摘要
目的:探讨低氧促进肺腺癌A549细胞迁移的机制。方法:培养肺腺癌A549细胞,转染慢病毒获得稳定敲低ACC1的A549细胞株,转染si-RNA获得敲低SREBP-1的A549细胞。分别以低氧(5%O_(2))联合低氧诱导因子1α(HIF-1α)抑制剂PX-478(25μmol)处理A549细胞,低氧联合亚油酸(LA)(20μmol)处理敲低ACC1的A549细胞,低氧处理敲低胆固醇调节原件结合蛋白1(SREBP-1)的A549细胞。Transwell实验检测细胞迁移,蛋白质印迹法检测HIF-1α、ACC1及上皮-间质转化(EMT)相关波形蛋白(Vimentin)及E-钙黏蛋白(E-Cadherin)的表达与SREBP-1的表达,实时荧光定量聚合酶链反应(RT-qPCR)检测低氧联合HIF-1α抑制剂PX-478(25μmol)处理A549细胞后ACC1及SREBP-1 mRNA水平变化。每项实验重复三次。结果:与常氧组相比,低氧组A549细胞迁移数增加,ACC1与HIF-1α表达上调(P均<0.01),SREBP-1表达上调(P<0.05);与低氧对照组相比,PX-478(25μmol)抑制A549细胞迁移,SREBP-1表达下调(P<0.05);低氧处理A549细胞后ACC1 mRNA上升(P<0.05),SREBP-1 mRNA水平上升(P<0.01);低氧并使用PX-478(25μmol)处理A549细胞24 h,ACC1 mRNA水平下降(P<0.05),SREBP-1 mRNA水平下降(P<0.01);转染si-RNA获得敲低SREBP-1的A549细胞,Transwell实验显示si-SREBP-1组细胞迁移数较常氧对照组减少(P<0.01);低氧处理si-SREBP-1组与si-NC组,与对照组相比si-SREBP-1组细胞迁移数减少(P<0.01)但与常氧组相比差异无统计学意义(P>0.05);Western blot检测到si-SREBP-1组ACC1表达较对照组下降(P<0.01);低氧处理si-SREBP-1组,ACC1表达较对照组下降(P<0.01);敲低ACC1抑制A549细胞迁移(P<0.05),敲低ACC1后A549细胞在常氧和5%O 2条件下细胞迁移数目差异无统计学意义(P>0.05);低氧处理敲低ACC1的A549细胞并给予LA(25μmol)促进A549细胞迁移(P<0.05)。结论:低氧通过HIF-1α/SREBP-1/ACC1途径调节脂肪酸代谢进而促进肺腺癌A549细胞迁移。
Objective:To investigate the mechanism that hypoxia promotes the migration of lung adenocarcinoma A549 cells.Methods:A549 cells were cultured and cells that knockdown of acetyl-CoA carboxylase 1(ACC1)were obtained by transfection with lentivirus,and cells that knockdown of sterol regulatory element-binding proteins-1(SREBP-1)were obtained by treated with si-RNA.A549 cells were treated with hypoxia combined with hypoxia inducible factor-1α(HIF-1α)inhibitor PX-478(25μmol);Hypoxia combined with linoleic acid(LA)(20μmol)treated A549 cells with ACC1 knockdown,and A549 cells with SREBP-1 knockdown were treated by hypoxia.Transwell migration assay was used to detect cell migration.Western blot was conducted to detect HIF-1α,ACC1 and epithelial mesenchymal transition(EMT)related proteins,Vimentin,E-Cadherin and SREBP-1;Real-time fluorescent quantitative polymerase chain reaction(RT-qPCR)was performed to detect the changes of ACC1 and SREBP-1 mRNA in A549 cells after hypoxia and HIF-1αinhibitor PX-478(25μmol)treatment.Each experiment was repeated three times.Results:Compared with the normoxic control group,hypoxia promoted the migration of A549 cells(P<0.01),and up-regulated the expressions of ACC1,HIF-1α(all P<0.01)and SREBP-1(P<0.05).PX-478(25μmol)inhibited the migration of A549 cells induced by hypoxia and down-regulated the expression of SREBP-1(all P<0.05).ACC1 mRNA and SREBP-1 mRNA levels were increased after hypoxia treatment of A549 cells(all P<0.05).The levels of ACC1 mRNA and SREBP-1 mRNA were decreased after A549 cells treated with hypoxia combined with PX-478(25μmol)for 24 h(P<0.05,P<0.01).Knockdown of SREBP-1 in A549 cells was obtained by transfection with si-RNA.Transwell migration assay showed the number of cell migration in si-SREBP-1 group was less than that in normoxia control group(P<0.01).The si-SREBP-1 group and the si-NC group were treated with hypoxia.Compared with the control group,the number of cell migration in the si-SREBP-1 group was decreased(P<0.01),however,the difference was not st
作者
金家豪
赵宝生
刘玉珍
JIN Jia-hao;ZHAO Bao-sheng;LIU Yu-zhen(Department of Thoracic Surgery,the First Affiliated Hospital of Xinxiang Medical University;Life Science Research Center,the First Affiliated Hospital of Xinxiang Medical University,Weihui 453100,China)
出处
《中国应用生理学杂志》
CAS
CSCD
北大核心
2022年第1期68-75,共8页
Chinese Journal of Applied Physiology
基金
河南省重点科技攻关项目(202102310103)
新乡医学院研究生科研创新支持计划项目(YJSCX201914Z)
新乡市科技攻关计划项目(GG 2020027)。
关键词
肺腺癌
低氧
乙酰辅酶A羧化酶1
低氧诱导因子1Α
固醇调节元件结合蛋白1
迁移
lung adenocarcinoma
hypoxia
acetyl-CoA carboxylase 1
hypoxia inducible factor 1α
sterol regulatory element binding protein 1
migration
