Background: The microRNAs (miRNAs) regulate gene expression at the posttranscriptional level. ATG16L1, an essential component for autophagy and a risk gene for Crohn’s disease, contains two binding sites in the 3’UTR for miR-17 family, including miRs-20a, -93, -106a, and -106b. The purpose of this study was to assess the effects of these miRNAs on ATG16L1 expression and autophagic activity in HCT116 cells.
Methods: The functional binding sites in the ATG16L1 3’UTR were evaluated by transfection of pMIR-GLO vectors bearing the wild type or mutant 3’UTR into cells for luciferase reporter assay. The miRNA regulation of ATG16L1 expression was determined by quantitative real-time polymerase chain reaction and Western blot. The miRNA regulation of autophagic activity was evaluated by examining LC3II formation using Western blot and confocal imaging.
Results: Both miR-106a and miR-106b mimics inhibited starvation-induced autophagy. The miR-106b mimic reduced ATG16L1 protein expression. Luciferase reporter assays showed that mutating the binding sequence at the positions 1036 to 1042 abrogated miR-106b regulation of ATG16L1 3’UTR luciferase activity. In addition, miR-106a and miR-106b overexpression inhibited the expression of several other autophagy genes, including ATG12.
Conclusions: miR-106b targets ATG16L1 and modulates autophagy, partially through the binding site at the 3’ end of ATG16L1 3’UTR. miR-106a regulates autophagy, possibly irrelevant to ATG16L1 regulation. Both miR-106a and miR-106b regulate multiple autophagy genes so that they may play an integral role in fine-tuning autophagy.
Article first published online 30 July 2013
Section of Gastroenterology, Department of Medicine, The University of Chicago, Chicago, Illinois.
Reprints: John H. Kwon, MD, PhD, Section of Gastroenterology, Department of Medicine, The University of Chicago, 900 East 57th Street, KCBD 9118, Chicago, IL 60637 (e-mail: email@example.com).
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Supported by the National Institute of Health Grant RO3 DK086549, NIH T32 Gastrointestinal training Grant DK07074, and the NIH DDRCC core Grant P30 DK42086.
The authors have no conflicts of interest to disclose.
Received March 13, 2013
Accepted May 31, 2013