羰基氰氯苯腙调节非经典自噬的机制研究

Mechanism of CCCP-induced non-canonical autophagy

目的探讨可以调控羰基氰氯苯腙(carbonyl cyanide m-chlorophenylhydrazone,CCCP)诱导的不依赖自噬关键基因的非经典自噬的影响因素。方法分别将野生型细胞和自噬关键基因敲除/敲低的细胞(Atg5-KO MEF,FIP-200 KO MEF,ULK1-KO MEF,Beclin 1-KO U251)用CCCP处理,观察GFP-LC3的分布聚集情况和LC3蛋白水平的表达。荧光定量PCR检测CCCP处理后LC3B mRNA水平变化。Western blot检测FIP200-KO MEF中CCCP处理时,水通道蛋白抑制剂对LC3蛋白水平的影响。结果在Atg5-KO MEF细胞中,和对照组相比,CCCP处理组免疫荧光实验不能产生GFP-LC3自噬斑点,Western blot结果显示LC3-I型不能转变为LC3-II。而在ULK1-KO MEF,FIP200-KO MEF,及Beclin1-KD U251细胞中,CCCP处理组免疫荧光实验能产生GFP-LC3自噬斑点,同时LC3-I型也能转变为LC3-II型。但CCCP处理不影响LC3B的mRNA水平。CCCP诱导的非经典自噬能被水通道蛋白抑制剂所抑制。结论 CCCP诱导的非经典自噬需要自噬关键基因Atg5参与,但不需要Beclin 1,ULK1,FIP200的参与,不影响LC3B的转录调控。渗透压不平衡可以调节CCCP诱导的自噬。

Objective To identify the factors those regulate CCCP-induced non-canonical autophagy. Methods Different cells expressing GFPLC3 were treated with or without CCCP（ 30 μM） for 6 h. Fluorescent images were taken and cell lysates were analyzed by western blot assay. Real-time PCR was used to measure the mRNA levels of LC3 B. FIP200-KO MEF cells were cultured and treated by 30 μM CCCP with or without water channel inhibitors,for 6 h. Cell lysates were analyzed by Western blot assay. Results CCCP could not induced autophagy in Atg5-KO MEF cells. CCCP could induce non-canonical autophagy in ULK1-KO MEF,FIP200-KO MEF,and Beclin1-KD U251. CCCP treatment in FIP200-KO MEF cells had no effect on the expression level of LC3 B mRNA. We also found two distinct aquaporin water channel inhibitors could inhibit the generation of LC3 which was induced by CCCP. Conclusion CCCP induced non-canonical autophagy was Atg5-dependent,but Beclin1-,ULK1- and FIP200-independent. Osmotic imbalance could regulate CCCP-induce non-canonical autophagy.