AMPKg在NSCLC组织中表达及对A549细胞增殖迁移机制探讨

目的探讨腺苷酸活化蛋白激酶(AMPK)亚基γ(AMPKg)在非小细胞肺癌(NSCLC)组织中的表达情况和对NSCLC细胞增殖、侵袭和迁移的影响及机制。方法选取2010-01-01-2014-01-01新疆医科大学附属肿瘤医院120例NSCLC患者临床和病理资料。免疫组化法检测NSCLC癌组织及癌旁组织AMPKg的表达,根据免疫评分分为高表达组和低表达组。采用NSCLC A549细胞株进行实验,慢病毒转染抑制NSCLC细胞中AMPKg的表达,分为shCtrl组(转染空病毒组)和shAMPKg组(慢病毒shRNA-AMPKg组)。细胞增殖实验、划痕实验和Transwell实验检测细胞增殖、迁移和侵袭能力。蛋白质印迹法检测蛋白激酶B/雷帕霉素靶蛋白(Akt/mTOR)信号通路相关分子变化。KaplanMeier绘制生存曲线,Log-rank检验AMPKg高表达和低表达组别总生存率的差异,单因素和多因素Cox风险回归模型分析影响NSCLC预后的独立危险因素。结果 120例NSCLC患者中,AMPKg阳性率为58.3%(70/120)。AMPKg的异常表达与NSCLC患者病理类型(χ²=7.792,P=0.005)和TNM分期(χ²=15.664,P<0.001)相关。单因素Cox分析中病理类型(HR=1.945,95%CI:0.521~1.548,P=0.046)、TNM分期(HR=1.677,95%CI:1.267~2.351,P=0.002)和AMPKg表达(HR=2.438,95%CI:1.706~4.581,P<0.001)是影响NSCLC患者预后的危险因素。多因素Cox分析中,TNM分期(HR=1.522,95%CI:1.032~2.166,P=0.004)和AMPKg表达(HR=2.093,95%CI:1.103~3.537,P=0.031)是影响NSCLC患者预后的主要独立危险因素。中位生存期AMPKg高表达组(38.6个月)低于低表达组(59.3个月),χ²=20.020,P<0.001,AMPKg高表达的患者预后较差。细胞实验结果显示抑制AMPKg表达后,shAMPKg组细胞增殖能力在第4天(F=83.070,P<0.001)、第5天(F=81.200,P<0.001)降低。shAMPKg组细胞划痕愈合率为(84.80±6.31)%,小于shCtrl组(78.08±6.53)%,t=6.106,P=0.003。shAMPKg组和shCtrl组A549细胞迁移数分别为(96.84±39.86)个和(423.30±31.91)个,差异有统计学意义,t=11.070,P=0.004。shAMPKg组细胞和shCtrl组A549细胞侵袭数分别为(119.23±21.76)个和(561.67±15.67)个,差异有统计学意义,t=28.580,P<0.001。Akt/mTOR信号通路中shAMPKg组p-mTOR、p-AKT、EIF4G蛋白表达减少。结论高表达AMPKg可能预测NSCLC患者的疾病进展和不良预后,AMPKg促进NSCLC细胞增殖、迁移和侵袭,其机制可能通过调节Akt/mTOR信号通路影响NSCLC的进展,提示AMPKg可能是NSCLC的一种潜在分子标志物。

AMPKg promoting the proliferation,invasion and migration of NSCLC cells

Objective To investigate the expression of AMP-activated protein kinase(AMPK)subunitγ(AMPKg)in nonsmall cell lung cancer(NSCLC)tissues and its effect on the proliferation,invasion and migration of NSCLC cells and its mechanism.Methods Retrospective analysis of the clinical and pathological data of 120 NSCLC patients in the Department of Pathology,Affiliated Tumor Hospital of Xinjiang Medical University from 2010-01-01 to 2014-01-01.Immunohistochemical method was used to detect the expression of AMPKg in NSCLC cancer tissues and adjacent tissues.According to the immune score,it was divided into high expression group and low expression group.Kaplan-Meier plotted survival curve,and Log-rank test of the difference in overall survival between AMPKg high expression and low expression groups.Univariate and multivariate Cox risk regression models to analyze independent risk factors affecting the prognosis of NSCLC.The NSCLC A549 cell line was used for experiments.Lentiviral transfection inhibited the expression of AMPKg in NSCLC cells.They were divided into shCtrl group(transfected with empty virus group)and shAMPKg group(lentiviral shRNA-AMPKg group).Cell proliferation test,scratch test and Transwell test detect cell proliferation,migration and invasion ability.Western blotting was used to detect the molecular changes related to protein kinase B/mammalian target of rapamycin(Akt/mTOR)signaling pathway.Results The positive rate of AMPKg in 120 NSCLC patients was 58.3%(70/120).The abnormal expression of AMPKg is related to the pathological type(χ²=7.792,P=0.005)and TNM stage(χ²=15.664,P<0.001)of NSCLC patients.In univariate Cox analysis,pathological type(HR=1.945,95%CI:0.521-1.548,P=0.046),TNM stage(HR=1.677,95%CI:1.267-2.351,P=0.002)and AMPKg expression(HR=2.438,95%CI:1.706-4.581,P<0.001)were risk factors affecting the prognosis of NSCLC patients.In the multivariate Cox analysis,TNM stage(HR=1.522,95%CI:1.032-2.166,P=0.004)and AMPKg expression(HR=2.093,95%CI:1.103-3.537,P=0.031)were the main independent risk factors affecting the prognosis of NSCLC patients.The median survival time of AMPKg high expression group(38.6 months)was lower than that of low expression group(59.3 months),χ²=20.020,P<0.001.The patients with high AMPKg expression had a worse prognosis.The cell experiment results showed that,the cell proliferation ability of shAMPKg group decreased on the 4 th day(F=83.070,P<0.001)and the 5 th day(F=81.200,P<0.001).The healing rate of cell scratches in shAMPKg group was(84.80±6.31)%,which was less than that of shCtrl group(78.08±6.53)%,t=6.106,P=0.003.The migration numbers of A549 cells in shAMPKg group and shCtrl group were 96.84±39.86 and 423.30±31.91,respectively,the difference was statistically significant,t=11.070,P=0.004.The invasion numbers of A549 cells in shAMPKg group and shCtrl group were 119.23±21.76 and 561.67±15.67,respectively,the difference was statistically significant,t=28.580,P<0.001.The expression of p-mTOR,p-AKT and EIF4 Gprotein in the shAMPKg group decreased in Akt/mTOR signaling pathway.Conclusions High expression of AMPKg may predict the disease progression and poor prognosis of NSCLC patients.AMPKg promotes the proliferation,migration and invasion of NSCLC cells,and its mechanism may affect the progression of NSCLC by regulating the Akt/mTOR signaling pathway,and suggesting that AMPKg may be a potential molecular marker of NSCLC.