ATG5敲低后抑制人肺癌细胞H1299自噬并增强南蛇藤素诱导的细胞凋亡

摘要

Objective To establish the lung cancer cell strain with low ATG5 expression and to 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 was measured by Western blot and RFP-LC3 transfection.Cell apoptosis of ATG5 normal expression group and of ATG5 low expression group of H1299 cells was detected by FACS.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 significantly decreased 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).ConclusionsThe 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.%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的(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.