MiR-20a-5p mediates hypoxia-induced autophagy by targeting ATG16L1 in ischemic kidney injury

Aims: Autophagy is a cellular homeostatic mechanism activated under stress conditions and might act as protective response for cell survival in ischemic kidney injury. The micro RNA (miRNA) network may be critically involved in the regulation of autophagy. The aim of this study was to evaluate whether miRNA regulates autophagy in ischemic kidney injury and renal proximal tubular cells under hypoxic conditions. Materials and methods: Ischemic kidney injury was performed by clamping bilateral renal pedicles for 60 min in male mice. Human kidney proximal tubular (HK-2) cells were exposed to in vitro hypoxic conditions. ATG16L1 is essential for autophagosome formation. Bioinformatics analyses were used to select the candidate miRNA, miR-20a-5p, which potentially targets ATG16L1. Gain-of-function and loss-of-function methods were employed to evaluate the effects of miRNA on autophagy. Chromatin immunoprecipitation analysis and promoter luciferase reporter assays were used to evaluate the interaction of transcriptional factors with miRNA. Key findings: Increased expression of punctate LC3 and ATG16L1, autophagy-related proteins, and down-expression of miR-20a-5p were detected in kidneys after ischemic injury and in HK-2 cells under hypoxic conditions. 3'-untranslated region luciferase reporter assays indicated that miR-20a-5p targeted ATG16L1 messenger RNA. Over-expression of miR-20a-5p reduced the expression of LC3-II and ATG16L1 in HK-2 cells under hypoxic conditions, whereas antagomiR-20a reversed the inhibition. Using RNAi against hypoxia-inducible factor-1 alpha (HIF-1 alpha) in HK-2 cells, we confirmed the inhibitory binding of HIF-1 alpha to miR-20a-5p. Significance: The signaling axis of HIF-1 alpha, miR-20a-5p, and ATG16L1 in autophagic process might be a critical adapting mechanism for ischemic kidney injury. (C) 2015 Elsevier Inc. All rights reserved.