AMPK在二甲双胍诱导结肠癌细胞SW48O自噬中的作用

摘要

Objective To study whether the activity of AMPK was involved in autophagy induced by Metformin (Met) in human colon carcinoma SW-480 cells,and discover the role of AMPK in Metforrnin-induced autophagy.Methods SW480 cells were treated with different doses (0,1,5,10 rmmol/L) of Met.Western blotting was used to detect the protein level of p-AMPK,AMPK,autophagy marker protein microtubule-associated protein 1 light chain 3 (LC3) and p62,and real-time PCR to detect the mRNA level of LC3.RNAi technology was applied to silence AMPK expression in SW480 cells,then cells were treated with 10 mmol/L Met for 24 h.Western blotting was used to detect the protein level of LC3 and p62,and real-time PCR to detect the rmRNA level of LC3.Results With different doses of Met (1,5,10 rmmol/L),the phosphorylation level of AMPK was raised,as well as autophagy related proteins LC3 and its rmRNA level,the protein levels of p62 were decreased.After SW480 cells were treated with Met and 3-MA,the protein and mRNA expression levels of LC3 were decreased,and the expression level of p62 reduced.When AMPK was silenced,the protein and mRNA expression levels of LC3 were decreased,and the expression level of p62 increased.Conclusions The activity of AMPK was upregulated by Met in human colon carcinoma SW-480 cells,suggesting AMPK may be involved in the autophagy induced by Met.%目的 研究二甲双胍是否影响人结肠癌SW480细胞中AMPK活性,并探讨AMPK在二甲双胍诱导自噬中的作用.方法 用不同剂量(0、1、5、10 mmol/L)二甲双胍处理SW480细胞24 h,Western blot检测AMPK、p-AMPK以及自噬相关蛋白LC3、p62蛋白表达;荧光定量PCR检测LC3 mRNA表达.二甲双胍与自噬抑制剂3-甲基腺嘌呤(3-Methyladenine,3-MA)单独或联合处理后,Western blot检测LC3、p62表达.沉默AMPK后,10 mmol/L二甲双胍处理SW480细胞24 h,Western blot检测LC3、p62蛋白表达,荧光定量PCR检测LC3mRNA表达情况.结果 不同浓度二甲双胍(1、5、10 mmol/L)处理SW480细胞后,AMPK磷酸化水平递增上升,LC3蛋白及mRNA递增表达增加,p62递减表达降低.二甲双胍与3-MA联合处理SW480细胞后,LC3表达降低,p62表达升高.3-MA有抑制二甲双胍的作用.沉默AMPK后,LC3蛋白及mRNA表达下降,p62蛋白表达增加.结论 二甲双胍可诱导SW480细胞中AMPK活化,AMPK的活化参与了二甲双胍诱导的自噬.