miR-106b Fine Tunes ATG16L1 Expression and Autophagic Activity in Intestinal Epithelial HCT116 Cells

• DOI: 10.1097/mib.0b013e31829e71cf
• Publication Date: 2013-07-31T14:07:06Z
• Authors: Zili Zhai; Feng Wu; Alice Y. Chuang; John H. Kwon
• Keywords: 0301 basic medicine; Reverse Transcriptase Polymerase Chain Reaction; Blotting, Western; Autophagy-Related Proteins; Real-Time Polymerase Chain Reaction; Gene Expression Regulation, Neoplastic; MicroRNAs; 03 medical and health sciences; Colonic Neoplasms; Autophagy; Tumor Cells, Cultured; Humans; RNA, Messenger; Carrier Proteins; 3' Untranslated Regions; Cell Proliferation

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.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.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.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.