中国癌症杂志 ›› 2021, Vol. 31 ›› Issue (4): 277-284.doi: 10.19401/j.cnki.1007-3639.2021.04.006

• 论著 • 上一篇    下一篇

H2AX磷酸化抑制肺癌细胞发生上皮-间质转化的作用机制

乔婷婷 1 ,葛淑静 2 ,罗 渊 1 ,陆承荣 1 ,段连宁 3   

  1. 1. 中国人民解放军空军特色医学中心临床医学实验室,北京 100142 ;
    2. 中国人民解放军空军特色医学中心干部病房,北京 100142 ;
    3. 中国人民解放军空军特色医学中心保健诊疗科,北京 100142
  • 出版日期:2021-04-30 发布日期:2021-04-29
  • 通信作者: 段连宁 E-mail: duanln@tom.com
  • 基金资助:
    国家自然科学基金(81272303)。

Mechanism of inhibition of epithelial-to-mesenchymal transition by phosphorylated H2AX in lung cancer cells

QIAO Tingting 1 , GE Shujing 2 , LUO Yuan 1 , LU Chengrong 1 , DUAN Lianning 3    

  1. 1. Clinical Medicine Laboratory, Air Force Medical Center of PLA, Beijing 100142, China; 2. Department of Cardre Ward, Air Force Medical Center of PLA, Beijing 100142, China; 3. Department of Healthcare, Air Force Medical Center of PLA, Beijing 100142, China
  • Online:2021-04-30 Published:2021-04-29
  • Contact: DUAN Lianning E-mail: duanln@tom.com

摘要: 背景与目的:上皮-间质转化(epithelial-to-mesenchymal transition,EMT)是肿瘤细胞发生转移的关键生物学过程,阻碍EMT将抑制肿瘤转移,因此阐明EMT的分子机制具有重要意义。抑癌蛋白H2AX调控肿瘤细胞凋亡相关基因表达,进而产生抗肿瘤作用。揭示H2AX与EMT的关系,探讨H2AX调控肺癌细胞EMT可能的作用机制。方法:利用5%胎牛血清诱导肺癌A549稳定株细胞(包括H2AX基因沉默、H2AX过表达和H2AX磷酸化位点突变)产生EMT。采用蛋白质印迹法(Western blot)和实时荧光定量聚合酶链反应(real-time fluorescence quantitative polymerase chain reaction,RTFQ-PCR)检测肿瘤细胞EMT标志性分子E-cadherin和vimentin的表达、以及其他EMT相关蛋白血管内皮生长因子受体2(vascular endothelial growth factor receptor 2,VEGFR2)等分子的表达。采用transwell实验分析肺癌A549细胞的迁移和侵袭能力。结果:基因沉默肺癌A549细胞H2AX导致E-cadherin蛋白和mRNA水平下调以及vimentin蛋白和mRNA水平的上调,并增强A549细胞的迁移和侵袭能力。肺癌A549细胞过表达H2AX则上调E-cadherin蛋白和mRNA、下调vimentin蛋白和mRNA,抑制A549细胞的迁移和侵袭;H2AX磷酸化位点突变则部分消除H2AX对E-cadherin上调和vimentin的下调作用以及H2AX对A549细胞EMT的抑制作用;肺癌A549细胞中H2AX基因沉默则刺激VEGFR-2表达,过表达H2AX则抑制VEGFR-2表达;H2AX磷酸化位点突变则消除H2AX对VEGFR-2表达的抑制作用;基因沉默A549细胞中VEGFR-2则抑制EMT作用;H2AX基因沉默刺激肺癌A549细胞EMT相关因子Slug和β-catenin的表达,过表达H2AX抑制Slug和β-catenin表达;H2AX磷酸化位点突变后则消除H2AX对Slug和β-catenin表达的抑制作用。结论:γH2AX通过下调VEGFR-2以及Slug和β-catenin表达进而抑制肺癌A549的EMT过程。

关键词: H2AX, 上皮-间质转化, 肺癌细胞, 侵袭, 迁移

Abstract: Background and purpose: Epithelial-to-mesenchymal transition (EMT) is the key biological process of tumor cell metastasis. Blocking EMT inhibits tumor metastasis, therefore it is of great significance to elucidate the molecular mechanism of EMT. Tumor suppressor protein H2AX regulates apoptosis-related gene expression of tumor cells, and then produces anti-tumor effect. This study aimed to reveal the relationship between H2AX and EMT, and to explore the possible mechanism of H2AX regulating EMT in lung cancer cells. Methods: About 5% fetal bovine serum was used to induce EMT in lung cancer A549 stable cells including H2AX gene silencing, H2AX overexpression and H2AX phosphorylation site mutation. Western blot and real- time fluorescent quantitative polymerase chain reaction (RTFQ-PCR) were used to detect the expressions of E-cadherin, vimentin, vascular endothelial growth factor receptor 2 (VEGFR2) and other EMT-related proteins. Transwell experiment was used to analyze the migration and invasion of A549 cells. Results: Knockdown of H2AX in lung cancer A549 cells resulted in the downregulation of E-cadherin and the upregulation of vimentin, and enhanced the migration and invasion ability of A549 cells. Overexpression of H2AX in lung cancer A549 cells upregulated E-cadherin, downregulated vimentin, and inhibited the migration and invasion of A549 cells. Mutation of H2AX phosphorylation site eliminated the regulation of E-cadherin and vimentin by H2AX, and the inhibition of EMT by H2AX. H2AX knockdown in A549 cells stimulated the expression of VEGFR2, while overexpression of H2AX inhibited VEGFR2 expression. Mutation of H2AX phosphorylation site eliminated the VEGFR2 expression inhibition caused by H2AX. Knockdown of VEGFR2 inhibited the EMT of A549. Knockdown of H2AX stimulated the expression of EMT-related factors Slug and β-catenin, whereas overexpression of H2AX inhibited the expression of Slug and β-catenin. Reducing H2AX phosphorylation eliminated the expression inhibition of Slug and β-catenin by H2AX. Conclusion: H2AX inhibits EMT of lung cancer A549 cells by downregulating the expressions of VEGFR2, Slug and β-catenin.

Key words:  H2AX, Epithelial-to-mesenchymal transition, Lung cancer cells, Invasion, Migration