PDK1对肺癌A549细胞生物学行为的影响  

Effect of PDK1 on biological characteristics of lung cancer A549 cells

作　　者：李苏霞[1] 何钎铖[1] 陈素秀[1] LI Su-xia;HE Qian-cheng;CHEN Su-xiu(Department of General Medicine,The First Affiliated Hospital of Wenzhou Medical Universit)

机构地区：[1]温州医科大学附属第一医院全科医学科,浙江温州325000

出　　处：《中国病理生理杂志》2018年第2期225-231,共7页Chinese Journal of Pathophysiology

基　　金：温州市科技计划项目(No.Y20130041)

摘　　要：目的:探讨3-磷酸肌醇依赖性蛋白激酶1(PDK1)对肺癌细胞株A549生物学特性的影响及其潜在的作用机制。方法:采用Western blot和real-time PCR检测肺正常上皮细胞BEAS-2B和肺癌细胞(H460、SPCA1和A549)中PDK1的表达水平。利用RNA干扰技术下调肺癌A549细胞中PDK1的表达,然后分别采用CCK-8法和流式细胞术检测细胞活力及凋亡的变化;Western blot检测增殖及周期相关蛋白的表达和Akt/Fox O1信号通路的活性。最后通过Akt信号通路特异性激动剂胰岛素样生长因子1(insulin-like growth factor-1,IGF-1)进一步验证PDK1和Akt/Fox O1信号通路的相互作用。结果:相较于肺正常上皮细胞BEAS-2B,肺癌细胞中的PDK1均呈高表达(P<0.05)。干扰A549细胞中PDK1的表达后,细胞活力显著降低,周期进程缓慢,而细胞凋亡显著增加。Western blot实验结果显示,干扰PDK1后,细胞中cyclin D1、CDK4、p-Rb、Bcl-2、p-Akt及胞浆中p-Fox O1的含量显著下降,P27、cleaved caspase-3及核内Fox O1的蛋白水平显著升高(P<0.05)。预先给予Akt激动剂IGF-1可部分逆转干扰PDK1对Akt/Fox O1信号通路的影响,并增加A549细胞的活力(P<0.05)。结论:干扰人肺癌细胞株A549的PDK1可通过抑制Akt/Fox O1信号通路而调控周期及凋亡相关因子的表达,发挥生长抑制及凋亡促进作用,提示PDK1可作为肺癌的潜在诊疗靶点。AIM： To investigate the effects of 3-phosphoinositide-dependent protein-1（ PDK1） on the biological characteristics of non-small-cell lung cancer cell line A549 and the underlying mechanisms. METHODS： The expression levels of PDK1 in lung normal epithelial cell line BEAS-2 B and different lung cancer cell lines H460,SPCA1 and A549 were determined by Western blot and real-time PCR. Small interfering RNA was used to down-regulated PDK1 expression in the A549 cells,and then cell viability and apoptosis were measured by CCK-8 assay and flow cytometry,respectively. The expression of cell cycle-and apoptosis-related molecules at protein level and the activation of Akt/Fox O1 pathway were measured by Western blot. Insulin-like growth factor-1（ IGF-1,one of the most potent Akt activators） was used to evaluate the interaction between PDK1 and Akt/Fox O1 pathway. RESULTS： Compared with lung normal epithelial cell line BEAS-2 B,PDK1 expression in the lung cancer cell lines was obviously increased（ P 〈 0. 05）. Knockdown of PDK1 suppressed cell viability and cell cycle,but promoted the apoptosis of the A549 cells. The results of Western blot showed that the protein levels of cyclin D1,CDK4,p-Rb,Bcl-2,p-Akt and cytoplasmic p-Fox O1 were significantly decreased after knockdown of PDK1,with increases in the protein levels of P27,cleaved caspase-3 and nuclear Fox O1. Pre-incubation with IGF-1 partly reversed the effect of PDK1 knockdown on Akt/Fox O1 pathway and increased the viability of A549 cells.CONCLUSION： In human non-small-cell lung cancer A549 cells,knockdown of PDK1 suppresses cell viability and promotes cell apoptosis by regulating the expression of cell cycle-and apoptosis-related molecules via Akt/Fox O1 pathway,suggesting that PDK1 may be a potential target for diagnosis and theatment of lung cancer.

关 键 词：3-磷酸肌醇依赖性蛋白激酶1 肺癌 细胞活力 细胞凋亡 RNA干扰 

分 类 号：R0[医药卫生] R730.23