侵袭及迁移出的细胞数量明显少于对照组(P<0.05)。siPAK6组A549细胞骨架应力纤维减少明显,肌动蛋白皱缩。结论:PAK6通过细胞骨架的重构影响非小细胞肺癌的侵袭及迁移能力。

AIM：To investigate the effects of p21-activated kinase 6 （PAK6） on the invasive and migratory abilities of human non-small-cell lung cancer A549 cells. METHODS：The expression of PAK6 mRNA in A549 cells, human bronchial epithelial （HBE） cells, non-small-cell lung cancer tissues and paired adjacent non-tumor tissues was measured by real-time PCR. After A549 cells were transfected with siRNA-PAK6 （siPAK6） or negative control （NC） for 48 h, the expression of PAK6 at mRNA and protein levels was measured by real-time PCR and Western blotting, respectively. The invasion and migration of A549 cells were detected by Matrigel invasion assay and Transwell migration assay. The cytoskeletal changes were observed with FITC-phalloidin staining under confocal microscope. RESULTS：The level of PAK6 mRNA in A549 cells was higher than that in HBE cells （3.50±1.16 vs 1.12±0.42, P〈0.05）. The level of PAK6 mRNA in non-small-cell lung cancer tissues was higher than that in paired adjacent non-tumor tissues （5.13±1.33 vs 1.08±0.37, P〈0.05）. The expression of PAK6 protein decreased by 72% in A549 cells transfected with siPAK6 （P〈0.05）, and the level of PAK6 mRNA significantly decreased in A549 cells transfected with siPAK6 （3.72±0.75 vs 0.69±0.21, P〈0.05）. Matrigel invasion assay and Transwell migration assay demonstrated that knockdown of PAK6 markedly attenuated the invasion and migration of A549 cells （P〈0.05）. The cytoskeletal actin remodeling and reduction of stress fibers in A549 cells transfected with siPAK6 were observed under confocal microscope. CONCLUSION：PAK6 may affect the invasive and migratory abilities of non-small-cell lung cancer cells by cytoskeletal actin remodeling.