title: MIR106B and MIR93 prevent removal of bacteria from epithelial cells by disrupting ATG16L1-mediated autophagy.
abstract: OBJECTIVE
  Variants in genes that regulate autophagy have been associated with Crohn's disease (CD). Defects in autophagy-mediated removal of pathogenic microbes could contribute to the pathogenesis of CD. We investigated the role of the microRNAs (miRs) MIR106B and MIR93 in induction of autophagy and bacterial clearance in human cell lines and the correlation between MIR106B and autophagy-related gene 16L1 (ATG16L1) expression in tissues from patients with CD.
  
  
  METHODS
  We studied the ability of MIR106B and MIR93 to regulate ATG transcripts in human cancer cell lines (HCT116, SW480, HeLa, and U2OS) using luciferase report assays and bioinformatics analyses; MIR106B and MIR93 mimics and antagonists were transfected into cells to modify levels of miRs. Cells were infected with LF82, a CD-associated adherent-invasive strain of Escherichia coli, and monitored by confocal microscopy and for colony-forming units. Colon tissues from 41 healthy subjects (controls), 22 patients with active CD, 16 patients with inactive CD, and 7 patients with chronic inflammation were assessed for levels of MIR106B and ATG16L1 by in situ hybridization and immunohistochemistry.
  
  
  RESULTS
  Silencing Dicer1, an essential processor of miRs, increased levels of ATG protein and formation of autophagosomes in cells, indicating that miRs regulate autophagy. Luciferase reporter assays indicated that MIR106B and MIR93 targeted ATG16L1 messenger RNA. MIR106B and MIR93 reduced levels of ATG16L1 and autophagy; these increased after expression of ectopic ATG16L1. In contrast, MIR106B and MIR93 antagonists increased formation of autophagosomes. Levels of MIR106B were increased in intestinal epithelia from patients with active CD, whereas levels of ATG16L1 were reduced compared with controls. Levels of c-Myc were also increased in intestinal epithelia of patients with active CD compared with controls. These alterations could impair removal of CD-associated bacteria by autophagy.
  
  
  CONCLUSIONS
  In human cell lines, MIR106B and MIR93 reduce levels of ATG16L1 and autophagy and prevent autophagy-dependent eradication of intracellular bacteria. This process also appears to be altered in colon tissues from patients with active CD.
authors.affiliation: Division of Hematology and Oncology, Comprehensive Cancer Center, University of Alabama at Birmingham, Birmingham, Alabama.
authors.forename: Changming
authors.initials: C
authors.lastname: Lu
authors.affiliation: Division of Hematology and Oncology, Comprehensive Cancer Center, University of Alabama at Birmingham, Birmingham, Alabama.
authors.forename: Jianfeng
authors.initials: J
authors.lastname: Chen
authors.affiliation: Division of Hematology and Oncology, Comprehensive Cancer Center, University of Alabama at Birmingham, Birmingham, Alabama.
authors.forename: Hua-Guo
authors.initials: HG
authors.lastname: Xu
authors.affiliation: Department of Pediatrics, University of Minnesota, Minneapolis, Minnesota; Departments of Pediatrics, Microbiology and Immunology, and Cell Biology and Anatomy, New York Medical College, Valhalla, New York.
authors.forename: Xianzheng
authors.initials: X
authors.lastname: Zhou
authors.affiliation: Department of Pathology, Xiangya School of Medicine, Central South University, Changsha, Hunan, China.
authors.forename: Qiongqiong
authors.initials: Q
authors.lastname: He
authors.affiliation: Key Laboratory of Pathobiology, Ministry of Education, Norman Bethune College of Medicine, Jilin University, Changchun, Jilin, China.
authors.forename: Yu-Lin
authors.initials: YL
authors.lastname: Li
authors.affiliation: Department of Surgery, 2nd Affiliated Hospital, Soochow University, Suzhou, Jiangsu, China.
authors.forename: Guoqing
authors.initials: G
authors.lastname: Jiang
authors.affiliation: Department of Surgery, 2nd Affiliated Hospital, Soochow University, Suzhou, Jiangsu, China.
authors.forename: Yuxi
authors.initials: Y
authors.lastname: Shan
authors.affiliation: Department of Surgery, 2nd Affiliated Hospital, Soochow University, Suzhou, Jiangsu, China.
authors.forename: Boxin
authors.initials: B
authors.lastname: Xue
authors.affiliation: Division of Hematology and Oncology, Comprehensive Cancer Center, University of Alabama at Birmingham, Birmingham, Alabama.
authors.forename: Rui-Xun
authors.initials: RX
authors.lastname: Zhao
authors.affiliation: Division of Hematology and Oncology, Comprehensive Cancer Center, University of Alabama at Birmingham, Birmingham, Alabama.
authors.forename: Yong
authors.initials: Y
authors.lastname: Wang
authors.affiliation: Division of Hematology and Oncology, Comprehensive Cancer Center, University of Alabama at Birmingham, Birmingham, Alabama.
authors.forename: Kaitlin D
authors.initials: KD
authors.lastname: Werle
authors.affiliation: Department of Dermatology, Boston University School of Medicine, Boston, Massachusetts.
authors.forename: Rutao
authors.initials: R
authors.lastname: Cui
authors.affiliation: Department of Systems Biology, University of Texas MD Anderson Cancer Center, Houston, Texas.
authors.forename: Jiyong
authors.initials: J
authors.lastname: Liang
authors.affiliation: Division of Hematology and Oncology, Comprehensive Cancer Center, University of Alabama at Birmingham, Birmingham, Alabama. Electronic address: zhixiangxu@uabmc.edu.
authors.forename: Zhi-Xiang
authors.initials: ZX
authors.lastname: Xu
mesh_terms: D001343:Autophagy / Q000235:genetics / Q000276:immunology*; D000071183:Autophagy-Related Proteins; D002352:Carrier Proteins / Q000276:immunology*; D016022:Case-Control Studies; D045744:Cell Line, Tumor; D003424:Crohn Disease / Q000235:genetics / Q000276:immunology*; D053487:DEAD-box RNA Helicases / Q000276:immunology; D004847:Epithelial Cells / Q000276:immunology*; D004926:Escherichia coli; D045325:HCT116 Cells; D006367:HeLa Cells; D006801:Humans; D007413:Intestinal Mucosa / Q000276:immunology / Q000378:metabolism; D035683:MicroRNAs / Q000235:genetics / Q000276:immunology*; D016271:Proto-Oncogene Proteins c-myc / Q000276:immunology / Q000378:metabolism; D043244:Ribonuclease III / Q000276:immunology
chemical_list: C518959:ATG16L1 protein, human; D000071183:Autophagy-Related Proteins; D002352:Carrier Proteins; C542541:MIRN106 microRNA, human; C543350:MIRN93 microRNA, human; D035683:MicroRNAs; D016271:Proto-Oncogene Proteins c-myc; C408087:DICER1 protein, human; D043244:Ribonuclease III; D053487:DEAD-box RNA Helicases
keywords: AIEC; ATG; Baf; CD; Cell Biology; Crohn's disease; FBS; GAPDH; GFP; Glyceraldehyde-3-phosphate dehydrogenase; IHC; IRGM1; Infection; Inflammatory Bowel Disease; LC3; UTR; adherent-invasive Escherichia coli; autophagy-related gene; bafilomycin; fetal bovine serum; green fluorescent protein; immunity-related guanosine triphosphatase family M protein 1; immunohistochemistry; light chain 3; mRNA; messenger RNA; miRNA/miR; microRNA; siRNA; small interfering RNA; untranslated region
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