RNAi沉默HIF-1α基因调控乏氧肺腺癌A549细胞放射敏感性和自噬能力  

Effect of HIF-1α expression inhibition by RNA interference on radiosensitivity and autophagy of hypoxic human lung adenocarcinoma cell line A549

作　　者：邹燕梅[1] 熊华[1] 肖志平[1] 于世英[1] 袁响林[1] 

机构地区：[1]华中科技大学同济医学院附属同济医院肿瘤中心,湖北武汉430030

出　　处：《中国癌症杂志》2013年第6期413-419,共7页China Oncology

基　　金：国家自然科学基金青年科学基金(No:81201779)

摘　　要：背景与目的:乏氧导致肿瘤细胞放射敏感性下降是引起肿瘤放疗抵抗、复发转移的根源。HIF-1α基因在乏氧调控中起关键作用,但HIF-1α基因在乏氧肺腺癌放射敏感性中的作用及其与自噬的关系尚未阐明。本研究建立利用RNAi沉默HIF-1α基因的乏氧肺腺癌A549细胞模型,以此探讨HIF-1α对乏氧细胞放射敏感性和自噬的影响。方法:构建靶向抑制HIF-1α的shRNA表达质粒,转染乏氧A549细胞,筛选稳定表达的克隆细胞,将其命名为A549/HIF-1α-shRNA,同时设阴性对照A549/Neg-shRNA。克隆形成实验检测细胞D0、SF2、SER等值。Western-blot检测细胞照射前后HIF-1α、LC3、c-parp蛋白的表达。结果:乏氧A549细胞的SF2为0.62,高于常氧A549细胞,SER为1.45;乏氧A549细胞照射后LC3Ⅱ增加,c-parp下调。乏氧A549细胞HIF-1α表达增加;A549/HIF-1α-shRNA细胞HIF-1α表达降低;A549/HIF-1α-shRNA细胞的SF2为0.45,SER为0.72,低于A549/Neg-shRNA细胞;A549/HIF-1α-shRNA细胞照射后LC3Ⅱ降低,c-parp上调。结论:稳定转染及RNAi技术建立的HIF-1α表达抑制克隆可以成为简单实用的细胞模型;shRNA抑制HIF-1α的表达可提高乏氧A549细胞的放射敏感性,降低自噬活性。Background and purpose： Hypoxia induced the decreased radiosensitivity of tumor cells, which was the cause of tumor radioresistance and relapse and metastasis. During the course, HIF-1α played the most important role in the regulation of hypoxia. However, it＇s still unknown about the effect of HIF-1α on the radiosensitivity of hypoxia tumor cells and the relationship with autophagy. This study was to inhibit HIF-1α expression in hypoxic lung adenocarcinoma cell line A549 with RNA interference （RNAi）, and explore its effect on hypoxic cell radiosensitivity and autophagy. Methods： Plasmids pHIF-1α-shRNA and Neg-shRNA （negative control） were constructed and transfected into hypoxic A549 cells, this positive clone was named A549/HIF-1α-shRNA. Clone formation array was applied to calculate the value of Do, SF2, SER. The expression of HIF-1α, LC3, c-parp was detected by Western blot. Results： The SF2 of hypoxic A549 cell was 0.62, which was higher than that of normoxic A549 cell, SER was 1.45. The level of LC3 1T increased significantly and the level of c-parp decreased after the radiation of hypoxic A549 cell. The level of HIF-1α increased in hypoxic A549 cells. The expression of HIF-1 α in hypoxic A549 cells was suppressed markedly after transfection of HIF-1 α-shRNA; this clone was named A549/HIF-1 α-shRNA. The SF2 and SER were significantly lower in A549/HIF-1α-shRNA cells, 0.45 and 0.72 respectively. Under the hypoxic condition and after the inhibition of HIF-1α, the expression of LC3 Ⅱ decreased significantly and the expression of c-parp increased. Conclusion： We successfully established a cell model that HIF-1α expression was suppressed almost completely byRNAi. The inhibition of HIF-1α by shRNA may raise the radiosensitivity and decrease the autophagy of hypoxic A549 cells in vitro.

关 键 词：乏氧 自噬 放射敏感性 HIF-1Α RNAi 

分 类 号：R734.2[医药卫生—肿瘤]