基础医学与临床 ›› 2017, Vol. 37 ›› Issue (4): 531-536.

• 研究论文 • 上一篇    下一篇

ATG5敲低后抑制人肺癌H1299细胞自噬并增强南蛇藤素诱导的细胞凋亡

曾晓刚1,葛明建2   

  1. 1. 重庆市公共卫生医疗救治中心
    2. 重庆医科大学附属第一医院
  • 收稿日期:2016-07-19 修回日期:2016-12-02 出版日期:2017-04-05 发布日期:2017-03-24
  • 通讯作者: 葛明建 E-mail:mingjian_ge@hotmail.com

Knock-down ATG5 gene inhibits autophagy and enhances celastrol-induced apoptosis in human lung cancer cell H1299

  • Received:2016-07-19 Revised:2016-12-02 Online:2017-04-05 Published:2017-03-24

摘要: 目的 构建自噬相关基因5(autophagy-related gene 5,ATG5)低表达肺癌细胞株,观察肺癌细胞自噬活性,检测南蛇藤素对肺癌细胞凋亡的影响。 方法 用ATG5 shRNA技术构建ATG5敲低的人肺癌H1299细胞株作为ATG5敲低组,未敲低ATG5的H1299细胞作为对照组;荧光定量PCR和Western blot检测肺癌细胞中ATG5的表达和自噬标志物微管相关轻链蛋白3(Microtubule-associated protein 1A/1B-light chain 3,LC3)及死骨片蛋白1(Sequestosome 1,P62)表达,并转染红色荧光标记的LC3质粒观察LC3斑点聚集情况;经南蛇藤素刺激后,以流式细胞术检测细胞凋亡,最后使用Western Blot检测cleaved caspase-3、Bcl-2和Bax等蛋白的表达。 结果 慢病毒感染组ATG5较对照组mRNA和蛋白表达水平明显降低(P < 0.05);ATG5敲低后LC3-II水平下降,P62水平上升,并且转染后RFP-LC3斑点聚集减少(P < 0.05)。相比于对照组,南蛇藤素明显促进ATG5敲低细胞凋亡(P < 0.01);ATG5敲低组中促凋亡分子Bax、cleaved caspase-3表达比对照组明显增加(P < 0.05),抗凋亡蛋白Bcl-2表达减少(P < 0.05)。 结论 ATG5敲低抑制肺癌细胞自噬后,南蛇藤素能够明显增强人肺癌细胞的凋亡,提示抑制肺癌细胞自噬可能为针对性处理肺癌细胞耐药提供新的思路。

关键词: ATG5, 自噬, 南蛇藤素, 肺癌, 凋亡

Abstract: Objective To establish the lung cancer cell strain of ATG5 low expression and detect the effect of celastrol on lung cancer cell apoptosis after downregulation of autophagy. Methods H1299 was infected by lentivirus-mediated ATG5 shRNA. RT-qPCR and Western blot assays were applied to confirm the effect of ATG5 knock down. Autophagy levels were measured by Western blot and RFP-LC3 transfection. Cell apoptosis between ATG5 normal expression group and ATG5 low expression group of H1299 cells was detected by FACS after cells were treated with celastrol. Finally, Western blot was used to detect the expression of apoptosis-related proteins Bcl-2, Bax and cleaved caspase-3. Results The expression of ATG mRNA and protein levels were decreased significantly after ATG5 knockdown in H1299 cells (P < 0.05). The autophagy marker of LC3-II level was downregulated and P62 expression was upregulated after inhibition of ATG5, and the RFP-LC3 puncta reduced significantly after ATG5 knockdown (P < 0.05). Compared with control group, the apoptosis rate in ATG5 downregulation group increased significantly after celastrol treatment (P < 0.01). Pro-apoptotic proteins of Bax and cleaved caspase-3 levels were upregulated and anti-apoptotic protein of Bcl-2 level decreased after ATG5 inhibition (P < 0.05). Conclusion The effect of celastrol-induced apoptosis of lung cancer cells was enhanced after downregulation of autophagy, demonstrating inhibition autophay may be a new target of lung cancer treatment.

Key words: ATG5, autophagy, celastrol, lung cancer, apoptosis