缺氧条件下HIF-1α/ROS对肺癌A549细胞凋亡和侵袭的作用及其机制  

Effects of HIF-1α/ROS on apoptosis and invasion of lung cancer A549 cells under hypoxia and its mechanism

作　　者：黄波[1] 丁洁[2] 郭红荣 王红娟[1] 徐建群 郑泉 HUANG Bo;DING Jie;GUO Hongrong;WANG Hongjuan;XU Jianqun;ZHENG Quan(Department of Respiratory and Critical Care Medicine,Wuhan Third Hospital,Hubei Province,Affiliated Tongren Hospital,Wuhan University,Wuhan 430070,China;Hemodialysis Room,Wuhan Third Hospital,Hubei Province,Affiliated Tongren Hospital,Wuhan University,Wuhan 430070,China)

机构地区：[1]湖北省武汉市第三医院武汉大学附属同仁医院呼吸与危重症医学科,湖北武汉430070 [2]湖北省武汉市第三医院武汉大学附属同仁医院血液透析室,湖北武汉430070

出　　处：《吉林大学学报（医学版）》2023年第3期682-690,共9页Journal of Jilin University：Medicine Edition

基　　金：湖北省卫健委科研项目(WJ2021F005);湖北省武汉市卫健委医学科研项目(WX21Q43);湖北省武汉市中青年医学骨干人才培养工程项目[武卫通(2019)87号]。

摘　　要：目的:探讨缺氧条件下缺氧诱导因子1α(HIF-1α)/活性氧(ROS)对非小细胞肺癌A549细胞凋亡和侵袭的作用,并阐明其相关机制。方法:将A549细胞置于缺氧条件下培养12、24、48和72h,构建缺氧细胞模型,实时荧光定量PCR(RT-qPCR)法检测不同处理时间细胞中HIF-1αmRNA表达水平,CCK-8法检测细胞增殖能力,鉴定缺氧细胞模型并确定最佳诱导时间。将A549细胞分为对照组(21%O2)、模型组(缺氧诱导)、N-乙酰半胱氨酸(NAC)组(20 mmol·L^(-1)NAC)、NAC+利非西呱(YC-1)组(20 mmol·L^(-1)NAC+100μmol·L^(-1)YC-1)和YC-1组(100μmol·L^(-1)YC-1)。RT-qPCR和Western blotting法检测各组细胞中HIF-1α和人甲酰肽受体1(FPR1)mRNA及蛋白表达水平,流式细胞术检测各组细胞中ROS水平,透射电镜观察各组细胞自噬体结构,免疫荧光法检测各组细胞中微管相关轻链3Ⅱ(LC3-Ⅱ)表达情况,流式细胞术检测各组细胞凋亡率,Transwell小室实验检测各组侵袭细胞数。结果:随着低氧诱导时间的延长,A549细胞中HIF-1αmRNA表达水平和细胞增殖能力均明显升高(P<0.05或P<0.01),最佳低氧诱导时间为24 h。RT-qPCR和Western blotting法检测,与对照组比较,模型组细胞中HIF-1α和FPR1 mRNA及蛋白表达水平明显升高(P<0.01);与模型组比较,NAC组、NAC+YC-1组和YC-1组细胞中HIF-1α和FPR1 mRNA及蛋白表达水平均明显降低(P<0.01);与NAC组比较,NAC+YC-1组细胞中HIF-1α和FPR1 mRNA及蛋白表达水平明显降低(P<0.01)。流式细胞术检测,与对照组比较,模型组细胞中ROS水平明显升高(P<0.01);与模型组比较,NAC组、NAC+YC-1组和YC-1组细胞中ROS水平均明显降低(P<0.01);与NAC组比较,NAC+YC-1组细胞中ROS水平明显降低(P<0.01)。透射电镜观察,对照组细胞中细胞器结构完整,未见自噬体结构;模型组细胞中可见大量自噬体和明显空泡,细胞中细胞器被降解;与模型组比较,NAC组、NAC+YC-1组和YC-1组细胞中自噬体减少,其中NAC+YC-1组细胞�Objective:To discuss the effect of hypoxia inducible factor-1α(HIF-1α)/reactive oxygen species(ROS)on the apoptosis and invasion of the non-small cell lung cancer A549 cells,and to clarify the related mechanism.Methods:The A549 cells were cultured under hypoxia condition for 12,24,48 and 72 h to construct the hypoxia cell model.The expression levels of HIF-1αmRNA in the cells at different treatment time were detected by real-time fluorescence quantitative PCR(RT-qPCR)method,and the proliferation abilities of the cells were detected by CCK-8 assay;the hypoxia cell model was verified,and the optimal induction time was determined.The A549 cells were divided into control group(21%O2),model group(hypoxia induction),NAC group(20 mmol·L^(-1) NAC),NAC+YC-1 group(20 mmol·L^(-1) NAC+100μmol·L^(-1) YC-1)and YC-1(100μmol·L^(-1) YC-1)group.The expression levels of HIF-1αand FPR1 mRNA and proteins were detected by RT-qPCR and Western blotting methods;the ROS levels were detected by flow cytometry;the autophagosome structure in the cells was observed by transmission electron microscope;the expressions of microtubule-associated protein light china 3Ⅱ(LC3-Ⅱ)in the cells in various groups were detected by immunofluorescence;the apoptotic rates of the cells in various groups were detected by flow cytometry;and the number of invasion cells was detected by Transwell chamber assay.Results:With the prolongation of hypoxia induction time,the HIF-1αmRNA expression levels in the A549 cells and cell proliferation abilities were increased(P<0.05 or P<0.01),and the optimal hypoxia induction time was 24 h.The results of RT-qPCR and Western blotting methods showed that compared with control group,the expression levels of HIF-1αand FPR1 mRNA and proteins in the cells in model group were significantly increased(P<0.01);compared with model group,the expression levels of HIF-1αand FPR1 mRNA and proteins in the cells in NAC group,NAC+YC-1 group and YC-1 group were significantly decreased(P<0.01).Compared with control group,the leve

关 键 词：缺氧 缺氧诱导因子1Α 活性氧 自噬 癌 非小细胞肺 

分 类 号：R734.2[医药卫生—肿瘤]