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

I. Kuan Wang, Kuo Ting Sun, Tsung Hsun Tsai, Chia Wen Chen, Shih Sheng Chang, Tung Min Yu, Tzung Hai Yen, Feng Yen Lin, Chiu Ching Huang, Chi Yuan Li

Research output: Contribution to journalArticle

22 Citations (Scopus)

Abstract

Abstract 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-1α (HIF-1α) in HK-2 cells, we confirmed the inhibitory binding of HIF-1α to miR-20a-5p. Significance The signaling axis of HIF-1α, miR-20a-5p, and ATG16L1 in autophagic process might be a critical adapting mechanism for ischemic kidney injury.

Original languageEnglish
Article number14437
Pages (from-to)133-141
Number of pages9
JournalLife Sciences
Volume136
DOIs
Publication statusPublished - Jul 27 2015

Fingerprint

Autophagy
MicroRNAs
Hypoxia-Inducible Factor 1
Kidney
Wounds and Injuries
Luciferases
Assays
3' Untranslated Regions
Bioinformatics
Chromatin
Cells
Hypoxia
Chromatin Immunoprecipitation
RNA Interference
Computational Biology
Messenger RNA
Constriction
Cell Survival
Proteins

Keywords

  • Acute kidney injury
  • Autophagy
  • Ischemia-reperfusion
  • MicroRNA

ASJC Scopus subject areas

  • Pharmacology, Toxicology and Pharmaceutics(all)
  • Biochemistry, Genetics and Molecular Biology(all)
  • Medicine(all)

Cite this

Wang, I. K., Sun, K. T., Tsai, T. H., Chen, C. W., Chang, S. S., Yu, T. M., ... Li, C. Y. (2015). MiR-20a-5p mediates hypoxia-induced autophagy by targeting ATG16L1 in ischemic kidney injury. Life Sciences, 136, 133-141. [14437]. https://doi.org/10.1016/j.lfs.2015.07.002

MiR-20a-5p mediates hypoxia-induced autophagy by targeting ATG16L1 in ischemic kidney injury. / Wang, I. Kuan; Sun, Kuo Ting; Tsai, Tsung Hsun; Chen, Chia Wen; Chang, Shih Sheng; Yu, Tung Min; Yen, Tzung Hai; Lin, Feng Yen; Huang, Chiu Ching; Li, Chi Yuan.

In: Life Sciences, Vol. 136, 14437, 27.07.2015, p. 133-141.

Research output: Contribution to journalArticle

Wang, IK, Sun, KT, Tsai, TH, Chen, CW, Chang, SS, Yu, TM, Yen, TH, Lin, FY, Huang, CC & Li, CY 2015, 'MiR-20a-5p mediates hypoxia-induced autophagy by targeting ATG16L1 in ischemic kidney injury', Life Sciences, vol. 136, 14437, pp. 133-141. https://doi.org/10.1016/j.lfs.2015.07.002
Wang, I. Kuan ; Sun, Kuo Ting ; Tsai, Tsung Hsun ; Chen, Chia Wen ; Chang, Shih Sheng ; Yu, Tung Min ; Yen, Tzung Hai ; Lin, Feng Yen ; Huang, Chiu Ching ; Li, Chi Yuan. / MiR-20a-5p mediates hypoxia-induced autophagy by targeting ATG16L1 in ischemic kidney injury. In: Life Sciences. 2015 ; Vol. 136. pp. 133-141.
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abstract = "Abstract 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-1α (HIF-1α) in HK-2 cells, we confirmed the inhibitory binding of HIF-1α to miR-20a-5p. Significance The signaling axis of HIF-1α, miR-20a-5p, and ATG16L1 in autophagic process might be a critical adapting mechanism for ischemic kidney injury.",
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AU - Wang, I. Kuan

AU - Sun, Kuo Ting

AU - Tsai, Tsung Hsun

AU - Chen, Chia Wen

AU - Chang, Shih Sheng

AU - Yu, Tung Min

AU - Yen, Tzung Hai

AU - Lin, Feng Yen

AU - Huang, Chiu Ching

AU - Li, Chi Yuan

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N2 - Abstract 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-1α (HIF-1α) in HK-2 cells, we confirmed the inhibitory binding of HIF-1α to miR-20a-5p. Significance The signaling axis of HIF-1α, miR-20a-5p, and ATG16L1 in autophagic process might be a critical adapting mechanism for ischemic kidney injury.

AB - Abstract 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-1α (HIF-1α) in HK-2 cells, we confirmed the inhibitory binding of HIF-1α to miR-20a-5p. Significance The signaling axis of HIF-1α, miR-20a-5p, and ATG16L1 in autophagic process might be a critical adapting mechanism for ischemic kidney injury.

KW - Acute kidney injury

KW - Autophagy

KW - Ischemia-reperfusion

KW - MicroRNA

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