中国癌症杂志 ›› 2013, Vol. 23 ›› Issue (6): 413-419.doi: 10.3969/j.issn.1007-3969.2013.06.003

• 论著 • 上一篇    下一篇

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

邹燕梅,熊华,肖志平,于世英,袁响林   

  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
  • 出版日期:2013-06-25 发布日期:2014-11-13

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

ZOU Yan-mei, XIONG Hua, XIAO Zhi-ping, YU Shi-ying, YUAN Xiang-lin   

  1. Cancer Center, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, Hubei, 430030, China
  • Published:2013-06-25 Online:2014-11-13
  • Contact: XIONG Hua E-mail: xionghua2001@yahoo.com.cn

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-1αshRNA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D0SF2SER等值。Western-blot检测细胞照射前后HIF-1αLC3c-parp蛋白的表达。结果:乏氧A549细胞的SF20.62,高于常氧A549细胞,SER1.45;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SF20.45SER0.72,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。

关键词: 乏氧, 自噬, 放射敏感性, HIF-1&alpha, RNAi

Abstract:

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-1a played the most important role in the regulation of hypoxia. However, it’s still unknown about the effect of HIF-1a 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 D0, 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 LC3increased significantly and the level of c-parp decreased after the radiation of hypoxic A549 cell. The level of HIF-1a increased in hypoxic A549 cells. The expression of HIF-1α in hypoxic A549 cells was suppressed markedly after transfection of HIF-1α-shRNA; this clone was named A549/HIF-1α-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 LC3decreased significantly and the expression of c-parp increased. Conclusion: We successfully established a cell model that HIF-1α expression was suppressed almost completely by RNAi. The inhibition of HIF-1α by shRNA may raise the radiosensitivity and decrease the autophagy of hypoxic A549 cells in vitro.

Key words: Hypoxia, Autophagy, Radiosensitivity, HIF-1α, RNAi